ES2364010B1 - MUTANTS OF MU POLYMERASE DNA - Google Patents

Abstract

Mutants of the DNA polymerase MU. # Mutant DNA polymerase mutants (Pol {mu}) are described that have a higher terminal deoxynucleotidyl transferase activity than the wild enzyme (wt) and improve their potential as a molecular tool. Said mutants can be used, among other applications, in polynucleotide labeling techniques or protruding and blunt end gathering techniques.

Description

Mutants of DNA polymerase mu.

Field of the Invention

The present invention relates to the identification of mutants of the human mu DNA polymerase (Polμ) and the uses thereof. These mutants have improved characteristics with respect to the wild version of said polymerase that improve its potential as a molecular tool.

Background of the invention

The stability of genetic information depends not only on the reliability of replication systems but also on the existence of multiple repair systems, which correct most types of damage that occur in DNA. In addition to these repair systems, the existence of alternative enzymatic reactions that somehow counteract the effects of the former is evident, generating variability in the DNA. One of the enzymes involved in this type of process is the terminal deoxynucleotidyl transferase (TdT) involved in the processes of generating diversity and selectively acting on those genes encoding antigen receptors (Komori et al. (1993). Science, 261, 1171-1175).

TdT is a 58 kDa enzyme that is normally only present in immature B and T lymphocytes. This enzyme catalyzes the addition of deoxynucleotides at the 3'OH terminal end of the oligonucleotide chains without the need for a strand of template DNA (terminal deoxynucleotidyl transferase activity), thereby generating diversity in the antigen receptors of the aforementioned defense cells of the immune system. This activity of TdT has allowed this polymerase to have been widely used as a molecular tool, especially in DNA mapping techniques.

One of these marking techniques is the TUNEL assay that is based on the principle that TdT incorporates labeled deoxyuridine (e.g. dUTP-biotin) at the 3’OH ends of single and double stranded DNA breaks. The incorporation of labeled dUTP acts as a signal that can be easily detected, for example, by fluorescence techniques. The more breaks the DNA has, the greater the resulting signal will be. This technique allows, among other applications, to identify samples that are undergoing apoptotic processes or measure the quality of a biological sample.

Since the discovery of TdT, there have been few enzymes with terminal deoxynucleotidyl transferase activity found in nature and, currently, this is the only one that is marketed on a large scale (e.g. Roche Applied Science, Promega, Invitrogen).

In 2000, a new polymerase with terminal deoxynucleotidyl transferase activity and belonging to the X family of DNA polymerases, polymerase mu (Polμ), was identified and sequenced for the first time (Domínguez et al, (2000) Embo J, 19, 1731-1742). However, although it can be said that there is a wide structural similarity between Polμ and TdT, the terminal deoxynucleotidyl transferase activity of this new enzyme is comparatively much lower than that of TdT. This fact has made it impossible to enter the market as a viable alternative to TdT, with the discovery or development of new enzymes that have this activity improved or increased. To date, only the single human Polμ mutant, R387K, has a terminal deoxynucleotidyl transferase activity superior to wild polymerase and similar to that of TdT (María José Martín et al. Gordon Research Conference on Mutagenesis 07/20/2008 -07/25/2008 at Magdalen College in Oxford. “Rate limited terminal transferase activity of human Pol μ: contribution to imprecise end-joining of non-complementary ends”).

Brief Description of the Invention

The authors of the present invention have developed Polμ mutants that exhibit a higher terminal deoxynucleotidyl transferase activity than the wild or wild enzyme (Polμ wt: UniProtKB / Swiss-Prot Q9NP87: SEQ ID NO: 17) and even than TdT itself. Specifically, the invention provides several Polμ mutants with a high terminal deoxynucleotidyl transferase activity, superior to that of Polμ wt, which can be used, among others, in polynucleotide or blunt end assembly techniques (End joining).

Thus, in a first aspect of the present invention, Polμ mutants (hereinafter, mutants of the invention) are provided which have at least about 10% more terminal deoxynucleotidyl transferase activity than Polμ wt, preferably human Polμ . Preferably, said activity is at least about 25%, 50%, 75%, 100%, 200%, 300%, 400%, 500% higher than that of the Polμ wt. Similarly, the mutants of the invention also have a terminal deoxynucleotidyl transferase activity equal to or greater than that of the R387K mutant (hereinafter, also referred to as an R mutant), preferably said activity is at least about 10% higher and, more preferably, at least about 25%, 50%, 75%, 100%, 200%, 300% higher. These mutants in combination or individually are capable of being used as molecular tools, preferably, in any of the techniques in which TdT is used, either as substitutes for it or in combination with it.

In a second aspect of the invention there is provided a polynucleotide sequence (hereinafter, polynucleotide sequence or polynucleotide of the invention) that encodes any of the mutants of the invention. Likewise, any part of the invention also comprises: i) vector comprising the polynucleotide of the invention or ii) host cell comprising the polynucleotide sequence of the invention or a vector according to the invention.

In a third aspect, the invention relates to a method for the production of the mutants of the invention which preferably comprises: i) the cultivation of a host cell according to the invention and ii) the isolation of the mutant produced.

A fourth aspect of the invention refers to the use of the mutants of the invention as a molecular tool, preferably, in all those techniques where TdT participates, either as substitutes for it or in combination with it. More specifically, the mutants of the invention in combination or individually are capable of being used in tests of: i) single-stranded (single-stranded or homopolymer) and double-stranded polynucleotide endpoints (ii) protruding or blunt ends, ii) gathering of protuberant and blunt ends, iii) mold-dependent or independent polymerization, iv) filling of gaps or gaps (gap-fi lling), v) detection of DNA breaks (mismatch detection; eg TUNEL), vi) mutagenesis or generation of variability, etc.

A fifth aspect of the invention provides kits comprising any of the mutants of the invention, preferably, for carrying out any of the tests referred to in the preceding paragraph. Additionally, in addition to the components necessary to launch the corresponding assay (e.g. buffers, primers, dNTPs, ddNTPs, markers, etc.), the kits may comprise TdT or mutants thereof. The use of such kits for the aforementioned applications, for example, in tests of: i) single-stranded (single-stranded or homopolymer) and double-stranded polynucleotide ends (protruding or blunt ends), ii) protruding ends and blunt, iii) mold-dependent or independent polymerization, iv) filling of gaps or gaps (gap-fi lling), v) DNA breakage detection (mismatch detection; eg TUNEL), vi) mutagenesis or generation of variability, etc., It constitutes an additional aspect of this invention.

De fi nitions

The term "terminal deoxynucleotidyl transferase activity" or "terminal transferase activity" is defined as the ability to carry out mold independent polymerization reactions, adding nucleotides to a 3'OH end without being selected by any criteria of base complementarity . This activity can be measured by different tests on different types of substrates, preferably on homopolymeric single stranded DNA (see Examples 1 and 3). Throughout the description, when it is indicated that this activity is increased or improved, it implies that it is at least 10% higher than that of the Polμ wt, preferably that of the human Polμ. Preferably, said activity is at least about 25%, 50%, 75%, 100%, 200%, 300%, 400%, 500% higher than that of Polμ wt, preferably that of human Polμ.

The term "identity" or "percentage percent (%) of identity" refers to two or more sequences or sub-sequences that have in common a specific percentage of amino acids or nucleotides, when they are compared and aligned to find maximum correspondence between the two. . This parameter can be measured by sequence comparison techniques well known in the state of the art, preferably by BLAST or FASTA algorithms, using the parameters loaded by default.

The term "vector" refers to a linear or circular polynucleotide (e.g. plasmids, bacterial chromosome) comprising a polynucleotide sequence, wherein said vector is preferably adapted for ampli fi cation, replication and / or expression of said polynucleotide. In addition, these vectors may contain regions that allow to control or enhance the amplification, expression and / or replication of the polynucleotide, as well as others that facilitate or promote the excretion or capture of the polypeptides resulting from the expression of the polynucleotide.

The term "host cell" refers to a cell or group of cells (eukaryotic or prokaryotic) that contains a vector or polynucleotide according to the present invention.

The term "polynucleotides" refers to both double and single stranded DNA and RNA polymers. Both polymers when in double chain form can have blunt or protruding ends.

