CtIP-BRCA1 complex and MRE11 maintain replication forks in the presence of chain terminating nucleoside analogs.

Mohiuddin Mohiuddin,Julian E Sale,Md Maminur Rahman,Christopher E Pearson

doi:10.1093/nar/gkz009

Abstract

Chain-terminating nucleoside analogs (CTNAs), which cannot be extended by DNA polymerases, are widely used as antivirals or anti-cancer agents, and can induce cell death. Processing of blocked DNA ends, like camptothecin-induced trapped-topoisomerase I, can be mediated by TDP1, BRCA1, CtIP and MRE11. Here, we investigated whether the CtIP-BRCA1 complex and MRE11 also contribute to cellular tolerance to CTNAs, including 2’,3’-dideoxycytidine (ddC), cytarabine (ara-C) and zidovudine (Azidothymidine, AZT). We show that BRCA1−/−, CtIPS332A/−/− and nuclease-dead MRE11D20A/− mutants display increased sensitivity to CTNAs, accumulate more DNA damage (chromosomal breaks, γ-H2AX and neutral comets) when treated with CTNAs and exhibit significant delays in replication fork progression during exposure to CTNAs. Moreover, BRCA1−/−, CtIPS332A/−/− and nuclease-dead MRE11D20A/− mutants failed to resume DNA replication in response to CTNAs, whereas control and CtIP+/−/− cells experienced extensive recovery of DNA replication. In summary, we provide clear evidence that MRE11 and the collaborative action of BRCA1 and CtIP play a critical role in the nuclease-dependent removal of incorporated ddC from replicating genomic DNA. We propose that BRCA1-CTIP and MRE11 prepare nascent DNA ends, blocked from synthesis by CTNAs, for further repair.

Highlights

Homologous recombination is initiated at double strand breaks (DSBs) by resection, a process in which DSB ends are converted into 3’-single-strand DNA overhangs
The BRCA1–CtIP complex contributes to cellular tolerance to various Chain-terminating nucleoside analogs (CTNAs), independently of its role in HR
Based upon the activity of the BRCA1–CtIP complex in removing 3’ adducts at DSBs induced by camptothecin [2], we tested the possibility that BRCA1 may act in the removal of chain terminating nucleoside analogs

Summary

Introduction

Homologous recombination is initiated at double strand breaks (DSBs) by resection, a process in which DSB ends are converted into 3’-single-strand DNA overhangs. Interaction of BRCA1 with CtIP is promoted by phosphorylation of Ser327 of CtIP by cyclin-dependent kinase 1 (CDK1) [1]. This finding suggested the attractive idea that the BRCA1–CtIP interaction is involved in DSB resection. The CtIPS327A mutation causes significant increases in cellular sensitivity to camptothecin [2], a Top poison, which stabilizes Top1-DNA-cleavage complex (Top1cc), a single-strand break (SSB) covalently associated with Top at the 3’ end of the break [4,5]. BRCA1−/− and BRCA1−/−/CtIPS332A/−/− DT40 cells show very similar sensitivity to camptothecin [2] These observations suggest that the BRCA1–CtIP complex facilitates removal of Top from Top1cc, a role played by Tyrosyl DNA phosphodiesterase 1 (TDP1), releasing Top together with covalently attached oligonucleotide. Since TDP1 can eliminate incorporated chain terminating nucleoside analogs [6], an interesting question is whether the BRCA1–CtIP complex can facilitate the removal of nucleoside analogs from the 3’ end of oligonucleotides

Methods

Results

Conclusion