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The role of BRCA2 in replication-coupled DNA interstrand cross-link repair in vitro

Nature Structural & Molecular Biology volume 13, pages 729–733 (2006)Cite this article

Abstract

Using a defined substrate DNA with a single psoralen interstrand cross-link (ICL), we studied the molecular mechanism of human ICL repair. In vitro ICL repair by human extracts is dependent on replication and is a largely error-free process. Extracts from a human BRCA2-defective mutant cell line, CAPAN-1, are severely compromised in ICL repair. Specifically, 'unhooked' but not fully repaired products accumulate in the reaction with CAPAN-1, and transient expression of BRCA2 in CAPAN-1 restores repair activity. Together, these results reveal that BRCA2 participates in repair of replication-mediated double-strand breaks generated when replication forks encounter ICLs. We also show that nucleotide excision repair is essential for the removal of the lesion left behind on one strand after unhooking. This study provides new mechanistic insights into the repair of ICLs in human cells.

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Figure 1: In vitro ICL repair is DNA replication dependent and error-free.
Figure 2: BRCA2 stimulates replication-coupled ICL repair.
Figure 3: Defining the roles of BRCA2 and NER in ICL repair.

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Acknowledgements

We thank J. Hearst for the psoralen-modified oligonucleotides, S. Powell (Washington University) for the HA-BRCA2 plasmid and J. Reardon, R. Lahue, R. Lewis and A. Tomkinson for critical reading of the manuscript. This work was supported by grant CA095291 from the US National Institutes of Health and grant DAMD17-03-1-0225 from the US Army Medical Research and Materiel Command.

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  1. The Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, 68198-6805, Nebraska, USA

    Lubos Cipak, Norifumi Watanabe & Tadayoshi Bessho

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Correspondence to Tadayoshi Bessho.

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Supplementary information

Supplementary Fig. 1

Defined substrate DNA used in the study (PDF 568 kb)

Supplementary Fig. 2

Control experiments for BRCA2-dependent, replication-coupled ICL repair (PDF 339 kb)

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Cipak, L., Watanabe, N. & Bessho, T. The role of BRCA2 in replication-coupled DNA interstrand cross-link repair in vitro. Nat Struct Mol Biol 13, 729–733 (2006). https://doi.org/10.1038/nsmb1120

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