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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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Single-base deletion mechanisms. On a deletion ‘hotspot’ containing a homopolymeric run of pyrimidines followed by a 5′G, DinB polymerases could (A) add the correct nucleotide (dGTP) or generate single-base deletions in the presence of the incorrect nucleotide (dCTP) by any of three proposed mechanisms: (B) template slippage (C) misincorporation–misalignment and (D) dNTP-stabilized misalignment (see text for more details). 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 phosphodiester bond formation.
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gkt179-F1: Single-base deletion mechanisms. On a deletion ‘hotspot’ containing a homopolymeric run of pyrimidines followed by a 5′G, DinB polymerases could (A) add the correct nucleotide (dGTP) or generate single-base deletions in the presence of the incorrect nucleotide (dCTP) by any of three proposed mechanisms: (B) template slippage (C) misincorporation–misalignment and (D) dNTP-stabilized misalignment (see text for more details). 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 phosphodiester bond formation.

Mentions: To date, three mechanisms have been suggested for generation of single-base deletions (Figure 1). On repetitive/iterative sequences, Streisinger slippage is thought to occur, where the intrinsic nature of the substrate allows DNA duplexes to readily misalign, leaving an extrahelical nucleotide in one strand (Figure 1B). After a round of replication, this can lead to addition or deletion of the unpaired nucleotide, depending on whether the unpaired nucleotide is in the primer or template strand. When this occurs in an open reading frame, Streisinger slippage results in +1 or −1 frameshift mutations (18,19). For deletions to occur by this mechanism, the polymerase must be able to tolerate the unpaired nucleotide that is skipped during replication in the template strand, causing the newly synthesized primer strand to be shorter than the template.Figure 1.


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)

Single-base deletion mechanisms. On a deletion ‘hotspot’ containing a homopolymeric run of pyrimidines followed by a 5′G, DinB polymerases could (A) add the correct nucleotide (dGTP) or generate single-base deletions in the presence of the incorrect nucleotide (dCTP) by any of three proposed mechanisms: (B) template slippage (C) misincorporation–misalignment and (D) dNTP-stabilized misalignment (see text for more details). 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 phosphodiester bond formation.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt179-F1: Single-base deletion mechanisms. On a deletion ‘hotspot’ containing a homopolymeric run of pyrimidines followed by a 5′G, DinB polymerases could (A) add the correct nucleotide (dGTP) or generate single-base deletions in the presence of the incorrect nucleotide (dCTP) by any of three proposed mechanisms: (B) template slippage (C) misincorporation–misalignment and (D) dNTP-stabilized misalignment (see text for more details). 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 phosphodiester bond formation.
Mentions: To date, three mechanisms have been suggested for generation of single-base deletions (Figure 1). On repetitive/iterative sequences, Streisinger slippage is thought to occur, where the intrinsic nature of the substrate allows DNA duplexes to readily misalign, leaving an extrahelical nucleotide in one strand (Figure 1B). After a round of replication, this can lead to addition or deletion of the unpaired nucleotide, depending on whether the unpaired nucleotide is in the primer or template strand. When this occurs in an open reading frame, Streisinger slippage results in +1 or −1 frameshift mutations (18,19). For deletions to occur by this mechanism, the polymerase must be able to tolerate the unpaired nucleotide that is skipped during replication in the template strand, causing the newly synthesized primer strand to be shorter than the template.Figure 1.

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