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WO2011039390A2 - Mutants de l'adn polymérase mu - Google Patents

Mutants de l'adn polymérase mu Download PDF

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WO2011039390A2
WO2011039390A2 PCT/ES2010/000402 ES2010000402W WO2011039390A2 WO 2011039390 A2 WO2011039390 A2 WO 2011039390A2 ES 2010000402 W ES2010000402 W ES 2010000402W WO 2011039390 A2 WO2011039390 A2 WO 2011039390A2
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seq
dna polymerase
ροΐμ
mutants
mutated
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WO2011039390A8 (fr
WO2011039390A3 (fr
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Luis BLANCO DÁVILA
Raquel JUÁRES SANTOS
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X-POL BIOTECH SL
X Pol Biotech SL
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X-POL BIOTECH SL
X Pol Biotech SL
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Definitions

  • the present invention relates to the identification of mutants of the human mu DNA polymerase ( ⁇ ) 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
  • TdT terminal deoxynucleotidyl transferase
  • 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 ⁇ terminal end of the oligonucleotide chains without the need for a strand of template DNA (terminal deoxynucleotidyl transferase activity), thus generating diversity in the antigen receptors of the aforementioned immune system defense cells . This activity of TdT has allowed this polymerase to have been widely used as a molecular tool, especially in DNA mapping techniques.
  • TUNEL assay is based on the principle that TdT incorporates labeled deoxyuridine (eg dUTP-biotin) at the 3 ⁇ ends of breaks in double and single strands of DNA.
  • labeled deoxyuridine eg dUTP-biotin
  • the incorporation of labeled dUTP acts as a signal that can be easily detected, for example, through 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.
  • the authors of the present invention have developed ⁇ mutants that exhibit a higher terminal deoxynucleotidyl transferase activity than the wild-type enzyme ( ⁇ wt: UniProtKB / Swiss-Prot Q9NP87: SEQ ID NO: 17) and even that TdT itself.
  • the invention provides several ⁇ mutants with a high terminal deoxynucleotidyl transferase activity, superior to that of ⁇ wt, which can be used, among others, in polynucleotide or blunt end assembly techniques (End joining).
  • mutants of the invention which have at least about 10% more terminal deoxynucleotidyl transferase activity than ⁇ wt, preferably human ⁇ .
  • said activity is at least about 25%, 50%, 75%, 100%, 200%, 300%, 400%, 500% higher than that of the ⁇ wt.
  • 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.
  • R 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.
  • a polynucleotide sequence (hereinafter, polynucleotide sequence or polynucleotide of the invention) encoding any of the mutants of the invention.
  • any part of the invention is also part of: 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.
  • the invention relates to a method for the production of the mutants of the invention that preferably comprises: i) the cultivation of a host cell according to the invention and ii) the isolation of the produced mutant.
  • 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) gaps or void filling (gap-filling), v) DNA breakage detection (mismatch detection; eg TUNEL), vi) mutagenesis or generation of variability, etc.
  • 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 (eg buffers, primers, dNTPs, ddNTPs, markers, etc.), the kits may comprise TdT or mutants thereof.
  • the components necessary to launch the corresponding assay eg buffers, primers, dNTPs, ddNTPs, markers, etc.
  • the kits may comprise TdT or mutants thereof.
  • 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 gaps or gaps (gap-filling), v) DNA break detection (mismatch detection; eg TUNEL), vi) mutagenesis or generation of variability, etc., constitutes an additional aspect of this invention
  • terminal deoxynucleotidyl transferase activity or “terminal transferase activity” is defined as the ability to carry out template independent polymerization reactions, adding nucleotides to a 3 ⁇ 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 ⁇ wt, preferably that of human ⁇ . Preferably, said activity is at least about 25%, 50%, 75%, 100%, 200%, 300%, 400%, 500% higher than that of ⁇ wt, preferably that of human ⁇ .
  • 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.
  • vector refers to a linear or circular polynucleotide (e.g. plasmids, bacterial chromosome) comprising a polynucleotide sequence, wherein said vector is preferably adapted for amplification, replication and / or expression of said polynucleotide.
  • these vectors may contain regions that allow the amplification, expression and / or replication of the polynucleotide to be controlled or enhanced, as well as others that facilitate or promote the excretion or capture of the polypeptides resulting from the expression of the polynucleotide.
  • host cell refers to a cell or group of cells (eukaryotic or prokaryotic) that contains a vector or polynucleotide according to the present invention.
