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Human polymerase kappa uses a template-slippage deletion mechanism, but can realign the slipped strands to favour base substitution mutations over deletions.

Mukherjee P, Lahiri I, Pata JD - Nucleic Acids Res. (2013)

Bottom Line: Here, we show that hPolκ uses a classical Streisinger template-slippage mechanism to generate -1 deletions in repetitive sequences, as do the bacterial and archaeal homologues.Strand realignment results in a base-substitution mutation, minimizing generation of more deleterious frameshift mutations.On non-repetitive sequences, we find that nucleotide misincorporation is slower if the incoming nucleotide can correctly basepair with the nucleotide immediately 5' to the templating base, thereby competing against the mispairing with the templating base.

View Article: PubMed Central - PubMed

Affiliation: Wadsworth Center, New York State Department of Health, University at Albany, School of Public Health, Albany, NY 12201-0509, USA.

ABSTRACT
Polymerases belonging to the DinB class of the Y-family translesion synthesis DNA polymerases have a preference for accurately and efficiently bypassing damaged guanosines. These DinB polymerases also generate single-base (-1) deletions at high frequencies with most occurring on repetitive 'deletion hotspot' sequences. Human DNA polymerase kappa (hPolκ), the eukaryotic DinB homologue, displays an unusual efficiency for to extend from mispaired primer termini, either by extending directly from the mispair or by primer-template misalignment. This latter property explains how hPolκ creates single-base deletions in non-repetitive sequences, but does not address how deletions occur in repetitive deletion hotspots. Here, we show that hPolκ uses a classical Streisinger template-slippage mechanism to generate -1 deletions in repetitive sequences, as do the bacterial and archaeal homologues. After the first nucleotide is added by template slippage, however, hPolκ can efficiently realign the primer-template duplex before continuing DNA synthesis. Strand realignment results in a base-substitution mutation, minimizing generation of more deleterious frameshift mutations. On non-repetitive sequences, we find that nucleotide misincorporation is slower if the incoming nucleotide can correctly basepair with the nucleotide immediately 5' to the templating base, thereby competing against the mispairing with the templating base.

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Structure of an hPolκ ternary complex. (A) View looking into the active site of the polymerase. (B) View looking at the template strand of DNA entering the active site, between the polymerase and polymerase-associated domains. The polymerase [PDB code 3IN5 (34)] is shown in surface representation, except for the N-clasp (yellow), which is shown in ribbons representation. The polymerase domain is composed of fingers (blue), palm (magenta) and thumb (green) subdomains and is connected to the polymerase-associated domain (orange) by a relatively unstructured polypeptide linker (white). DNA is coloured white, except for the templating base and nucleotides at positions −1 through −4 on the 3′ side of the templating base. The first residue visible in the structure (amino acid 25) is marked with a yellow asterisk.
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gkt179-F6: Structure of an hPolκ ternary complex. (A) View looking into the active site of the polymerase. (B) View looking at the template strand of DNA entering the active site, between the polymerase and polymerase-associated domains. The polymerase [PDB code 3IN5 (34)] is shown in surface representation, except for the N-clasp (yellow), which is shown in ribbons representation. The polymerase domain is composed of fingers (blue), palm (magenta) and thumb (green) subdomains and is connected to the polymerase-associated domain (orange) by a relatively unstructured polypeptide linker (white). DNA is coloured white, except for the templating base and nucleotides at positions −1 through −4 on the 3′ side of the templating base. The first residue visible in the structure (amino acid 25) is marked with a yellow asterisk.

Mentions: Most polymerases show a strong dependence of deletion frequency on homopolymeric run length. Y-family polymerases differ from other polymerase families in not having a very strong dependence of deletion frequency on run length, with a run of just two nucleotides already resulting in a high deletion frequency. The structure of hpolκ (Figure 6) shows that nucleotides 3′ of the templating base are adjacent to a large solvent-accessible gap between the polymerase and polymerase-associated domains of the enzyme, suggesting that bulged bases can readily be accommodated in this area. Crystal structures of the archaeal DinB polymerases Dbh and Dpo4 show how extrahelical nucleotides at positions −3 and −4 interact with the protein in this region (27,28). In contrast, polymerases from other families have much tighter constraints around the DNA duplex, which would suppress nucleotides from adopting extrahelical conformations.Figure 6.