The term "nucleotides" refers to ribonucleotides, deoxyribonucleotides (dNTPs) or dideoxynucleotides (ddNTPs) that, preferably, are labeled or modified to allow their identification and / or follow-up. Preferably, nucleotide mapping is performed with fl uorophores (e.g. Cy3, Cy5, fl uorescein) or biotin. Throughout the description when nucleotides are labeled with fluorophores, they are referred to as fluorescent nucleotides.

The term "nucleotide analogs" refers to compounds or molecules that, for a specific application, behave in a similar or analogous way to a nucleotide and that do not have a particular or specific structure, although preferably their structure allows the detection of their incorporation into polynucleotides and / or stopping the polymerization reaction. Examples of these compounds can be found in other applications such as WO95 / 07920, WO2005 / 051530, WO2008 / 016909.

The term "ortholog" refers to proteins or genes that code for said proteins, with terminal transferase activity, which share at least about 40% identity with Polμ wt, preferably with human Polμ, and in successively more preferred embodiments. at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%.

The term "mutation" preferably refers to substitutions, insertions or deletions that occur at the level of polypeptides or polynucleotides that express said polypeptides. These mutations when they occur at the level of a single amino acid or triplet that encodes it are referred to as point. Within the point mutations can be differentiated between conservative and non-conservative, the former being understood as those that occur when an amino acid is replaced by another of similar characteristics, according to Table 1.

TABLE 1

The term "around", as used throughout the description, refers to the variation between ± 5% that a given value (eg, identity or activity) may present, preferably between ± 3% and more preferably ± 1%.

The term "substantially", as used throughout the description, refers to the variation between ± 10% that a given value (eg activity) may present, preferably between ± 5% and more preferably ± one%.

The terms "fragments" or "subsequences" refer to portions of polypeptides or polynucleotides that code for said polypeptides, which maintain the increased or substantially increased terminal transferase activity.

Brief description of the fi gures

Figure 1 shows the terminal transferase activity of each of the mutants [μ-R387K-F389G, μQ275M, μR387K-Q275M, μR387K-Chloop1, μ-N457D, μ-S458N and μ-N457D-S458N (Example 1)], in comparison with that of the Polμ wt and that of the commercial TdT.

Figure 2 shows the terminal transferase activity of the double mutants (μ-R387K / Q275M and μ-R387K / Chloop1) at decreasing doses of polymerase (400 nM, 200 nM and 100 nM).

Figure 3 shows the terminal transferase activity of the double mutants (μ-R387K / Q275M and μ-R3 87K / Chloop1) and of the simple mutants (μ-R387K, μ-Chloop1 and μ-Q275M and compared to that of the Polμ wt, with each of the 4 dNTPs separately.

Figure 4 shows the terminal transferase activity of the double mutants (μ-R387K / F389G, μ-R387K / Q275M and μ-R387K / Chloop1) and of the simple mutants (μ-R387K and μ-Q275M), compared to of the Polμ wt, with each 4 dNTPS in the presence of Co2 + as activating metal.

Figure 5 shows the results of the test performed to compare whether the double mutants (M3 = μ-R387K / Q275M; M7 = μ-R387K / Chloop1) and simple (R = μ-R387K; Ch = μ-Chloop1; M2 = μ -Q275M) have affected their ability of polymerization dependent on mold with respect to that of Polμ wt.

Figure 6 shows the mold dependence of double mutants (M3 and M7) and simple mutants (R, Ch and M2) in the context of a 1 nucleotide DNA gap with phosphate.

Figure 7 shows the results of the dye fluorescent DNA or ddNTPs labeling assay, with the indicated mutants of Polμ (M1-M7), together with Polμ wt and TdT on single chain polynucleotides.

Figure 8 shows the results of the dye fluorescent DNA or ddNTPs mapping test, with the indicated mutants of Polμ (R, Ch, M2, M3 and M7), on single chain polynucleotides.

Figure 9 shows the results of the screening test with fluorescent derivatives of DNA or ddNTPs, with the indicated mutants of Polμ (R, Ch, M2, M3 and M7), on single chain polynucleotides.

Figure 10 shows the results of the screening test with fluorescent derivatives of DNA or ddNTPs, with the indicated mutants of Polμ (R, Ch, M2, M3 and M7), on single chain polynucleotides.

Figure 11 shows how the tidal reaction with M3 and M7 is highly effective with the 4 fluorescent ddNTPs, both in the presence of Mn2 + and Co2 +.

Figure 12 shows the alignment of the Polμ wt polypeptide sequences of Mus musculus, Rattus norvegicus and Bos taurus with the M3 mutant. At the top, the arrow indicates amino acid 275 of human Polμ wt. At the bottom, the arrow indicates amino acid 387 of human Polμ wt.

Figure 13 shows the alignment of the polypeptide sequences of human Polμ wt and TdT of human, murine and bovine origin. The arrows show the position of amino acids 275, 387, 457 and 458 in the human Polμ wt.

Figure 14 shows the three-dimensional structure of human Polμ wt. A) the arrow indicates the position of amino acid R387, B) the arrow indicates the position of amino acid Q275, C) the arrow indicates the position of amino acid N457 and D) the arrow indicates the position of amino acid S458.

Detailed description of the invention

The TdT enzyme, also known as terminal deoxynucleotidyl transferase or terminal transferase, is a specialized DNA polymerase that is primarily expressed in immature B and T lymphocytes, in addition to certain types of tumors. Until the discovery of Polμ in 2000 by the laboratory of Dr. Luis Blanco (Dominguez et al, (2000)), TdT was the only known polymerase with the ability to add or bind nucleotides to a 3 'OH end of polynucleotides without requiring a mold chain. However, as mentioned above, the low terminal deoxynucleotidyl transferase activity of Polμ wt compared to TdT has made it impossible for this enzyme to date to become a viable alternative to TdT.

In the present invention, a series of Polμ wt mutants are presented that increase the terminal deoxynucleotidyl transferase activity of wild Polμ and even TdT. Preferably, said mutants are derived from or derived from human Polμ wt, although they can also be obtained from the Polμ of other vertebrates (orthologous Polμ polymerases), preferably mammals, such as Mus musculus, Rattus norvegicus, Bos taurus, among others .

Obtaining mutants, from polymerases orthologous to human Polμ wt (or the gene that encodes them) and the teaching of the present invention, can be carried out simply by a person skilled in the art, as explained in Example 12. Similarly, new mutants with increased terminal transferase activity, as with the mutants of the present invention, can also be readily obtained, as shown in Example 13.

Polμ mutants and polynucleotides of the invention

TdT and Polμ wt are two enzymes that belong to the X family of DNA polymerase that are characterized by being small (between 39 and 66 KDa), both being generally quite inaccurate during DNA synthesis. They are distributive enzymes with little capacity to synthesize more than a few bases before dissociating from DNA. In addition to belonging to the same family and having terminal deoxynucleotidyl transferase activity, these polymerases have a BRCT domain (C-terminal domain of BRCA1), involved in the protein-protein interaction, which is not present in other members of family X like polymerase beta (Polβ). When this domain is eliminated together with the rest of the amino terminus (NH2-BRCT-) and even with part of the 8 KDa domain (NH2-BRCT-KDa-) the TdT activity of these enzymes is not affected (see table 2).

The 8 KDa domain is also characteristic of the enzymes of this family, being present in both Polμ wt and TdT. This domain gives these polymerases the ability to anchor the gaps that occur in the DNA, allowing them to effectively perform their biological activity. Even, the distribution of amino acids between the different domains of these two enzymes is quite similar, as can be seen in Table 2.

Despite the clear structural similarities between the two proteins, the degree of identity they share is around 40% and, therefore, they have a large number of different or non-conserved amino acids (approximately 300). By aligning and comparing sequences between TdT and Polμ wt, the authors of the present invention identified and selected a group of amino acids conserved in Polμ wt and TdT, but different between both enzymes. These amino acids were reversed in Polμ wt towards the amino acid of TdT in order to obtain a series of single and double mutants with a potential better terminal deoxynucleotidyl transferase activity. In addition to these point mutants, another mutant was generated that included a point mutation and the substitution of the loop-1 subdomain with the same TdT subdomain (see Table 2). The assay of these mutants has offered surprising results in terms of their terminal deoxynucleotidyl transferase activity, such that the present invention provides a series of Polμ mutants that exhibit a greater terminal transferase activity than Polμ wt and even that TdT itself.