  • 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.
  • nucleotides refers to ribonucleotides, deoxyribonucleotides (dNTPs) or dideoxynucleotides (ddNTPs) that, preferably, are labeled or modified to allow their identification and / or monitoring.
  • nucleotide mapping is performed with fluorophores (eg Cy3, Cy5, fluorescein) or biotin.
  • fluorophores eg Cy3, Cy5, fluorescein
  • biotin e.g Cy3, Cy5, fluorescein
  • nucleotides are labeled with fluorophores, they are referred to as fluorescent nucleotides.
  • 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.
  • ortholog refers to proteins or genes encoding said proteins, with terminal transferase activity, which share at least about 40% identity with the ⁇ wt, preferably with the human ⁇ , 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%.
  • 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
  • substantially refers to the variation between ⁇ 10% that a given value (eg activity) may present, preferably between ⁇ 5% and more preferably ⁇ one%.
  • fragments refer to portions of polypeptides or polynucleotides encoding said polypeptides, which maintain the increased or substantially increased terminal transferase activity.
  • Figure 1 shows the terminal transferase activity of each of the mutants [ ⁇ - ⁇ 387 ⁇ - F389G, ⁇ ( ⁇ 275 ⁇ , ⁇ 387 ⁇ - (2275 ⁇ , ⁇ .387 ⁇ - ⁇ 1 ⁇ 1, ⁇ - ⁇ 457 ⁇ , ⁇ -8458 ⁇ and ⁇ - ⁇ 457 ⁇ -8458 ⁇ (Example 1)], compared to that of the ⁇ wt and that of the commercial TdT.
  • Figure 2 shows the terminal transferase activity of the double mutants ⁇ -R387K / Q275M and ⁇ - ⁇ 1387 ⁇ : ⁇ 1) at decreasing doses of polymerase (400 nM, 200 nM and 100 nM).
  • Figure 3 shows the terminal transferase activity of the double mutants ⁇ -R387K / Q275M and ⁇ - ⁇ 387 ⁇ / ⁇ 1 ⁇ 1) and of the simple mutants ( ⁇ - ⁇ 1387 ⁇ , ⁇ -Chloopl and ⁇ - (3275 ⁇ and compared to that of ⁇ wt, with each of the 4 dNTPs separately
  • Figure 4 shows the terminal transferase activity of the double mutants ⁇ -R387K / F389G, ⁇ - ⁇ 087 ⁇ / ⁇ ) 275 ⁇ and ⁇ - ⁇ 387 ⁇ / ⁇ 1 ⁇ 1) and of the simple mutants ( ⁇ - ⁇ 387 ⁇ and ⁇ - ⁇ > 275 ⁇ ), compared to that of ⁇ wt, with each 4 dNTPS in the presence of Co 2+ as the activating metal.
  • Figure 6 shows the mold dependence of the double mutants (M3 and M7) and the simple mutants (R, Ch and M2) in the context of a 1 nucleotide DNA gap with phosphate.
  • Figure 7 shows the results of the fluorescent derivative assay of DNA or ddNTPs, with the indicated mutants of ⁇ (M1-M7), together with ⁇ wt and TdT on single chain polynucleotides.
  • Figure 8 shows the results of the fluorescent derivative assay of DNA or ddNTPs, with the indicated mutants of ⁇ (R, Ch, M2, M3 and M7), on single chain polynucleotides.
  • Figure 9 shows the results of the fluorescent derivative assay of DNA or ddNTPs, with the indicated mutants of ⁇ (R, Ch, M2, M3 and M7), on single chain polynucleotides.
  • Figure 10 shows the results of the fluorescent derivative assay of DNA or ddNTPs, with the indicated mutants of ⁇ (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 Mn 2+ and Co 2+ .
  • Figure 12 shows the alignment of the ipe ⁇ 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 ⁇ wt. At the bottom, the arrow indicates amino acid 387 of human ⁇ wt.
  • Figure 13 shows the alignment of the human ⁇ wt polypeptide sequences and the TdT of human, murine and bovine origin.
  • the arrows show the position of amino acids 275, 387, 457 and 458 in human ⁇ wt.
  • Figure 14 shows the three-dimensional structure of the human ⁇ 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
  • D) the arrow indicates the position of amino acid S458.
  • 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.
  • TdT was the only known polymerase with the ability to add or bind nucleotides to an end 3 ⁇ of polynucleotides without requiring a template chain.