Human polymerase kappa uses a template-slippage deletion mechanism, but can realign the slipped strands to favour base substitution mutations over deletions.

Mukherjee P, Lahiri I, Pata JD - Nucleic Acids Res. (2013)

Structure of an hPolκ ternary complex. (A) View looking into the active site of the polymerase. (B) View looking at the template strand of DNA entering the active site, between the polymerase and polymerase-associated domains. The polymerase [PDB code 3IN5 (34)] is shown in surface representation, except for the N-clasp (yellow), which is shown in ribbons representation. The polymerase domain is composed of fingers (blue), palm (magenta) and thumb (green) subdomains and is connected to the polymerase-associated domain (orange) by a relatively unstructured polypeptide linker (white). DNA is coloured white, except for the templating base and nucleotides at positions −1 through −4 on the 3′ side of the templating base. The first residue visible in the structure (amino acid 25) is marked with a yellow asterisk.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3643592&req=5

gkt179-F6: Structure of an hPolκ ternary complex. (A) View looking into the active site of the polymerase. (B) View looking at the template strand of DNA entering the active site, between the polymerase and polymerase-associated domains. The polymerase [PDB code 3IN5 (34)] is shown in surface representation, except for the N-clasp (yellow), which is shown in ribbons representation. The polymerase domain is composed of fingers (blue), palm (magenta) and thumb (green) subdomains and is connected to the polymerase-associated domain (orange) by a relatively unstructured polypeptide linker (white). DNA is coloured white, except for the templating base and nucleotides at positions −1 through −4 on the 3′ side of the templating base. The first residue visible in the structure (amino acid 25) is marked with a yellow asterisk.
Mentions: Most polymerases show a strong dependence of deletion frequency on homopolymeric run length. Y-family polymerases differ from other polymerase families in not having a very strong dependence of deletion frequency on run length, with a run of just two nucleotides already resulting in a high deletion frequency. The structure of hpolκ (Figure 6) shows that nucleotides 3′ of the templating base are adjacent to a large solvent-accessible gap between the polymerase and polymerase-associated domains of the enzyme, suggesting that bulged bases can readily be accommodated in this area. Crystal structures of the archaeal DinB polymerases Dbh and Dpo4 show how extrahelical nucleotides at positions −3 and −4 interact with the protein in this region (27,28). In contrast, polymerases from other families have much tighter constraints around the DNA duplex, which would suppress nucleotides from adopting extrahelical conformations.Figure 6.

Bottom Line: Here, we show that hPolκ uses a classical Streisinger template-slippage mechanism to generate -1 deletions in repetitive sequences, as do the bacterial and archaeal homologues.Strand realignment results in a base-substitution mutation, minimizing generation of more deleterious frameshift mutations.On non-repetitive sequences, we find that nucleotide misincorporation is slower if the incoming nucleotide can correctly basepair with the nucleotide immediately 5' to the templating base, thereby competing against the mispairing with the templating base.

View Article: PubMed Central - PubMed

Affiliation: Wadsworth Center, New York State Department of Health, University at Albany, School of Public Health, Albany, NY 12201-0509, USA.

ABSTRACT
Polymerases belonging to the DinB class of the Y-family translesion synthesis DNA polymerases have a preference for accurately and efficiently bypassing damaged guanosines. These DinB polymerases also generate single-base (-1) deletions at high frequencies with most occurring on repetitive 'deletion hotspot' sequences. Human DNA polymerase kappa (hPolκ), the eukaryotic DinB homologue, displays an unusual efficiency for to extend from mispaired primer termini, either by extending directly from the mispair or by primer-template misalignment. This latter property explains how hPolκ creates single-base deletions in non-repetitive sequences, but does not address how deletions occur in repetitive deletion hotspots. Here, we show that hPolκ uses a classical Streisinger template-slippage mechanism to generate -1 deletions in repetitive sequences, as do the bacterial and archaeal homologues. After the first nucleotide is added by template slippage, however, hPolκ can efficiently realign the primer-template duplex before continuing DNA synthesis. Strand realignment results in a base-substitution mutation, minimizing generation of more deleterious frameshift mutations. On non-repetitive sequences, we find that nucleotide misincorporation is slower if the incoming nucleotide can correctly basepair with the nucleotide immediately 5' to the templating base, thereby competing against the mispairing with the templating base.

Show MeSH
Related in: MedlinePlus