Thus, in a first aspect of the present invention, the mutants of the invention have at least about 10% more terminal deoxynucleotidyl transferase activity than Polμ wt, preferably human Polμ wt. Preferably, said activity is at least about 25%, 50%, 75%, 100%, 200%, 300%, 400%, 500% higher than that of the Polμ wt. Similarly, the mutants of the invention also have a terminal deoxynucleotidyl transferase activity equal to or greater than that of the R mutant, preferably said activity is at least about 10% higher and, more preferably, at least about 25% , 50%, 75%, 100%, 200%, 300% higher. These mutants, in combination or individually, are likely to be used as molecular tools, preferably, in any of the techniques in which TdT is used, as substitutes for it or in combination with it. In a preferred embodiment of this aspect of the invention the mutants comprise at least two mutations.

In a preferred embodiment, the Polμ mutants comprise a polypeptide sequence with at least 60% identity with the sequence SEQ ID NO: 17 (Polμ wt: UniProtKB / Swiss-Prot Q9NP87), or with any of its SEQ ID NO sub-sequences : 1 and SEQ ID NO: 2, or with fragments thereof, wherein said mutants comprise at least two mutations and maintain an increased terminal transferase activity. Preferably, said mutations are (i) a first point mutation at position 387 and (ii) a second mutation consisting of:

a) a point mutation in a conserved amino acid of Polμ, where preferably said point mutation consists of a reversal towards the homologous amino acid present in TdT, or

b) a mutation in the subdomain loop-1.

In more preferred embodiments the degree of identity of the Polμ mutants with the sequence Q9NP87, any of its sub-sequences SEQ ID NO: 1 and SEQ ID NO: 2, or fragments thereof is about at least 65, 70% , 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%.

Also, preferably, the first point mutation (i) of the Polμ mutants consists in replacing the amino acid found in position 387 with any of the amino acids selected from the group comprising: Glutamine, Asparagine, Cysteine, Threonine, Serine, Methionine, Lysine, Arginine, Histidine or the like thereof, although even more preferably said point mutation consists of R387K substitution or reversal (non-conservative mutation).

Preferably, the second mutation (ii) corresponding to the point mutation (a) is located at position 275. Preferably, this mutation consists in replacing the amino acid found in said position (275) with any of the amino acids selected from the group comprising: Glutamine, Asparagine, Cysteine, Threonine, Serine, Methionine or the like thereof, although even more preferably said point mutation consists of the Q275M substitution (conservative mutation).

In another also preferred embodiment, the second mutation (ii) consists of (b) a mutation in the loop-1 subdomain, such as the replacement or modification of the loop-1 subdomain or fragments thereof by: i) a sequence with at least about 10% identity with SEQ ID NO: 3 (loop-1 of Polμ), preferably, with at least about 15%, 20%, 25%, 30%, 50%, 60%, 70% , 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or ii) by a sequence with at least about 10% identity with SEQ ID NO: 4 (loop -1 of TdT), preferably, with at least about 15%, 20%, 25%, 30%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%. In a particular embodiment, said sequence is SEQ ID NO: 4, which shares about 20% identity with SEQ ID NO: 3.

More specifically, the mutants of the invention have the sequences SEQ ID NO: 5, which comprises the point mutations R387K and Q275M (hereinafter, mutant M3), or SEQ ID NO: 6, which comprises the point mutation R387K and the insertion of the TdT loop-1 in place of Polμ loop-1 (hereinafter, M7 mutant). In the same way, the invention also comprises fragments or sub-sequences of the sequences SEQ ID NO: 5 or SEQ ID NO: 6 comprising said mutations and, preferably, substantially maintaining their increased terminal deoxynucleotidyltransferase activity. Preferably said fragments have the sequences SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

In accordance with the invention, it also provides a mutant comprising a polypeptide sequence with at least 60% identity with the sequence Q9NP87 (Polμ wt human: SEQ ID NO: 17) and at least one point mutation at position 275, wherein said mutant exhibits enhanced terminal deoxynucleotidyl transferase activity

or increased. This activity is at least about 10%, 25%, 50%, 75%, 100%, 200%, 300% higher than that of the Polμ wt. Preferably, said mutation at position 275 consists in the substitution of the amino acid found in said position by Glutamine, Asparagine, Cysteine, Threonine, Serine, Glycine, Alanine, Valine, Leucine, Isoleucine, Methionine, Proline, Phenylalanine and Tryptophan, although even more preferably said point mutation consists in the Q275M substitution. More specifically, this mutant has the sequence SEQ ID NO: 11 (hereinafter, mutant M2) or fragments thereof that comprise the mutation at position 275 and, preferably, substantially maintain its increased terminal transferase activity. Examples of these fragments or subsequences are the sequences SEQ ID NO: 12 and SEQ ID NO: 13.

A second aspect of the invention provides a polynucleotide sequence encoding any of the mutants of the invention. Likewise, they also form part of the invention: (i) a vector, comprising any of the polynucleotide sequences according to the invention, and (ii) a host cell comprising any of the polynucleotide sequences or vectors of the invention.

More specifically, the polynucleotide sequence of the invention comprises any of the sequences SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16, which encode respectively for mutants M3, M7 and M2. Also part of the invention are the sub-sequences or fragments of the polynucleotide sequence of the invention that code for any of the sub-sequences or fragments of the mutants of the invention. All these sequences and sub-sequences can be modified by silent mutations (eg point mutations, deletions, insertions or substitutions) that have no effect on the activity of the mutant in question or do not modify its activity substantially or that, simply, due to the degeneracy of the code genetic, do not modify its sequence and consequently its activity. Thus, any sequence with at least about 40% identity with any of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO.16, which, preferably, does not modify, is also part of the present invention. their activity or not substantially modify it. Preferably, said identity is at least about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%.

Production methods of Polμ mutants

In order to produce the mutants of the invention, the polynucleotide sequences of the invention are preferably incorporated into a vector comprising a promoter or regulatory sequence operatively related to the sequence encoding the mutant of the invention. Thus, a third aspect of the invention also refers to a method for the production of the mutants of the invention comprising: i) the cultivation of a host cell comprising any of the polynucleotides or vectors of the invention and ii) Isolation of the mutant produced. Preferably, the cell culture is performed under conditions that promote the growth of the host cell and / or the expression of the polynucleotide of the invention. Virtually any host cell that allows the expression of the polynucleotide of the invention can be used (e.g., bacteria, yeasts, etc.). Similarly, the invention also comprises the expression or production of the mutants of the invention by chemical synthesis or other cell-free expression systems ("Cell-Free Expression Systems") well known in the state of the art.

Uses of Polμ mutants

A fourth aspect of the invention refers to the use of the mutants of the invention as a molecular tool, preferably, in all those techniques where TdT is used. Mutants can be used in these types of techniques, as substitutes for TdT or in combination with it. More specifically, the mutants of the invention, in combination or individually, are likely to be used preferably in: i) single-stranded polynucleotide (heteropolymer or homopolymer) and double-stranded (double-ended (bulging or blunt), ii) assembly tests of protuberant and blunt ends, iii) mold-dependent or independent polymerization, iv) filling of gaps (gap-fi lling), v) DNA break detection (mismatch detection; eg TUNEL), vi) mutagenesis, etc.

Thus, this aspect provides a method for elongation of a target polynucleotide comprising: i) contacting a target polynucleotide with the mutant of the invention and nucleotides or nucleotide analogs (reaction mixture) and ii) subjecting the reaction mixture at conditions that favor the insertion of at least one nucleotide or nucleotide analogs at the 3'OH end of the target polynucleotide. In a preferred embodiment the number of nucleotides or nucleotide analogs inserted is at least 1, 2, 3, 4 or 5 nucleotides and in successively more preferred embodiments between 1 and 20.3 and 20.5 and 20.1 and 10.3 and 10.5 and 10.

The invention also provides methods for polynucleotide mapping comprising i) contacting a target polynucleotide with the mutant of the invention and labeled nucleotides or nucleotide analogues (reaction mixture), ii) subjecting the reaction mixture to conditions that favor the insertion of at least one nucleotide or analog thereof at the 3'OH end of the target polynucleotide, and iii) detect the presence or not of insertion. In a preferred embodiment the number of nucleotides or labeled nucleotide analogs inserted is at least 1, 2, 3, 4 or 5 nucleotides and in successively more preferred embodiments between 1 and 20, 3, 20, 5, 20, 1, 10, 10, 10, and 10.

