Single-nucleotide base excision repair DNA polymerase activity in C. elegans in the absence of DNA polymerase β

Kenjiro Asagoshi; Wade Lehmann; Elena K. Braithwaite; Lucas Santana-Santos; Rajendra Prasad; Jonathan H. Freedman; Bennett Van Houten; Samuel H. Wilson

doi:10.1093/nar/gkr727

Single-nucleotide base excision repair DNA polymerase activity in C. elegans in the absence of DNA polymerase β

Kenjiro Asagoshi, Wade Lehmann, Elena K. Braithwaite, Lucas Santana-Santos, Rajendra Prasad, Jonathan H. Freedman, Bennett Van Houten, Samuel H. Wilson^*

^*Corresponding author for this work

Pathology

Research output: Contribution to journal › Article › peer-review

23 Scopus citations

Abstract

The base excision DNA repair (BER) pathway known to occur in Caenorhabditis elegans has not been well characterized. Even less is known about the DNA polymerase (pol) requirement for the gap-filling step during BER. We now report on characterization of in vitro uracil-DNA initiated BER in C. elegans. The results revealed single-nucleotide (SN) gap-filling DNA polymerase activity and complete BER. The gap-filling polymerase activity was not due to a DNA polymerase β (pol β) homolog, or to another X-family polymerase, since computer-based sequence analyses of the C. elegans genome failed to show a match for a pol β-like gene or other X-family polymerases. Activity gel analysis confirmed the absence of pol β in the C. elegans extract. BER gap-filling polymerase activity was partially inhibited by both dideoxynucleotide and aphidicolin. The results are consistent with a combination of both replicative polymerase(s) and lesion bypass/BER polymerase pol θ contributing to the BER gap-filling synthesis. Involvement of pol θ was confirmed in experiments with extract from pol θ null animals. The presence of the SN BER in C. elegans is supported by these results, despite the absence of a pol β-like enzyme or other X-family polymerase.

Original language	English (US)
Pages (from-to)	670-681
Number of pages	12
Journal	Nucleic acids research
Volume	40
Issue number	2
DOIs	https://doi.org/10.1093/nar/gkr727
State	Published - Jan 2012

Funding

We thank Bonnie Mesmer for editorial assistance, Julie A. Hall, Julie R. Rice, Kathryn L. Haas and Windy A. Boyd for their assistance with nematode genotyping and culturing and anonymous reviewers for suggestions. Nematodes strains used in this work were provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources. The Intramural Research Program of the National Institutes of Health, National Institute of Environmental Health Sciences (grants Z01ES050158 and Z01ES050159 to S.H.W., partial); Z01-ES061060 and Z01ES102045 to J.H.F., partial); (R01-ES019566 to B.V.H., partial). Funding for open access charge: U.S. Government funds.

ASJC Scopus subject areas

Genetics

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10.1093/nar/gkr727

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title = "Single-nucleotide base excision repair DNA polymerase activity in C. elegans in the absence of DNA polymerase β",

abstract = "The base excision DNA repair (BER) pathway known to occur in Caenorhabditis elegans has not been well characterized. Even less is known about the DNA polymerase (pol) requirement for the gap-filling step during BER. We now report on characterization of in vitro uracil-DNA initiated BER in C. elegans. The results revealed single-nucleotide (SN) gap-filling DNA polymerase activity and complete BER. The gap-filling polymerase activity was not due to a DNA polymerase β (pol β) homolog, or to another X-family polymerase, since computer-based sequence analyses of the C. elegans genome failed to show a match for a pol β-like gene or other X-family polymerases. Activity gel analysis confirmed the absence of pol β in the C. elegans extract. BER gap-filling polymerase activity was partially inhibited by both dideoxynucleotide and aphidicolin. The results are consistent with a combination of both replicative polymerase(s) and lesion bypass/BER polymerase pol θ contributing to the BER gap-filling synthesis. Involvement of pol θ was confirmed in experiments with extract from pol θ null animals. The presence of the SN BER in C. elegans is supported by these results, despite the absence of a pol β-like enzyme or other X-family polymerase.",

author = "Kenjiro Asagoshi and Wade Lehmann and Braithwaite, {Elena K.} and Lucas Santana-Santos and Rajendra Prasad and Freedman, {Jonathan H.} and {Van Houten}, Bennett and Wilson, {Samuel H.}",

note = "Funding Information: We thank Bonnie Mesmer for editorial assistance, Julie A. Hall, Julie R. Rice, Kathryn L. Haas and Windy A. Boyd for their assistance with nematode genotyping and culturing and anonymous reviewers for suggestions. Nematodes strains used in this work were provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources. Funding Information: The Intramural Research Program of the National Institutes of Health, National Institute of Environmental Health Sciences (grants Z01ES050158 and Z01ES050159 to S.H.W., partial); Z01-ES061060 and Z01ES102045 to J.H.F., partial); (R01-ES019566 to B.V.H., partial). Funding for open access charge: U.S. Government funds.",

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doi = "10.1093/nar/gkr727",

language = "English (US)",

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AU - Asagoshi, Kenjiro

AU - Lehmann, Wade

AU - Braithwaite, Elena K.

AU - Santana-Santos, Lucas

AU - Prasad, Rajendra

AU - Freedman, Jonathan H.

AU - Van Houten, Bennett

AU - Wilson, Samuel H.

N1 - Funding Information: We thank Bonnie Mesmer for editorial assistance, Julie A. Hall, Julie R. Rice, Kathryn L. Haas and Windy A. Boyd for their assistance with nematode genotyping and culturing and anonymous reviewers for suggestions. Nematodes strains used in this work were provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources. Funding Information: The Intramural Research Program of the National Institutes of Health, National Institute of Environmental Health Sciences (grants Z01ES050158 and Z01ES050159 to S.H.W., partial); Z01-ES061060 and Z01ES102045 to J.H.F., partial); (R01-ES019566 to B.V.H., partial). Funding for open access charge: U.S. Government funds.

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N2 - The base excision DNA repair (BER) pathway known to occur in Caenorhabditis elegans has not been well characterized. Even less is known about the DNA polymerase (pol) requirement for the gap-filling step during BER. We now report on characterization of in vitro uracil-DNA initiated BER in C. elegans. The results revealed single-nucleotide (SN) gap-filling DNA polymerase activity and complete BER. The gap-filling polymerase activity was not due to a DNA polymerase β (pol β) homolog, or to another X-family polymerase, since computer-based sequence analyses of the C. elegans genome failed to show a match for a pol β-like gene or other X-family polymerases. Activity gel analysis confirmed the absence of pol β in the C. elegans extract. BER gap-filling polymerase activity was partially inhibited by both dideoxynucleotide and aphidicolin. The results are consistent with a combination of both replicative polymerase(s) and lesion bypass/BER polymerase pol θ contributing to the BER gap-filling synthesis. Involvement of pol θ was confirmed in experiments with extract from pol θ null animals. The presence of the SN BER in C. elegans is supported by these results, despite the absence of a pol β-like enzyme or other X-family polymerase.

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Single-nucleotide base excision repair DNA polymerase activity in C. elegans in the absence of DNA polymerase β

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