Homologous Recombination but Not Nucleotide Excision Repair Plays a Pivotal Role in Tolerance of DNA-Protein Cross-links in Mammalian Cells

Seung Pil Pack,Tomohiro Tsuboi,Hiroshi Tauchi,Takahiro Tatsumoto,Hiroshi Ide,Toshiaki Nakano,Deborah L Croteau,Kenta Iijima,Atsushi Katafuchi,Keisuke Makino,Hiroaki Terato,Bennett Van Houten,Mayumi Matsubara,Tasuku Masuda

doi:10.1074/jbc.m109.019174

Abstract

DNA-protein cross-links (DPCs) are unique among DNA lesions in their unusually bulky nature. The steric hindrance imposed by cross-linked proteins (CLPs) will hamper DNA transactions, such as replication and transcription, posing an enormous threat to cells. In bacteria, DPCs with small CLPs are eliminated by nucleotide excision repair (NER), whereas oversized DPCs are processed exclusively by RecBCD-dependent homologous recombination (HR). Here we have assessed the roles of NER and HR for DPCs in mammalian cells. We show that the upper size limit of CLPs amenable to mammalian NER is relatively small (8-10 kDa) so that NER cannot participate in the repair of chromosomal DPCs in mammalian cells. Moreover, CLPs are not polyubiquitinated and hence are not subjected to proteasomal degradation prior to NER. In contrast, HR constitutes the major pathway in tolerance of DPCs as judged from cell survival and RAD51 and gamma-H2AX nuclear foci formation. Induction of DPCs results in the accumulation of DNA double strand breaks in HR-deficient but not HR-proficient cells, suggesting that fork breakage at the DPC site initiates HR and reactivates the stalled fork. DPCs activate both ATR and ATM damage response pathways, but there is a time lag between two responses. These results highlight the differential involvement of NER in the repair of DPCs in bacterial and mammalian cells and demonstrate the versatile and conserved role of HR in tolerance of DPCs among species.

Highlights

From the ‡Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan, the §Department of Biotechnology and Bioinformatics, Korea University, Jochiwon, Chungnam 339-700, Korea, the ¶Institute of Advanced Energy, Kyoto University, Gokasho, Uji 611-0011, Japan, the ʈLaboratory of Molecular Genetics, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, and the **Department of Environmental Sciences, Faculty of Science, Ibaraki University, Bunkyo 2-1-1, Mito, Ibaraki 310-8512, Japan
We show that the upper size limit of cross-linked proteins (CLPs) amenable to mammalian nucleotide excision repair (NER) is relatively small (8 –10 kDa) so that NER cannot participate in the repair of chromosomal DNA-protein cross-links (DPCs) in mammalian cells
It was confirmed that 5Ј incisions for DPCs were attributable to NER activity; the 5Ј incision activity for DPCs was not observed with cell-free extracts (CFEs) from NER-deficient human xeroderma pigmentosum XPA or XPF cells, but it was restored when CFEs from both cells were combined

Summary

Homologous Recombination but Not Nucleotide Excision

Repair Plays a Pivotal Role in Tolerance of DNA-Protein Cross-links in Mammalian Cells*□S. DPCs activate both ATR and ATM damage response pathways, but there is a time lag between two responses These results highlight the differential involvement of NER in the repair of DPCs in bacterial and mammalian cells and demonstrate the versatile and conserved role of HR in tolerance of DPCs among species. CLPs attenuate the binding of the damage recognition protein (UvrB) involved in bacterial nucleotide excision repair (NER) in a size-dependent manner [7] It has been largely elusive how cells circumvent the genotoxic effects of DPCs. We recently showed that NER and homologous recombination (HR) play pivotal roles in mitigating the genotoxic effects of DPCs in bacteria [7].

EXPERIMENTAL PROCEDURES

RESULTS

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