The mutants of the invention are also capable of being employed in gap fi ling methods or techniques comprising: i) contacting a target polynucleotide, comprising at least one gap or gap of at least one nucleotide, with the mutant of the invention and nucleotides or nucleotide analogs (reaction mixture), and ii) subjecting the reaction mixture to conditions that favor the insertion of at least one nucleotide or nucleotide analog at the 3'OH end of the gap. Additionally, this method comprises iii) detecting whether or not the gap has been filled in and / or iv) the addition of enzymes (eg nucleases, ligases), which bind to nucleotides or nucleotide analogs introduced into the target polynucleotide, or Hydrolyze the target polynucleotide. In a more preferred embodiment the gap size is at least 1, 2, 3, 4 or 5 nucleotides and, more preferably, between 1 and 10 nucleotides. These reactions can be further enhanced by the presence of a phosphate group in the 5'P regions of the target polynucleotide.

Because the mutants of the invention have the ability to incorporate nucleotides independently of template, they are excellent candidates for use in end-gathering techniques, whether they are bulging or blunt. Thus, the invention also provides a method for gathering double-stranded polynucleotide ends comprising: i) contacting a double-stranded target polynucleotide with the mutant of the invention and at least two complementary nucleotides or nucleotide analogs ( reaction mixture), and ii) placing the reaction mixture under conditions that favor the introduction of a sufficient number of nucleotides or nucleotide analogs at each end of the target polynucleotide for the meeting to occur. Additionally, this method comprises iii) the addition of enzymes (e.g. ligases) that bind the ends once they are combined. In a preferred embodiment the number of nucleotides or nucleotide analogs inserted is at least 1, 2, 3, 4 or 5 nucleotides and in successively more preferred embodiments between 1 and 20, 3 and 20, 5 and 20, 1 and 10, 3 and 10, 5 and 10.

Another of the important applications of the TdT activity is its use in the TUNEL trials (TdT-mediated dUTPbiotin nick end labeling) that allow the detection of biological samples that are undergoing apoptosis (Gavrieli,

Y. et al. (1992) Identi fi cation of programmed cell death in situ via speci fi c labeling of nuclear DNA fragmentation. J. Cell. Biol. 119, 493,501). When the apoptosis signals are triggered in the cell a breakage process occurs

or DNA fragmentation. A polymerase with terminal deoxynucleotidyl transferase activity has the ability to introduce nucleotides or nucleotide analogs into such breaks thus making the apoptotic process visible. Thus, the present invention provides a method for the detection of apoptotic samples, preferably in their early stages. Said method comprises i) contacting a sample, containing genetic material of potentially apoptotic cells, with the mutant of the invention and at least one nucleotide or nucleotide analogue (reaction mixture), ii) subjecting the reaction mixture to conditions that favor the insertion of nucleotides or analogs thereof, and iii) detect the presence or not of insertion, preferably by fluorescence, where the detection of insertion is indicative of the presence of fragmented genetic material and, consequently, of the beginning of the apoptotic process In the sample. This same method can also be used in techniques aimed at checking the viability of a biological sample, such as sperm analysis in assisted reproduction techniques, where in the sperm with normal DNA only background fluorescence is detected, while the Spermatozoa with fragmented DNA (multiple 3'OH terminals) are stained with intense fluorescence. The fluorescence can be detected, preferably, both by flow cytometry and through fluorescent microscopy.

In any of the mentioned methods in which it is necessary to detect the presence or absence of insertion of nucleotides or analogs thereof, this detection can be done by multiple methodologies well known in the state of the art, such as fluorescence. Preferably, the samples to be labeled or detected are compared with control samples, so that the test sample is considered positive when it has at least about 5% more insertion than the control sample and in successively more preferred embodiments at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%.

As mentioned above, in any of the methods described the mutants of the invention can be found in combination with TdT or mutants thereof, as a way to improve the efficiency of the techniques. Even several mutants of the invention can be combined with TdT with the same purpose. Another way of increasing the efficiency of the mentioned methods comprises the addition of ions, preferably Mn2 + oCo2 +, to the reaction mixtures, since these are capable of increasing the catalytic efficiency of the mutants of the invention.

Kits

A fifth aspect of the invention relates to kits that incorporate any of the mutants of the invention, preferably, to carry out any of the methods mentioned above. Additionally, in addition to the components necessary to launch the corresponding assay (eg buffers, primers, dNTPs, ddNTPs, hNTPs, nucleotide analogs, ions, etc.), the kits may also comprise TdT or mutants thereof as a way to improve the efficiency of these tests.

The use of such kits for the aforementioned applications, for example, in tests of: i) single-stranded (single-stranded or homopolymer) and double-stranded polynucleotide ends (protruding or blunt ends), ii) protruding ends and blunt, iii) mold-dependent or independent polymerization, iv) filling of gaps or gaps (gap-fi lling), v) DNA breakage detection (mismatch detection; eg TUNEL), vi) mutagenesis or generation of variability, etc., It constitutes an additional aspect of this invention.

Example 1

Terminal Transferase Reaction

This assay shows the terminal transferase activity of each of the mutants, compared to the human Polμ wt [SEQ ID NO: 17] and the commercial TdT of Promega (Fig. 1). The maximum extent is analyzed with each of the 4 dNTPS separately, on a 3'OH end of single chain homopolymeric DNA (PoliT). Initially, those mutants that produced a striking stimulation of terminal transferase activity with respect to Polμ wt were selected for the conduct of DNA 3'OH end-labeling assays.

Materials and methods

The fluorescent oligonueleotide used to evaluate terminal transferase activity was PoliT-Cy5 (PoliT15-CY5), which was purchased from Sigma. The dNTPs (dATP, dCTP, dGTP and dTTP) were purchased from GE Healthcare. The polymerization reactions were carried out in a volume of 10 μl, in the presence of 1 mM MnCl 2, 50 mM TrisHCl (pH 7.5), 1 mM dithiothreitol (DTT), 4% glycerol, 0.1 mg / ml bovine serum albumin ( BSA), 20 nM of the fluorescent oligonucleotide indicated in each case, 100 μM of the dNTP indicated in each case, and 600 nM of the protein indicated in each case, except for the commercial TdT of Promega (1 unit). After a 30 min incubation at 37 ° C, the reactions were stopped by adding 5.6 µl of loading buffer (95% formamide, 10 mM EDTA (ethylenediaminetetraacetic acid)). These samples were analyzed by electrophoresis in 20% polyacrylamide gels and 8 M urea and subsequent fluorescent signal reading using a Typhoon 9410 (GE Healthcare) device.

Results (see Figure 1)

-

The combined mutant R387K-F389G (M1) has greatly reduced the intrinsic terminal transferase activity of Polμ wt.

-

The Q275M (M2) mutation alone seems to slightly stimulate the terminal transferase with respect to Polμ wt.

-

The changes R387K-Q275M (M3) and R387K-Chloop1 (M7) are the truly spectacular ones, since they consume all the starting substrate (which is very applicable to 3'OH end marking since it can be a near tidal efficiency 100%) and extend to large sizes (which can be applied in “tailing” reactions).

-

The changes N457D (M4), S458N (M5) and their combination also stimulate terminal transferase activity.

The point mutations Q275M, N457D, S458N and R387K correspond to reversals (substitutions) towards the amino acid that is conserved in TdT as can be seen in Figure 13. The first three mutations are conservative mutations, since the amino acid to which reversed belongs to the same group (see Table 1), and instead the R387K mutation is non-conservative.

Example 2

Comparative terminal transferase test between the two double mutants in the series: μ-R387K / Q275M and μ-R387K / Chloop1

A reaction similar to that of Example 1 was carried out, but at decreasing doses of polymerase (400 nM, 200 nM and 100 nM). At 400 nM protein, the extension of about 100% of the original starting substrate was maintained. At lower doses of protein, although terminal transferase activity remained strongly stimulated, the extent of the original starting substrate was not as effective (Figure 2).