  • the low terminal deoxynucleotidyl transferase activity of ⁇ wt compared to TdT has made it impossible for this enzyme to date to become a viable alternative to TdT.
  • mutants that increase the terminal deoxynucleotidyl transferase activity of wild ⁇ and even TdT.
  • said mutants are derived from or derived from human ⁇ wt, although they can also be obtained from the ⁇ of other vertebrates (orthologous ras ⁇ de ⁇ polymerases), preferably mammals, such as Mus musculus, Rattus norvegicus, Bos taurus, among others .
  • mutants from orthologous polymerases to human ⁇ 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 is explained in Example 12. Similarly, new mutants with increased terminal transferase activity, as with the mutants of the present invention, can also be easily obtained, as shown in Example 13. ⁇ mutants and polynucleotides of the invention
  • TdT and ⁇ 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 beta polymerase ( ⁇ ). 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 ⁇ 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.
  • the mutants of the invention have at least about 10% more terminal deoxynucleotidyl transferase activity than ⁇ wt, preferably human ⁇ wt.
  • said activity is at least about 25%, 50%, 75%, 100%, 200%, 300%, 400%, 500% higher than that of the ⁇ wt.
  • 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.
  • 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.
  • the mutants comprise at least two mutations.
  • the ⁇ mutants comprise a polypeptide sequence with at least 60% identity with the sequence SEQ ID NO: 17 ( ⁇ wt: UniProtKB / Swiss-Prot Q9NP87), or with any of its SEQ ID NO sub-sequences : 1 and SEQ ⁇ ) NO: 2, or with fragments thereof, wherein said mutants comprise at least two mutations and maintain an increased terminal transferase activity.
  • said mutations are (i) a first point mutation at position 387 and (ii) a second mutation consisting of:
  • the degree of identity of the ⁇ mutants with the sequence Q9NP87, any of its sub-sequences SEQ ID NO: l 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%, or 100%.
  • the first point mutation (i) of the ⁇ mutants consists in replacing the amino acid found at position 387 with any of the amino acids selected from the group comprising: Glutamine, Asparagine, Cistern, 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).
  • the second mutation (ii) corresponding to the point mutation (a) is located at position 275.
  • 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).
  • 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 ⁇ ), 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%.
  • said sequence is SEQ ID NO: 4, which shares about 20% identity with SEQ ID NO: 3.
  • 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 loop-1 of TdT in the place of loop-1 of ⁇ (hereinafter, mutant M7).
  • the invention also comprises fragments or sub-sequences of the sequences SEQ ED NO: 5 or SEQ ID NO: 6 comprising said mutations and, preferably, substantially maintaining their increased terminal deoxynucleotidyl transferase activity.
  • said fragments have the sequences SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.
  • an imitant comprising a polypeptide sequence with at least 60% identity with the sequence Q9NP87 (human w ⁇ wt: SEQ ID NO: 17) and at least one point mutation at position 275, wherein said mutant has an enhanced or increased terminal deoxynucleotidyl transferase activity.
  • This activity is at least about 10%, 25%, 50%, 75%, 100%, 200%, 300% higher than that of the ⁇ wt.
  • 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 walls of the invention.
  • 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.
  • 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 the mimics M3, M7 and M2.
  • any sequence with at least about 40% identity with any of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 that preferably does not modify are also part of the present invention. its activity or do not modify it substantially.
  • said identity is at least about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%.
  • a third aspect of the invention also relates 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) the isolation of the mutant produced.
  • the cell flow is performed under conditions that promote the growth of the host cell and / or the expression of the polynucleolide of the invention.
  • 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 the mutants ⁇
  • a fourth aspect of the invention relates 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 protuberant and blunt ends, iii) mold-dependent or independent polymerization, iv) gaps filling (gap-filling), v) DNA break detection mismatch detection; e.g. TUNEL), vi) mutagenesis, etc.
  • 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 ⁇ end of the target polynucleotide.
  • 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 analogs (reaction mixture), ii) subjecting the reaction mixture at conditions that favor the insertion of at least one nucleotide or analogue thereof at the 3 ⁇ end of the target polynucleotide, and iii) detect the presence or not of insertion.
  • 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 and 20, 5 and 20, 1 and 10 , 3 and 10, 5 and 10.
  • the mutants of the invention are also capable of being employed in gap filing 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 ⁇ end of the gap.
  • 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.
  • enzymes eg nucleases, ligases
  • the gap size is at least 1, 2, 3, 4 or 5 nucleotides and, more preferably, between 1 and 10 nucleotides.
