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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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Nucleotide incorporation by hPolκ on repetitive sequence containing deletion hotspot. In all, 15 µM of hPolκ was pre-incubated with 50 nM DNA before initiating the reaction with addition of 1 mM dNTP (final concentrations). (A) On 4C-G (substrate 1), containing a run of four C’s followed by a 5′ G, hPolκ adds dGTP ∼4-fold faster than dCTP. (B) Modification of the nucleotide 5′ to the templating position (i.e. +1 position) from G to A [4C-A, substrate 6] results in reduction in overall kobs for incorporation of dCTP by 6-fold from 0.72 ± 0.04 s−1 to 0.12 ± 0.01 s−1. (C) Pre-steady-state assays were performed under single turnover conditions using DNA templates containing unpaired T’s at every C position in the homopolymeric run of 4 C-G (substrates 2–5) to test whether hPolκ can tolerate the presence of an extrahelical nucleotide at these positions. The dCTP incorporation by hPolκ on 1T-G, 2T-G, 3T-G and 4T-G substrates is shown. The hPolκ extends the various bulged templates with the -3T bulge (3T-G, substrate 4) being used most efficiently. (D) Modification of the +1 G position of the 1T-G substrate to an A [1T-A, substrate 7] resulted in a 3-fold increase in kobs from 0.44 ± 0.03 s−1 to 1.14 ± 0.09 s−1.
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gkt179-F2: Nucleotide incorporation by hPolκ on repetitive sequence containing deletion hotspot. In all, 15 µM of hPolκ was pre-incubated with 50 nM DNA before initiating the reaction with addition of 1 mM dNTP (final concentrations). (A) On 4C-G (substrate 1), containing a run of four C’s followed by a 5′ G, hPolκ adds dGTP ∼4-fold faster than dCTP. (B) Modification of the nucleotide 5′ to the templating position (i.e. +1 position) from G to A [4C-A, substrate 6] results in reduction in overall kobs for incorporation of dCTP by 6-fold from 0.72 ± 0.04 s−1 to 0.12 ± 0.01 s−1. (C) Pre-steady-state assays were performed under single turnover conditions using DNA templates containing unpaired T’s at every C position in the homopolymeric run of 4 C-G (substrates 2–5) to test whether hPolκ can tolerate the presence of an extrahelical nucleotide at these positions. The dCTP incorporation by hPolκ on 1T-G, 2T-G, 3T-G and 4T-G substrates is shown. The hPolκ extends the various bulged templates with the -3T bulge (3T-G, substrate 4) being used most efficiently. (D) Modification of the +1 G position of the 1T-G substrate to an A [1T-A, substrate 7] resulted in a 3-fold increase in kobs from 0.44 ± 0.03 s−1 to 1.14 ± 0.09 s−1.

Mentions: To determine whether hPolκ is error prone on the ‘deletion hotspot’ sequence characterized for other DinB polymerases (11,12,14,27,28), we used DNA substrate 4C-G, which contains a homopolymeric run of four C’s followed by a 5′ G on the template strand (3′-CCCCG-5′, Table 1). On this substrate, hPolκ adds the correct incoming nucleotide, dGTP, at a rate that is only 3.6-fold faster (kobs of 2.62 ± 0.39 s−1) than the rate of incorrect nucleotide, dCTP (kobs of 0.72 ± 0.04 s−1) addition (Figure 2A), indicating that deletions can be initiated on this substrate at a very high frequency.Figure 2.


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)

Nucleotide incorporation by hPolκ on repetitive sequence containing deletion hotspot. In all, 15 µM of hPolκ was pre-incubated with 50 nM DNA before initiating the reaction with addition of 1 mM dNTP (final concentrations). (A) On 4C-G (substrate 1), containing a run of four C’s followed by a 5′ G, hPolκ adds dGTP ∼4-fold faster than dCTP. (B) Modification of the nucleotide 5′ to the templating position (i.e. +1 position) from G to A [4C-A, substrate 6] results in reduction in overall kobs for incorporation of dCTP by 6-fold from 0.72 ± 0.04 s−1 to 0.12 ± 0.01 s−1. (C) Pre-steady-state assays were performed under single turnover conditions using DNA templates containing unpaired T’s at every C position in the homopolymeric run of 4 C-G (substrates 2–5) to test whether hPolκ can tolerate the presence of an extrahelical nucleotide at these positions. The dCTP incorporation by hPolκ on 1T-G, 2T-G, 3T-G and 4T-G substrates is shown. The hPolκ extends the various bulged templates with the -3T bulge (3T-G, substrate 4) being used most efficiently. (D) Modification of the +1 G position of the 1T-G substrate to an A [1T-A, substrate 7] resulted in a 3-fold increase in kobs from 0.44 ± 0.03 s−1 to 1.14 ± 0.09 s−1.
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gkt179-F2: Nucleotide incorporation by hPolκ on repetitive sequence containing deletion hotspot. In all, 15 µM of hPolκ was pre-incubated with 50 nM DNA before initiating the reaction with addition of 1 mM dNTP (final concentrations). (A) On 4C-G (substrate 1), containing a run of four C’s followed by a 5′ G, hPolκ adds dGTP ∼4-fold faster than dCTP. (B) Modification of the nucleotide 5′ to the templating position (i.e. +1 position) from G to A [4C-A, substrate 6] results in reduction in overall kobs for incorporation of dCTP by 6-fold from 0.72 ± 0.04 s−1 to 0.12 ± 0.01 s−1. (C) Pre-steady-state assays were performed under single turnover conditions using DNA templates containing unpaired T’s at every C position in the homopolymeric run of 4 C-G (substrates 2–5) to test whether hPolκ can tolerate the presence of an extrahelical nucleotide at these positions. The dCTP incorporation by hPolκ on 1T-G, 2T-G, 3T-G and 4T-G substrates is shown. The hPolκ extends the various bulged templates with the -3T bulge (3T-G, substrate 4) being used most efficiently. (D) Modification of the +1 G position of the 1T-G substrate to an A [1T-A, substrate 7] resulted in a 3-fold increase in kobs from 0.44 ± 0.03 s−1 to 1.14 ± 0.09 s−1.
Mentions: To determine whether hPolκ is error prone on the ‘deletion hotspot’ sequence characterized for other DinB polymerases (11,12,14,27,28), we used DNA substrate 4C-G, which contains a homopolymeric run of four C’s followed by a 5′ G on the template strand (3′-CCCCG-5′, Table 1). On this substrate, hPolκ adds the correct incoming nucleotide, dGTP, at a rate that is only 3.6-fold faster (kobs of 2.62 ± 0.39 s−1) than the rate of incorrect nucleotide, dCTP (kobs of 0.72 ± 0.04 s−1) addition (Figure 2A), indicating that deletions can be initiated on this substrate at a very high frequency.Figure 2.

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