Materials and methods

The fluorescent oligonucleotide used to evaluate terminal transferase activity was PoliT-Cy5 (PoliT15-CY5), which was purchased from Sigma. The dNTPs were purchased from GE Healthcare. The polymerization reactions were carried out in a volume of 10 μl, in the presence of 1 mM MnCl 2, 50 mM TrisHCl (pH 7.5), 1 mM DTT, 4% glycerol, 0.1 mg / ml BSA, 20 nM of the fluorescent oligonucleotide indicated in each case, 100 μM of the dNTP indicated in each case, and the protein dose indicated in each case. After a 30 min incubation at 37 ° C, the reactions were stopped by adding 5.6 µl of loading buffer (95% formamide, 10 mM EDTA). These samples were analyzed by electrophoresis in 20% polyacrylamide and urea8M gels and subsequent fluorescent signal reading using Typhoon 9410 (GE Healthcare) equipment.

Example 3

Terminal transferase activity assay of double mutants (μ-R387K / 0275M and μ-R387K / Chloop1) compared to the simple mutants of each of them

The maximum extent is analyzed with each of the 4 dNTPS separately on a 3'OH end of single-stranded homopolymeric DNA (Poly T). It is determined whether single mutations are sufficient by themselves to stimulate terminal transferase activity to the extent that it has been proven, or the combination of several mutations is necessary to achieve the potent activity achieved in the previous tests.

Materials and methods

The fluorescent oligonueleotide used to evaluate terminal transferase activity was PoliT-Cy5 (PoliT15-CY5), which was purchased from Sigma. The dNTPs were purchased from GE Healthcare. The polymerization reactions were carried out in a volume of 10 μl, in the presence of 1 mM MnCl 2, 50 mM TrisHCl (pH 7.5), 1 mM DTT, 4% glycerol, 0.1 mg / ml BSA, 20 nM of the fluorescent oligonueleotide indicated in each case, 100 μM of the dNTP indicated in each case, and 600 nM of the protein indicated in each case, except for the commercial TdT of Promega (1 unit). After a 30 min incubation at 37 ° C, the reactions were stopped by adding 5.6 µl of loading buffer (95% formamide, 10 mM EDTA). These samples were analyzed by electrophoresis in 20% polyacrylamide and urea8M gels and subsequent fluorescent signal reading using Typhoon 9410 (GE Healthcare) equipment.

Results (see Figure 3)

-

The simple mutant R387K (R) achieves an effective increase in the intrinsic terminal transferase activity of Polμ wt, especially with dT and dC. However, it is far from reaching the levels reached by any of the combined mutants (M3 and M7).

-

The simple mutant μ-Chloop1 (Ch) achieves a change in the pattern of insertion of dNTPs with respect to Polμ wt, but does not imply a particularly striking stimulus of its terminal transferase activity.

-

The mutant Q275M (M2) achieves a certain stimulus of terminal transferase activity compared to Polμ wt.

The combination of the two mutations that make up each selected mutant (μ-R387K / Q275M and μ-R387K / Chloop1) results in spectacular levels of terminal transferase activity. Each of the simple mutations separately implies a certain improvement over Polμ wt in this regard.

Example 4

Test of terminal transferase activity of double mutants (μ-R387K / Q275M and μ-R387K / Chloop1) compared to the simple mutants of each of them and in the presence of Co2 + as activating metal

In this case, although the insertion pattern of dNTPs is somewhat different from that of Mn2 +, and in general it is less advanced, it is clear that the double mutants (μ-R387K / Q275M and μ-R387K / Chloop1) stand out clearly with respect to the simple mutants in terms of terminal transferase activity.

Materials and methods

The fluorescent oligonucleotide used to evaluate terminal transferase activity was PoliT-Cy5 (PoliT15-CY5), which was purchased from Sigma. The dNTPs were purchased from GE Healthcare. The polymerization reactions were carried out in a volume of 10 μl, in the presence of 100 mM of cacodylate buffer (pH 6.8), 1 mM CoCl2, 0.1 mM DTT, 20 nM of the fluorescent oligonucleotide indicated in each case, 100 μM of the dNTP indicated in each case, and 600 nM of the protein indicated in each case, with the exception of the commercial TdT of Promega (1 unit). After a 30 min incubation at 37 ° C, the reactions were stopped by adding 5.6 µl of loading buffer (95% formamide, 10 mM EDTA). These samples were analyzed by electrophoresis in 20% polyacrylamide gels and 8 M urea and subsequent fluorescent signal reading using a Typhoon 9410 (GE Healthcare) device.

Results (see Figure 4)

The combination of the two mutations that make up each selected mutant (μ-R387K / Q275M and μ-R387K / Chloop1) achieves spectacular levels of terminal transferase activity. Each of the simple mutations separately implies a certain improvement over Polμ wt in this regard.

Example 5

Mold Dependency Test

With this test it is checked if the double mutants have been affected by their ability to mold dependent polymerization with respect to Polμ wt. It is convenient that this activity be maintained within certain levels, since this would allow its application in dizzying reactions with double-stranded DNA templates or with template.

Materials and methods

The fluorescent oligonucleotide SP1C-FLO (GATCACAGTGAGTAC-FLO) was hybridized with oligonucleotide T13 (G) (AGAAGTGTATCTGGTACTCACTGTGATC) to generate the DNA substrate indicated in the upper part of Figure 5. Both oligonucleotides were purchased from Sigma. The dNTPs were purchased from GE Healthcare. The polymerization reactions were carried out in a volume of 10 μl, in the presence of 2.5 mM MgCl2.50 mM TrisHCl (pH 7.5), 1 mM DTT, 4% glycerol, 0.1 mg / ml BSA, 10 nM of the fluorescent DNA hybrid, the dose of dNTP / dNTPs indicated in each case, and 100 nM of the protein indicated in each case. After a 30 min incubation at 37 ° C, the reactions were stopped by adding 5.6 µl of loading buffer (95% formamide, 10 mM EDTA). These samples were analyzed by electrophoresis in 20% polyacrylamide gels and 8 M urea and subsequent fluorescent signal reading using a Typhoon 9410 (GE Healthcare) device.

Results

As can be seen in the left part of Figure 5, the 4 dNTPs are supplied, in order to try to verify the maximum extent that the protein can carry out on the DNA substrate indicated in the upper part. Only the simple Chloop1 mutation seems to produce a small decrease in elongation, without the polymerization capacity disappearing. The M7 mutant, which contains the Chloop1 mutation is also affected to the same degree. The M2 and M3 mutants even seem to improve the elongation capacity of Polμ wt itself, which is very positive.

A similar reaction is carried out on the right side of Figure 5, but supplying only the complementary nucleotide to the first position in the mold. The step to +1 is done very efficiently with all cases.

Example 6

Mold Dependency Test

In this assay, mold dependence was verified with double mutants (M3 and M7) (see Figure 6) and simple mutants in the context of a 1 nucleotide DNA gap with phosphate.

Materials and methods

The fluorescent oligonucleotide SP1C-FLO (GATCACAGTGAGTAC-FLO) was hybridized with oligonueleotide T13 (G) (AGAAGTGTATCTGGTACTCACTGTGATC) and Dgl-P (AGATACACTTCT-P) to generate the DNA substrate indicated in Figure 6. They were bought from Sigma. The dNTP was purchased from GE Healthcare. The polymerization reactions were carried out in a volume of 10 μl, in the presence of 2.5 mM MgCl 2, 50 mM TrisHCl (pH 7.5), 1 mM DTT, 4% glycerol, 0.1 mg / ml BSA, 10 nM of the fluorescent DNA hybrid, the dose of dNTP / dNTPs indicated in each case, and 100 nM of the protein indicated in each case. After a 30 min incubation at 37 ° C, the reactions were stopped by adding 5.6 µl of loading buffer (95% formamide, 10 mM EDTA). These samples were analyzed by electrophoresis in 20% polyacrylamide and urea8M gels and subsequent fluorescent signal reading using Typhoon 9410 (GE Healthcare) equipment.

Results

As can be seen in Figure 6, in this type of DNA context all mutants show a similar insertion efficiency.

Example 7

3’OH End Marking Test

The complete series of Polμ mutants was tested, together with Polμ wt and TdT in a 3'OH end reaction reaction of a single chain oligonucleotide. A single chain oligonucleotide labeled with Cy5 at its 5 ′ end was used as a substrate for monitoring. Fluorescein-labeled ddATP (FLO) capable of being incorporated by polymerase at the 3OH ′ end of the DNA was supplied. The DNA channel allows you to track the original DNA, and check the proportion of oligo that has been marked (position +1) against the unmarked remainder (position 0). The ddNTP channel allows you to check the entry at position +1 of the ddNTP marked with FLO.

