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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

Mechanism of multiple dCTP incorporations. (A) Schematic representation of the two possible ways in which hPolκ can efficiently add a second dCTP on 4C-G (substrate 1). 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. ‘*’ indicates the +2 templating position. (B) Gels showing the unique pattern of multiple incorporations of dCTP on (i) 4C-G (substrate 1) and (ii) 4C-GA (substrate 9) to test for mispair formation. (iii) Gel showing the incorporation of dC to extend from a C-C mispair (substrate 8) suggests hPolκ’s ability to realign. (C) Plot shows that mechanism of multiple dC additions is distinct from dG additions on 4C-G substrate. First dCTP addition reaches a maximum of ∼40% extension, after which increase in second dCTP incorporation occurs exponentially, implying that most of the primer extended by one nucleotide is rapidly extended by two. (D) Overall dCTP incorporation on the 4C-G, 4C-GA and C-C mispair substrates shown with respect to time along with percent of second dCTP addition as seen for the 4C-G and 4C-GA substrates. Second dCTP incorporation traces are shown as dotted lines.
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gkt179-F4: Mechanism of multiple dCTP incorporations. (A) Schematic representation of the two possible ways in which hPolκ can efficiently add a second dCTP on 4C-G (substrate 1). 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. ‘*’ indicates the +2 templating position. (B) Gels showing the unique pattern of multiple incorporations of dCTP on (i) 4C-G (substrate 1) and (ii) 4C-GA (substrate 9) to test for mispair formation. (iii) Gel showing the incorporation of dC to extend from a C-C mispair (substrate 8) suggests hPolκ’s ability to realign. (C) Plot shows that mechanism of multiple dC additions is distinct from dG additions on 4C-G substrate. First dCTP addition reaches a maximum of ∼40% extension, after which increase in second dCTP incorporation occurs exponentially, implying that most of the primer extended by one nucleotide is rapidly extended by two. (D) Overall dCTP incorporation on the 4C-G, 4C-GA and C-C mispair substrates shown with respect to time along with percent of second dCTP addition as seen for the 4C-G and 4C-GA substrates. Second dCTP incorporation traces are shown as dotted lines.

Mentions: In contrast to the slow rate of addition of a second dG, we found that addition of a second dC is considerably faster and followed an entirely different pattern (Figure 3A and B versus Figure 4B and C). As the aforementioned data indicate, the first addition of dC occurs predominantly by template slippage, allowing the proper base pairing of the dCTP opposite the +1G. We can envision two possibilities for the addition of the second dC (Figure 4A). One possibility is that the next dCTP is misincorporated opposite the templating C at +2 to generate a C-dCMP ‘mispair’. A second possibility is that the extrahelical nucleotide generated during the first dCTP addition is ‘rearranged’ such that now there is a C-C mispair generated at the p/t junction. As hPolκ is an efficient extender of mispairs (16), it could tolerate the mispair to add the second dCTP correctly paired opposite the +1G.Figure 4.


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 multiple dCTP incorporations. (A) Schematic representation of the two possible ways in which hPolκ can efficiently add a second dCTP on 4C-G (substrate 1). 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. ‘*’ indicates the +2 templating position. (B) Gels showing the unique pattern of multiple incorporations of dCTP on (i) 4C-G (substrate 1) and (ii) 4C-GA (substrate 9) to test for mispair formation. (iii) Gel showing the incorporation of dC to extend from a C-C mispair (substrate 8) suggests hPolκ’s ability to realign. (C) Plot shows that mechanism of multiple dC additions is distinct from dG additions on 4C-G substrate. First dCTP addition reaches a maximum of ∼40% extension, after which increase in second dCTP incorporation occurs exponentially, implying that most of the primer extended by one nucleotide is rapidly extended by two. (D) Overall dCTP incorporation on the 4C-G, 4C-GA and C-C mispair substrates shown with respect to time along with percent of second dCTP addition as seen for the 4C-G and 4C-GA substrates. Second dCTP incorporation traces are shown as dotted lines.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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gkt179-F4: Mechanism of multiple dCTP incorporations. (A) Schematic representation of the two possible ways in which hPolκ can efficiently add a second dCTP on 4C-G (substrate 1). 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. ‘*’ indicates the +2 templating position. (B) Gels showing the unique pattern of multiple incorporations of dCTP on (i) 4C-G (substrate 1) and (ii) 4C-GA (substrate 9) to test for mispair formation. (iii) Gel showing the incorporation of dC to extend from a C-C mispair (substrate 8) suggests hPolκ’s ability to realign. (C) Plot shows that mechanism of multiple dC additions is distinct from dG additions on 4C-G substrate. First dCTP addition reaches a maximum of ∼40% extension, after which increase in second dCTP incorporation occurs exponentially, implying that most of the primer extended by one nucleotide is rapidly extended by two. (D) Overall dCTP incorporation on the 4C-G, 4C-GA and C-C mispair substrates shown with respect to time along with percent of second dCTP addition as seen for the 4C-G and 4C-GA substrates. Second dCTP incorporation traces are shown as dotted lines.
Mentions: In contrast to the slow rate of addition of a second dG, we found that addition of a second dC is considerably faster and followed an entirely different pattern (Figure 3A and B versus Figure 4B and C). As the aforementioned data indicate, the first addition of dC occurs predominantly by template slippage, allowing the proper base pairing of the dCTP opposite the +1G. We can envision two possibilities for the addition of the second dC (Figure 4A). One possibility is that the next dCTP is misincorporated opposite the templating C at +2 to generate a C-dCMP ‘mispair’. A second possibility is that the extrahelical nucleotide generated during the first dCTP addition is ‘rearranged’ such that now there is a C-C mispair generated at the p/t junction. As hPolκ is an efficient extender of mispairs (16), it could tolerate the mispair to add the second dCTP correctly paired opposite the +1G.Figure 4.

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