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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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Related in: MedlinePlus

The hPolκ misincorporates efficiently on 1T-G substrate. (A) Gel and (B) graph showing multiple incorporations of dCTP on the 1T-G substrate. First addition, second addition and overall addition. (C) Schematic showing possible mechanisms of dCTP addition on the 1T-G substrate. (D) Gel and (E) graph showing multiple incorporations of dCTP on the 1T-A substrate. (F) Schematic showing possible mechanisms of dCTP addition on the 1T-A substrate. In (C) and (F), 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-F5: The hPolκ misincorporates efficiently on 1T-G substrate. (A) Gel and (B) graph showing multiple incorporations of dCTP on the 1T-G substrate. First addition, second addition and overall addition. (C) Schematic showing possible mechanisms of dCTP addition on the 1T-G substrate. (D) Gel and (E) graph showing multiple incorporations of dCTP on the 1T-A substrate. (F) Schematic showing possible mechanisms of dCTP addition on the 1T-A substrate. In (C) and (F), 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: The ability to incorporate a single nucleotide multiple times was also observed on the 1T-G and 1T-A substrates (Figure 5A and D). As demonstrated earlier in the text, the addition of the first dCTP on 1T-G substrate occurred by misincorporation, as modification of the +1G to A in the 1T-A substrate resulted in ∼3-fold increase in overall rate. Although this clearly indicated that dNTP-stabilized misalignment was not the predominant incorporation mechanism, the increase in rate was puzzling. To understand this better, we calculated the amount of extension for the first and second additions separately (Figure 5B and E) and found that different patterns and rates of incorporation on the two substrates provided further information to decipher the mechanism.Figure 5.


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)

The hPolκ misincorporates efficiently on 1T-G substrate. (A) Gel and (B) graph showing multiple incorporations of dCTP on the 1T-G substrate. First addition, second addition and overall addition. (C) Schematic showing possible mechanisms of dCTP addition on the 1T-G substrate. (D) Gel and (E) graph showing multiple incorporations of dCTP on the 1T-A substrate. (F) Schematic showing possible mechanisms of dCTP addition on the 1T-A substrate. In (C) and (F), 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-F5: The hPolκ misincorporates efficiently on 1T-G substrate. (A) Gel and (B) graph showing multiple incorporations of dCTP on the 1T-G substrate. First addition, second addition and overall addition. (C) Schematic showing possible mechanisms of dCTP addition on the 1T-G substrate. (D) Gel and (E) graph showing multiple incorporations of dCTP on the 1T-A substrate. (F) Schematic showing possible mechanisms of dCTP addition on the 1T-A substrate. In (C) and (F), 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: The ability to incorporate a single nucleotide multiple times was also observed on the 1T-G and 1T-A substrates (Figure 5A and D). As demonstrated earlier in the text, the addition of the first dCTP on 1T-G substrate occurred by misincorporation, as modification of the +1G to A in the 1T-A substrate resulted in ∼3-fold increase in overall rate. Although this clearly indicated that dNTP-stabilized misalignment was not the predominant incorporation mechanism, the increase in rate was puzzling. To understand this better, we calculated the amount of extension for the first and second additions separately (Figure 5B and E) and found that different patterns and rates of incorporation on the two substrates provided further information to decipher the mechanism.Figure 5.

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