  • 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.
  • 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.
  • this method comprises iii) the addition of enzymes (eg ligases) that bind the ends once they are together.
  • enzymes eg ligases
  • 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 important application of the TdT activity is its use in the TUNEL assays (TdT-mediated dUTP-biotin nick end labeling) that allow the detection of biological samples that are undergoing apoptosis (Gavrieli, Y . et al. (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J.
  • 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 Sperm with fragmented DNA (multiple 3 ⁇ terminals) are stained with intense fluorescence.
  • the fluorescence can be detected, preferably, both by flow cytometry and by fluorescent microscopy.
  • 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%.
  • 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 for the same purpose.
  • Another way to increase the efficiency of the mentioned methods comprises the addition of ions, preferably Mn 2+ or Co 2+ 'to the reaction mixtures, since these are capable of increasing the catalytic efficiency of the mutants of the invention.
  • 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 such tests.
  • the components necessary to launch the corresponding assay eg buffers, primers, dNTPs, ddNTPs, hNTPs, nucleotide analogs, ions, etc.
  • 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 gaps or gaps (gap-fdling), v) DNA breakage detection ⁇ mismatch detection; e.g. TUNEL), vi) mutagenesis or generation of variability, etc., constitutes an additional aspect of this invention
  • This assay shows the terminal transferase activity of each of the mutants, compared to the human ⁇ wt [SEQ ED 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 ⁇ end of single-chain homopolymeric DNA (PoliT). Initially, those mutants that produced a striking stimulation of the terminal transferase activity with respect to ⁇ wt were selected for the conduct of DNA 3-end labeling assays. Materials and methods
  • the fluorescent oligonucleotide 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 ⁇ , in the presence of 1 mM MnC ⁇ , 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 ⁇ 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).
  • the combined mutant R387 -F389G (MI) has greatly reduced the intrinsic terminal transferase activity of ⁇ wt.
  • R387K-Q275M (M3) and R387K-Chloopl (M7) are the truly spectacular ones, since they consume the entire starting substrate (which is very applicable to 3 ⁇ end marking as it can be a near tidal efficiency 100%) and extend to large sizes (which can be applied in "tailing" reactions).
  • 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 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).
  • 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 ⁇ , 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 ⁇ of the dNTP indicated in each case, and the protein dose indicated in each case.
  • 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 ⁇ , 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 ⁇ 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).
  • the simple mutant R387K (R) achieves an effective increase in the intrinsic terminal transferase activity of ⁇ 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 ⁇ -Chloopl (Ch) achieves a change in the pattern of insertion of dNTPs with respect to ⁇ w ⁇ , but does not imply a special call for its terminal transferase activity.
  • the Q275M mutant (M2) achieves a certain stimulus of terminal transferase activity compared to ⁇ wt.
  • 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 ⁇ , in the presence of 100 mM of cacodylate buffer (pH 6.8), 1 mM CoCl 2 , 0.1 mM DTT, 20 nM of the fluorescent oligonucleotide indicated in each case, 100 ⁇ 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).
  • the fluorescent oligonucleotide SP1C-FLO (GATCACAGTGAGTAC-FLO) was hybridized with oligonucleotide TI 3 (G) (AGAAGTGTATCTGGTACTCACTGTGATC) to generate the DNA substrate indicated in the upper part of Figure 5.
  • G oligonucleotide TI 3
  • Both oligonucleotides were bought from Sigma.
  • the dNTPs were purchased from GE Healthcare.
  • the polymerization reactions were carried out in a volume of 10 ⁇ , 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 ⁇ 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 Typhoon 9410 (GE Healthcare) equipment. Results
  • 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 Chloopl mutation seems to produce a small decrease in elongation, without the polymerization capacity disappearing.
  • the M7 mutant, which contains the Chloopl mutation is also affected to the same degree.
  • the M2 and M3 mutants even seem to improve the elongation capacity of ⁇ 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.
  • the fluorescent oligonucleotide SP1C-FLO (GATCACAGTGAGTAC-FLO) was hybridized with oligonucleotide TI 3 (G) (AGAAGTGTATCTGGTACTCACTGTGATC) and Dgl-P (AGATACACTTCT-P) to generate the DNA substrate indicated in the upper part of Figure 6.
  • the oligonucleotides were purchased from Sigma.
  • the dNTP was purchased from GE Healthcare
  • the polymerization reactions were carried out in a volume of 10 ⁇ , 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.
