Impact of ribonucleotide incorporation by DNA polymerases beta and lambda on oxidative base excision repair (original) (raw)
Oxidative stress is a very frequent source of DNA damage. Many cellular DNA polymerases (Pols) can incorporate ribonucleotides (rNMPs) during DNA synthesis. However, whether oxidative stress-triggered DNA repair synthesis contributes to genomic rNMPs incorporation is so far not fully understood. Human specialized Pols b and l are the important enzymes involved in the oxidative stress tolerance, acting both in base excision repair and in translesion synthesis past the very frequent oxidative lesion 7,8-dihydro-8-oxoguanine (8-oxo-G). We found that Pol b, to a greater extent than Pol l can incorporate rNMPs opposite normal bases or 8-oxo-G, and with a different fidelity. Further, the incorporation of rNMPs opposite 8-oxo-G delays repair by DNA glycosylases. Studies in Pol band l-deficient cell extracts suggest that Pol b levels can greatly affect rNMP incorporation opposite oxidative DNA lesions. | www.nature.com/naturecommunications *The meaning of the kinetic parameters K m , k cat and k cat /K m and their calculations are described in the Methods section. Values are the means of two independent estimates ±s.d. w f inc , relative incorporation frequencies for the different nucleotide pairs, defined as the ratio of the respective k cat /K m values. Figure 4 | Incorporation of rNMPs opposite 8-oxo-G inhibits DNA repair and is reduced in the absence of DNA polymerase b. (a) Time course of the excision products accumulation generated by hOGG1 in the presence of a 8-oxo-G:dC (empty circles) or 8-oxo-G:rC (filled triangles) base pairs. The k app values refer to the apparent rates for the exponential phase. Values are the mean of three independent replicates, error bars represent ±s.d. A representative experiment is shown in (b) Quantification of the excision products generated by MutYH in the presence of a 8-oxo-G:dA (grey bars) or 8-oxo-G:rA (black bars) mismatch. Values are the mean of three independent replicates as the one presented in , error bars represent ± s.d. The P values were calculated by two-tailed Student's t-test. (c) Increasing amounts of the different cell extracts were titrated in the presence of the 1 nt-gap template bearing an 8-oxo-G and 200 mM rCTP. Lanes 13 and 14, control reactions in the absence of extracts. (d) The rCMP incorporation activity (expressed as pmols of rCTP incorporated opposite 8-oxo-G per mg of proteins of each extract) was normalized to the total DNA polymerase activity (expressed as pmols of dCMP incorporated opposite undamaged dG per mg of proteins of each extract). Values are the mean of three independent replicates like the one shown in c, error bars represent ±s.d. The P values were calculated by two-tailed Student's t-test.
Sign up for access to the world's latest research.
checkGet notified about relevant papers
checkSave papers to use in your research
checkJoin the discussion with peers
checkTrack your impact
Loading Preview
Sorry, preview is currently unavailable. You can download the paper by clicking the button above.