Materials and methods

The fluorescent oligonucleotide used was: SP1C-Cy5 (GATCACAGTGAGTAC-Cy5), which was purchased from Sigma. The ddATP-Cy5 was purchased from Perkin-Elmer. The reactions were carried out in a volume of 10 μl, in the presence of 1 mM MnCl 2, 50 mM TrisHCl (pH 7.5), 1 mM DTT, 4% glycerol and 0.1 mg / ml BSA (except the channel of TdT, which carried 100 mM of cacodylate buffer, 1 mM CoCl2 and 0.1 mM DTT), 10 nM of the fluorescent oligo of DNA, 1 μM ddATP-FLO, and 600 nM of the indicated protein in each case. After a 30 min incubation at 37 ° C, the reactions were stopped by adding 5.6 µl of loading buffer (95% formamide, 10 mM EDTA). These samples were analyzed by electrophoresis in 20% polyacrylamide and urea8M gels and subsequent fluorescent signal reading using Typhoon 9410 (GE Healthcare) equipment.

Results

As can be seen in Figure 7, Polμ wt is capable of marking around 50% of the oligo supplied. The mutants M4, M5 and M6 (μ-N457D-S458N) have a similar behavior. The MI mutant sees its ability to mareaje to Polμ wt. The M2 mutant represents an improvement over Polμ wt although it does not reach 100% of the starting substrate. The M3 and M7 are capable of marking 100% of the original starting substrate, even exceeding the levels achieved with the commercial TdT of Promega.

Example 8

3’OH end tide test

Test similar to that performed in Example 7, in which the effectiveness of the M3 and M7 mutants in tidal reactions is compared with respect to the simple mutations that make up each of the mutants.

Materials and methods

The fluorescent oligonueleotide used was: SP1C-Cy5 (GATCACAGTGAGTAC-Cy5), which was purchased from Sigma. The ddATP-Cy5 was purchased from Perkin-Elmer. The reactions were carried out in a volume of 10 μl, in the presence of 1 mM MnCl 2, 50 mM TrisHCl (pH 7.5), 1 mM DTT, 4% glycerol and 0.1 mg / ml BSA (except the channel of TdT, which carried 100 mM of cacodylate buffer, 1 mM CoCl2 and 0.1 mM DTT), 10 nM of the fluorescent oligo of DNA, 1 μM ddATP-FLO, and 600 nM of the indicated protein in each case. After a 30 minute incubation at 37 ° C, the reactions were stopped by adding 5.6 µl of loading buffer (95% formamide, 10 mM EDTA). These samples were analyzed by electrophoresis in 20% polyacrylamide and urea8M gels and subsequent fluorescent signal reading using Typhoon 9410 (GE Healthcare) equipment.

Results (see Figure 8)

-

The simple mutations R387K (R) and Ch-loop-1 (Ch) do not achieve any improvement over Polμ wt in such reactions.

-

The mutant M2, as already seen in Figure 7, does mean an improvement since it is capable of ending almost the entire starting substrate, although not 100%.

-

M3 and M7 do not achieve 100% marking of the starting substrate.

Example 9

3’OH End Marking Test

Test similar to that performed in Example 8, in which the effectiveness of the M3 and M7 mutants in tidal reactions is compared, with respect to the simple mutations that make up each of the mutants. In this case, a different single stranded DNA (PoliT) substrate is used to rule out that the result is sequence dependent.

Materials and methods

The fluorescent oligonucleotide used was: PoliT-Cy5 (PoliT 15-Cy5), which was purchased from Sigma. The ddATP-Cy5 was purchased from Perkin-Elmer. The reactions were carried out in a volume of 10 μl, in the presence of 1 mM MnCl 2, 50 mM TrisHCl (pH 7.5), 1 mM DTT, 4% glycerol and 0.1 mg / ml BSA (except the channel of TdT, which carried 100 mM of cacodylate buffer, 1 mM CoCl2 and 0.1 mM DTT), 10 nM of the fluorescent oligo of DNA, 1 μM ddATP-FLO, and 600 nM of the indicated protein in each case. After a 30 min incubation at 37 ° C, the reactions were stopped by adding 5.6 µl of loading buffer (95% formamide, 10 mM EDTA). These samples were analyzed by electrophoresis in 20% polyacrylamide and urea8M gels and subsequent fluorescent signal reading using Typhoon 9410 (GE Healthcare) equipment.

Results (see Figure 9)

-

The simple mutant Ch does not achieve any improvement over Polμ wt in such reactions.

-

The mutant R in this particular case improves slightly with respect to Polμ wt, which may be due to a sequence effect.

-

The mutant M2, as already seen in Figure 8, does mean an improvement, but does not achieve 100% marking of the starting substrate, as it does in the cases of M3 and M7.

Example 10

3’OH end tide test

In this test, similar to that shown in Example 9, the efficacy of the M3 and M7 mutants in mareaje reactions is compared with respect to the simple mutations that make up each of the mutants. In this case, a different single-stranded DNA (PolyA) substrate is used to contrast with the previous ones.

Materials and methods

The fluorescent oligonueleotide used was: PoliA-Cy5 (PoliA15-Cy5), which was purchased from Sigma. The ddATP-Cy5 was purchased from Perkin-Elmer. The reactions were carried out in a volume of 10 μl, in the presence of 1 mM MnCl 2, 50 mM TrisHCl (pH 7.5), 1 mM DTT, 4% glycerol and 0.1 mg / ml BSA (except the channel of TdT, which carried 100 mM of cacodylate buffer, 1 mM CoCl2 and 0.1 mM DTT), 10 nM of the fluorescent oligo of DNA, 1 μM ddATP-FLO, and 600 nM of the indicated protein in each case. After a 30 min incubation at 37 ° C, the reactions were stopped by adding 5.6 µl of loading buffer (95% formamide, 10 mM EDTA). These samples were analyzed by electrophoresis in 20% polyacrylamide and urea8M gels and subsequent fluorescent signal reading using Typhoon 9410 (GE Healthcare) equipment.

Results (see Figure 10)

-

SimpleRyChno mutants achieve no improvement over Polμ wt in such reactions. It seems to be confirmed that the effect of improvement with R seen in the previous figure was sequence dependent.

-

The mutant M2, as already seen in previous examples, does mean an improvement, but does not achieve 100% marking of the starting substrate, as it does in the cases of M3 and M7.

Example 11

3 ’End Marking Test with the 4 fluorescent ddNTPs

This test shows that the tidal reaction with M3 and M7 is highly effective with the 4 fluorescent ddNTPs, both in the presence of Mn2 + and Co2 + (Fig. 11).

Materials and methods

The fluorescent oligonueleotide used was: SP1C-Cy5 (GATCACAGTGAGTAC-Cy5), which was purchased from Sigma. Fluorescent dideoxynucleotides (ddATP-FLO, ddUTP-FLO, ddCTP-FLO, ddGTP-FLO) were purchased from Perkin-Elmer. The reactions were carried out in a volume of 10 µl, in the presence of 1 mM MnCl2.50mM TrisHCl (pH 7.5), 1 mM DTT, 4% glycerol and 0.1 mg / ml BSA (in the case of reactions with 1 mM Mn2 +) and 100 mM of cacodylate buffer, 1 mM CoCl2 and 0.1 mM DTT (in the case of reactions with 1 mM Co2 +), 10 nM of the fluorescent oligo of DNA, 1 μM ddATP-FLO, and 600 nM of the protein indicated in each case. After a 30 min incubation at 37 ° C, the reactions were stopped by adding 5.6 µl of loading buffer (95% formamide, 10 mM EDTA). These samples were analyzed by electrophoresis in 20% polyacrylamide gels and 8 M urea and subsequent fluorescent signal reading using a Typhoon 9410 (GE Healthcare) device.

Example 12

Obtaining new homologous mutants

As shown in Figure 12, the alignment by FASTA, using the parameters established by default, of the sequences corresponding to the Polμ wt of different mammals, more specifically, Mus musculus, Rattus norvegicu and Bos taurus with the M3 mutant, provides those amino acids homologous to 275 and 387 of M3 (or human Polμ wt).

