Holliday junction trap shows how cells use recombination and a junction-guardian role of RecQ helicase

Abstract

DNA repair by homologous recombination (HR) underpins cell survival and fuels genome instability, cancer, and evolution. However, the main kinds and sources of DNA damage repaired by HR in somatic cells and the roles of important HR proteins remain elusive. We present engineered proteins that trap, map, and quantify Holliday junctions (HJs), a central DNA intermediate in HR, based on catalytically deficient mutant RuvC protein of Escherichia coli. We use RuvCDefGFP (RDG) to map genomic footprints of HR at defined DNA breaks in E. coli and demonstrate genome-scale directionality of double-strand break (DSB) repair along the chromosome. Unexpectedly, most spontaneous HR-HJ foci are instigated, not by DSBs, but rather by single-stranded DNA damage generated by replication. We show that RecQ, the E. coli ortholog of five human cancer proteins, nonredundantly promotes HR-HJ formation in single cells and, in a novel junction-guardian role, also prevents apparent non-HR-HJs promoted by RecA overproduction. We propose that one or more human RecQ orthologs may act similarly in human cancers overexpressing the RecA ortholog RAD51 and find that cancer genome expression data implicate the orthologs BLM and RECQL4 in conjunction with EME1 and GEN1 as probable HJ reducers in such cancers. Our results support RecA-overproducing E. coli as a model of the many human tumors with up-regulated RAD51 and provide the first glimpses of important, previously elusive reaction intermediates in DNA replication and repair in single living cells.

DOI: 10.1126/sciadv.1601605

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Cite this paper

@inproceedings{Xia2016HollidayJT, title={Holliday junction trap shows how cells use recombination and a junction-guardian role of RecQ helicase}, author={Jun Xia and Li-Tzu Chen and Qian G. Mei and Chien-Hui Ma and Jennifer A. Halliday and Hsin-Yu Lin and David Magnan and John P Pribis and Devon M Fitzgerald and Holly M Hamilton and Megan Richters and Ralf Bernd Nehring and Xi Hong Shen and Lei Li and David W Bates and P J Hastings and Christophe Herman and Makkuni Jayaram and Susan M Rosenberg}, booktitle={Science advances}, year={2016} }