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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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Mechanism of second dGTP incorporation on 4C-G substrate. (A) Pre-steady-state assays under single turnover conditions were performed. (i) and (ii) show sequencing gels depicting multiple dG additions on substrates 4C-G and 4G, respectively. (B) Plot of percentage extension versus time for both 4C-G and 4G substrates. Addition of first dG plateaus at ∼60% followed by slow misincorporation of the second dGTP. Rate of second dG incorporation on the 4C-G corresponds well with misincorporation of dGTP on the 4G substrate that has the first dGTP already added. (C) Schematic showing the probable mechanism of second dGTP addition on 4C-G substrate. The incoming nucleotide and newly added base are shown in italics (bold). The base positions assigned are shown with respect to the templating position defined as 0. The black oval represents base-pairing before bond formation.
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gkt179-F3: Mechanism of second dGTP incorporation on 4C-G substrate. (A) Pre-steady-state assays under single turnover conditions were performed. (i) and (ii) show sequencing gels depicting multiple dG additions on substrates 4C-G and 4G, respectively. (B) Plot of percentage extension versus time for both 4C-G and 4G substrates. Addition of first dG plateaus at ∼60% followed by slow misincorporation of the second dGTP. Rate of second dG incorporation on the 4C-G corresponds well with misincorporation of dGTP on the 4G substrate that has the first dGTP already added. (C) Schematic showing the probable mechanism of second dGTP addition on 4C-G substrate. The incoming nucleotide and newly added base are shown in italics (bold). The base positions assigned are shown with respect to the templating position defined as 0. The black oval represents base-pairing before bond formation.

Mentions: We observed that hPolκ performs multiple additions on most DNA substrates examined, even when provided with just a single nucleotide. On the 4C-G substrate, this was true for both dGTP (correct) and dCTP (incorrect) incorporations [Figures 3A, panel (i), and 4B, panel (i)], but to differing extents. Interestingly, we found that multiple addition of dGTP followed a very different pattern than that observed for dCTP, indicating dissimilarity in mechanism. To better understand the relevance of this observation and ascertain its mechanistic implications, we decided to investigate how hPolκ performs multiple nucleotide additions on the 4C-G substrate.Figure 3.


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)

Mechanism of second dGTP incorporation on 4C-G substrate. (A) Pre-steady-state assays under single turnover conditions were performed. (i) and (ii) show sequencing gels depicting multiple dG additions on substrates 4C-G and 4G, respectively. (B) Plot of percentage extension versus time for both 4C-G and 4G substrates. Addition of first dG plateaus at ∼60% followed by slow misincorporation of the second dGTP. Rate of second dG incorporation on the 4C-G corresponds well with misincorporation of dGTP on the 4G substrate that has the first dGTP already added. (C) Schematic showing the probable mechanism of second dGTP addition on 4C-G substrate. The incoming nucleotide and newly added base are shown in italics (bold). The base positions assigned are shown with respect to the templating position defined as 0. The black oval represents base-pairing before bond formation.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3643592&req=5

gkt179-F3: Mechanism of second dGTP incorporation on 4C-G substrate. (A) Pre-steady-state assays under single turnover conditions were performed. (i) and (ii) show sequencing gels depicting multiple dG additions on substrates 4C-G and 4G, respectively. (B) Plot of percentage extension versus time for both 4C-G and 4G substrates. Addition of first dG plateaus at ∼60% followed by slow misincorporation of the second dGTP. Rate of second dG incorporation on the 4C-G corresponds well with misincorporation of dGTP on the 4G substrate that has the first dGTP already added. (C) Schematic showing the probable mechanism of second dGTP addition on 4C-G substrate. The incoming nucleotide and newly added base are shown in italics (bold). The base positions assigned are shown with respect to the templating position defined as 0. The black oval represents base-pairing before bond formation.
Mentions: We observed that hPolκ performs multiple additions on most DNA substrates examined, even when provided with just a single nucleotide. On the 4C-G substrate, this was true for both dGTP (correct) and dCTP (incorrect) incorporations [Figures 3A, panel (i), and 4B, panel (i)], but to differing extents. Interestingly, we found that multiple addition of dGTP followed a very different pattern than that observed for dCTP, indicating dissimilarity in mechanism. To better understand the relevance of this observation and ascertain its mechanistic implications, we decided to investigate how hPolκ performs multiple nucleotide additions on the 4C-G substrate.Figure 3.

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