Mutation of the homologous amino acids of the Polμ wt of Mus musculus, Rattus norvegicu and Bos taurus corresponding to positions 275 and 387 of M3, would result in the obtaining of new mutants, which would potentially have the enhanced deoxynucleotidyl transferase activity due to high degree of identity existing between the different polymerases (Table 3). As can be seen from Figure 12, homologous amino acids are not necessarily in the same position in relation to their sequence.

TABLE 3

Example 13

Obtaining new mutants with increased terminal transferase activity

The structural data of Polμ wt used conveniently can be used to obtain new mutants with increased terminal transferase activity, by identifying and mutating amino acids that are at or near the catalytic site of the enzyme, preferably on the surface, this It is, in direct interaction with DNA. Thus, for example, three-dimensional analysis of the polymerase structure can lead to the identification of residues that allow the creation of a salt bridge, the introduction of a hydrophobic group or the creation of any other type of interaction that allows a better interaction. with the DNA in order to obtain mutants with increased terminal transferase activity.

Certain laboratory techniques, such as X-ray crystallography and nuclear magnetic resonance imaging (NMR), which allow us to know the three-dimensional structure of proteins, are well known for the identification of candidate amino acids. In addition to these types of techniques, there are many computer programs that make it possible, from the primary structure of the enzyme, to predict its spatial configuration.

(e.g. Swiss PDB Viewer). Thus, it is easy by visual inspection of the structures to determine which amino acids may be candidates for mutation.

The use of these types of techniques in combination with the comparison of the primary structures of homologous enzymes and / or similar activities allows even more refinement in the selection of those mutants that are potentially going to have an increased activity.

As an example, the compilation of all known Polμ and TdT sequences of different species, their alignment and comparison of the primary structure allows the identification of those amino acids that are conserved in both families of polymerases and that are different from each other. When this process is carried out, some of the candidate amino acids of Polμ that could be selected are the Q275, R387, N457 and S458, which after a visual analysis of the three-dimensional structure of the enzyme is found to be on the surface and close to the center catalytic (Figure 14). The mutation of these amino acids to the homologous amino acid of TdT (Q275M, R387K, N457D and S458N) and, optionally, the combination of these mutations results in obtaining mutants of Polμ with an increased terminal transferase activity, as can be Check throughout the description.

Claims (23)

one.

Mutated DNA polymerase mu (Polμ) having a terminal deoxynucleotidyl transferase activity at least about 10% higher than the terminal deoxynucleotidyl transferase activity of mutated R387K mutated DNA polymerase.

2. Mutated DNA polymerase according to claim 1, comprising at least two mutations.

3.

Mutated DNA polymerase according to any one of claims 1 or 2, comprising a sequence with at least 60% identity with SEQ ID NO: 17, or with any of its sub-sequences SEQ ID NO: 1 and SEQ ID NO: 2 , or with a fragment of any of said sequences that keeps the terminal transferase activity increased or substantially increased.

Four.

Mutated DNA polymerase according to claim 3, comprising at least two mutations consisting of (i) a first point mutation at position 387 and (ii) a second mutation consisting of:

(to)

a point mutation in a conserved amino acid of Polμ wt and other than the homologous amino acid present in the TdT enzyme, or

(b)

a mutation in the loop-1 subdomain of Polμ.

5.

Mutated DNA polymerase according to claim 4, wherein the first point mutation (i) at position 387 consists of the substitution of the amino acid found in that position by any of the amino acids selected from the following group: Glutamine, Asparagine, Cysteine, Threonine, Serine, Methionine, Lysine, Arginine, Histidine or the like thereof.

6.

Mutated DNA polymerase according to claim 5, wherein the second point mutation (a) is a point mutation in the amino acid that is in position 275 and consists of the substitution of the amino acid that is in that position with any of the amino acids selected from the following group: Glutamine, Asparagine, Cysteine, Threonine, Serine, Methionine or the like thereof.

7.

Mutated DNA polymerase according to any of the preceding claims, wherein the second mutation (b) consists of the deletion and replacement of the Pol-1 loop or a fragment thereof with:

i) a sequence with at least about 10% identity with SEQ ID NO: 3 (loop-1 of Polμ), or ii) by a sequence with at least about 10% identity with SEQ ID NO: 4 (loop-1 of TdT).

8.

Mutated DNA polymerase according to any of the preceding claims, whose sequence is selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, fragments or sub-sequences thereof that substantially maintain their terminal transferase activity.

9.

Polynucleotide encoding a mutated DNA polymerase, or a fragment or sub-sequence thereof, according to any of claims 1-8.

10.

Polynucleotide according to claim 9, wherein its sequence is selected from the group comprising the sequences SEQ ID NO: 14 and SEQ ID NO: 15.

11. Vector comprising a polynucleotide according to any of claims 9 or 10.

12.

Host cell comprising a polynucleotide according to any one of claims 9 or 10, or a vector according to claim 11.

13.

Method for the production of a mutated mu DNA polymerase according to any one of claims 1 to 8, comprising: i) culturing a host cell according to claim 12 under conditions that promote expression of the polynucleotide according to any of claims 9 or 10 , and ii) isolation of the mutant produced.

14.

Method for elongation of a target polynucleotide comprising: i) contacting the target polynucleotide with the mutated DNA polymerase according to any one of claims 1 to 8 and nucleotides or nucleotide analogs (reaction mixture), and ii) subjecting the reaction mixture at conditions that favor the insertion of at least one nucleotide or an analog thereof at the 3'OH end of the target polynucleotide.

fifteen.

Method for filling voids comprising: i) contacting a target polynucleotide, comprising at least one gap or gap of at least one nucleotide, with the mutated DNA polymerase according to any one of claims 1 to 8 and nucleotides or nucleotide analogues (reaction mixture), and ii) subjecting the reaction mixture to conditions that favor the insertion of at least one nucleotide or nucleotide analogue at the 3'OH end of the gap.

16. A method for the detection of apoptotic samples comprising: i) contacting a sample, containing genetic material from potentially apoptotic cells, with the mutated mutated DNA polymerase according to any of claims 1-8 and minus a nucleotide or nucleotide analogue (mixture of reaction), ii) subject the reaction mixture to conditions that favor the insertion of nucleotides or analogs thereof, and iii) detect the presence or not of insertion, where the detection of insertion is indicative that the sample is apoptotic. SEQUENCE LIST <110> XPOL Biotech, S.L. <120> Mutants of DNA polymerase mu <130> P5299ES00 -XPOL-01 <160> 17 <170> PatentIn version 3.5 <210> 1 <211> 373 <212> PRT <213> Homo Sapiens (Pol mu NH2-BRCT-) <400> 1 <210> 2 <211> 357 <212> PRT <213> Homo sapiens (Pol mu NH2-BRCT-8 KDa) <400> 2 <210> 3 <211> 19 <212> PRT <213> Homo sapiens (Ch-loop-1 Pol mu) <400> 3 <210> 4 <211> 20 <212> PRT <213> Homo sapiens (Ch-loop-1 TdT) <400> 4 <210> 5 <211> 494 <212> PRT <213> Homo sapiens (Pol mu, mutant M3) <400> 5 <210> 6 <211> 493 <212> PRT <213> Homo sapiens (Pol mu, mutant M7) <400> 6 <210> 7 <211> 375 <212> PRT <213> Homo sapiens (M3: NH2-BRCT-) <400> 7 <210> 8 <211> 357 <212> PRT <213> Homo sapiens (M3: NH2-BRCT-8 KDa-) <400> 8 <210> 9 <211> 374 <212> PRT <213> Homo sapiens (M7: NH2-BRCT-) <400> 9 <210> 10 <211> 356 <212> PRT <213> Homo sapiens (M7: NH2-BRCT-8 KDa-) <400> 10 <210> 11 <211> 494 <212> PRT <213> Homo sapiens (Mutant M2) <400> 11 <210> 12 <211> 375 <212> PRT <213> Homo sapiens (M2: NH2-BRCT-)

<400>

12

<210> 13 <211> 357 <212> PRT <213> Homo sapiens (M2: NH2-BRCT-8 KDa-)

<400>

13

<210> 14 <211> 1485 <212> DNA <213> Homo sapiens (M3 mutant) <400> 14 <210> 15 <211> 1488 <212> DNA <213> Homo sapiens (M7 mutant) <400> 15 <210> 16 <211> 1485 <212> DNA <213> Homo sapiens (Mutant M2) <400> 16 <210> 17 <211> 494 <212> PRT <213> Homo sapiens <400> 17 SPANISH OFFICE OF THE PATENTS AND BRAND Application no .: 200901943 SPAIN Date of submission of the application: 02.10.2009 Priority Date: REPORT ON THE STATE OF THE TECHNIQUE 51 Int. Cl.: C12N15 / 54 (2006.01) C12N9 / 12 (2006.01) RELEVANT DOCUMENTS