  • the reactions were stopped by adding 5.6 ⁇ 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 Typhoon 9410 (GE Healthcare) equipment. Results
  • the complete series of mutants of ⁇ was tested, together with ⁇ wt and TdT in a 3 ma end reaction reaction of a single stranded 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 30H 'end of the DNA was supplied.
  • the DNA channel allows 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.
  • the fluorescent oligonucleotide used was: SPlC-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 ⁇ , in the presence of 1 mM ( ⁇ (3 ⁇ 4 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 CoCl 2 and 0.1 mM DTT), 10 nM of the fluorescent DNA oligo, 1 ⁇ ddATP-FLO, and 600 nM of the indicated protein in each case.
  • ⁇ wt is capable of marking around 50% of the oligo supplied.
  • the mutants M4, M5 and M6 ( ⁇ - ⁇ 457 ⁇ -8458 ⁇ ) have a similar behavior.
  • the mutant MI sees its ability to mareaje very low compared to ⁇ wt.
  • the mutant M2 represents an improvement over ⁇ 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.
  • the fluorescent oligonucleotide used was: SPlC-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 ⁇ , in the presence of 1 mM MnC ⁇ , 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 C0CI 2 and 0.1 mM DTT), 10 nM of the fluorescent DNA oligo, ⁇ ⁇ ddATP-FLO, and 600 nM of the indicated protein in each case.
  • the simple mutations R387 (R) and Ch-loop-1 (Ch) do not achieve any improvement over ⁇ wt in such reactions.
  • the mutant M2 does mean an improvement since it is capable of destroying almost the entire starting substrate, although not 100%.
  • M3 and M7 do not achieve 100% marking of the starting substrate.
  • the fluorescent oligonucleotide used was: PoliT-Cy5 (PoliT15-Cy5), which was purchased from Sigma.
  • the ddATP-Cy5 was purchased from Perkin-Elmer.
  • the reactions were carried out in a volume of 10 ⁇ , 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 CoCl 2 and 0.1 mM DTT), 10 nM of the fluorescent DNA oligo, 1 ⁇ ddATP-FLO, and 600 nM of the indicated protein in each case.
  • the simple mutant Ch does not achieve any improvement over ⁇ wt in such reactions.
  • the mutant R in this particular case improves slightly with respect to ⁇ wt, which may be due to a sequence effect.
  • the mutant M2 does mean an improvement, but it does not achieve 100% marking of the starting substrate, as it does in the cases of M3 and
  • the fluorescent oligonucleotide 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 ⁇ , 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 CoCl 2 and 0.1 mM DTT), 10 nM of the fluorescent DNA oligo, 1 ⁇ ddATP-FLO, and 600 nM of the indicated protein in each case.
  • the mutant M2 does mean an improvement, but it does not achieve 100% marking of the starting substrate, as it does in cases of
  • the fluorescent oligonucleotide used was: SPlC-Cy5 (GATCACAGTGAGTAC-Cy5), which It was bought 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 ⁇ , in the presence of 1 mM MnC, 50 mM TrisHCl (pH 7.5), 1 mM DTT, 4% glycerol and 0.1 mg / ml BSA (in the case of reactions with ImM Mn 2+ ) and 100 mM of cacodylate buffer, 1 mM C0CI 2 and 0.1 mM DTT (in the case of reactions with ImM Co 2+ ), 10 nM of the fluorescent DNA oligo, 1 ⁇ ddATP-FLO , and 600 nM of the protein indicated in each case.
  • the structural data of ⁇ 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.
  • the 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.

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Abstract

L'invention concerne des mutants de l'ADN polymérase mu (Polμ) présentant une activité désoxynucléotidyl-transférase terminale accrue par comparaison avec l'enzyme de type sauvage (wt), et améliorant sa potentialité en tant qu'outil moléculaire. Lesdits mutants peuvent être utilisés, entre autres applications, dans des techniques de marquage de polynucléotides ou de jonction d'extrémités protubérantes et franches.
PCT/ES2010/000402 2009-10-02 2010-10-02 Mutants de l'adn polymérase mu Ceased WO2011039390A2 (fr)

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ES200901943A ES2364010B1 (es) 2009-10-02 2009-10-02 Mutantes de la adn polimerasa mu
ESP200901943 2009-10-02

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WO2001064909A1 (fr) * 2000-03-03 2001-09-07 Consejo Superior De Investigaciones Cientificas ADN POLYMERASE ν ET SES UTILISATIONS

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