Category

Documents cited Claims Affected

Y

ANDRADE, P. et al., "Limited terminal transferase in human DNA polymerase mu define the required balance between accuracy and efficiency in NHEJ.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Sep 2009, Vol. 106 , No. 38, pages 16203-16208, ISSN: 0027-8424. Epub:, Results; Discussion. 1-16

Y

JUÁREZ, R, et al., 'A specific loop in human DNA polymerase mu allows switching between creative and DNA-instructed synthesis.', NUCLEIC ACIDS RESEARCH, 2006, Vol. 34, No. 16, pages 4572-4582, ISSN: 0305-1048, Materials and Methods; Results 1-16

TO

ROMAIN, F. et al., “Conferring a template-dependent polymerase activity to terminal deoxynucleotidyltransferase by mutations in the Loop1 region.”, NUCLEIC ACIDS RESEARCH, Aug 2009, Vol. 37, No.14, pages 4642-4656, ISSN: 0305-1048, Epub:, Results; Discussion. 1-16

TO

MOON, A.F. et al., ‘Structural insight into the substrate specificity of DNA Polymerase mu.’, NATURE STRUCTURAL & MOLECULAR BIOLOGY, 2007, Vol. 14, No. 1, pages 45-53, ISSN: 1545-9985, the entire document. 1-16

Category of the documents cited X: of particular relevance Y: of particular relevance combined with other / s of the same category A: reflects the state of the art O: refers to unwritten disclosure P: published between the priority date and the date of priority submission of the application E: previous document, but published after the date of submission of the application

This report has been prepared • for all claims • for claims no:

Date of realization of the report 20.07.2011

Examiner J. Vizán Arroyo Page 1/5

SPANISH OFFICE OF THE PATENTS AND BRAND Application no .: 200901943 SPAIN Date of submission of the application: 02.10.2009 Priority Date: REPORT ON THE STATE OF THE TECHNIQUE 51 Int. Cl.: C12N15 / 54 (2006.01) C12N9 / 12 (2006.01) RELEVANT DOCUMENTS

Category

Documents cited Claims Affected

TO

DOMÍNGUEZ, O. et al., "DNA polymerase mu (Pol mu), homologous to TdT, could act as a DNA mutator in eukaryotic cells.", THE EUROPEAN MOLECULAR BIOLOGY ORGANIZATION JOURNAL, 2000, Vol. 19, No. 7, pages 1731-1742, ISSN: 0261-4189, the entire document. 1-16

TO

MOON, A.F. et al., ‘The X family portrait: structural insights into biological functions of X family polymerases.’, DNA REPAIR, 2007, Vol. 6, No. 12, pages 1709-1725, ISSN: 1568-7864, the entire document. 1-16

TO

WO 01/64909 A1 (SUPERIOR SCIENTIFIC RESEARCH COUNCIL), the entire document. 1-16

Category of the documents cited X: of particular relevance Y: of particular relevance combined with other / s of the same category A: reflects the state of the art O: refers to unwritten disclosure P: published between the priority date and the date of priority submission of the application E: previous document, but published after the date of submission of the application

This report has been prepared • for all claims • for claims no:

Date of realization of the report 20.07.2011

Examiner J. Vizán Arroyo Page 2/5

REPORT OF THE STATE OF THE TECHNIQUE Application number: 200901943 Minimum documentation sought (classification system followed by classification symbols) C12N Electronic databases consulted during the search (name of the database and, if possible, terms of search used) INVENES, EPODOC, WPI, BIOSIS, EMBASE, MEDLINE, EMBL-EBI State of the Art Report Page 3/5  WRITTEN OPINION Application number: 200901943 Date of Written Opinion: 20.07.2011 Statement

Novelty (Art. 6.1 LP 11/1986)

Claims Claims 1-16 IF NOT

Inventive activity (Art. 8.1 LP11 / 1986)

Claims Claims 1-16 IF NOT

The application is considered to comply with the industrial application requirement. This requirement was evaluated during the formal and technical examination phase of the application (Article 31.2 Law 11/1986).  Opinion Base.- This opinion has been made on the basis of the patent application as published. State of the Art Report Page 4/5  WRITTEN OPINION Application number: 200901943 1. Documents considered.- The documents belonging to the state of the art taken into consideration for the realization of this opinion are listed below.

Document

Publication or Identification Number publication date

D01

Andrade, P. et al., Proc. Natl Acad. Sci. U S A., (2009 Sep), 106 (38): 16203-8.  

D02

Juárez, R. et al., Nucleic Acids Res., (2006), 34 (16): 4572-82. 2006

D03

Romain, F. et al., Nucleic Acids Res., (2009 Aug), 37 (14): 4642-56.  

D04

Moon, A.F. et al., Nat. Struct. Mol. Biol., (2007), 14 (1): 45-53. 2007

D05

Dominguez, O. et al., EMBO J., (2000), 19 (7): 1731-42. 2000

D06

Moon, A.F. et al., DNA Repair, (2007), 6 (12): 1709-25. 2007

D07

WO 01/64909 A1  

In D1 the activity deoxynucleotidyl transferase activity of different point mutants of the DNA polymerase mu (Polµ) is described. In D2-D3, the Loop 1 subdomain of Polµ and the Deoxynucleotidyl transferase (TdT) subdomain are structurally and functionally analyzed. Different members of the Polymerase X family (TdT, Pol�, PolA, Poll) are characterized in D4-D7. 2. Statement motivated according to articles 29.6 and 29.7 of the Regulations for the execution of Law 11/1986, of March 20, on Patents on novelty and inventive activity; quotes and explanations in support of this statement 1. NEW (Art. 4.1. And Art. 6.1. Of the Patent Law)

1.1.

 The present invention satisfies the criteria established in Art. 4.1. of the Patent Law because the object of the invention, defined in claims 1-16, is new in accordance with Art. 6.1. of the Patent Law.

2.

 INVENTIVE ACTIVITY (Art. 4.1. And Art. 8.1. Of the Patent Law).

2.1. Independent claims 1-16. 2.1.1. Documents D1 and D2 are considered to be the closest state of the art. D1 describes the mutant Polµ (R387K) characterized by having increased terminal transferase activity with respect to that of the wild allele Pol µ (cf. D1: Results; Table 1). In D2 the enzymatic activity of the Polµ chimeric construct (Ch-loop1) is analyzed, obtained by replacing the sequence of the subdomain subdomain of human Polµ with that of the corresponding subdomain loop 1 of human TdT (cf. D2: Materials and Methods; Results, Figure 5). 2.1.2. The technical problem to be solved by the object of independent claim 1 can therefore be considered as the provision of new Polµ mutants with a terminal transferase activity greater than that of the Polµ mutant (R387K). 2.1.3. The proposed solution is the mutant enzyme Polµ (M7) characterized by the sequence SEQ ID NO 6. Said mutant comprises the R387K mutation and the substitution of the loop subdomain sequence of Polµ by the corresponding loop sequence 1 of TdT (cf page 11 , lines 32-33). In order to properly assess the inventive activity of this solution, it is necessary to consider whether, at the date of filing the patent application, based on the closest state of the art, the person skilled in the art would try to apply said solution with a reasonable expectation of success. In addition, it is necessary to consider whether non-foreseeable technical difficulties arose during the implementation of this procedure. On the basis of the closest state of the art, represented by D1 and D2, together with the knowledge of usual use and application in this field of biotechnology, collected in D3-D7 it is concluded that the solution proposed by the patent application to Technical problem raised would be apparent to the person skilled in the art. For this reason, the object of claim 1, as regards the Polµ mutant (M7) (SEQ ID NO: 6, SEQ ID NO: 9 and SEQ ID NO: 10), is considered to be non-inventive. Likewise, it is considered that the object of dependent claims 2-16 is also not inventive. 2.2. The present invention does not meet the criteria established in Art. 4.1. of the Patent Law because the object of the invention, defined in claims 1-16, does not imply inventive activity in accordance with Art. 8.1. of the Patent Law. State of the Art Report Page 5/5