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Sci Adv. 2016 Nov 18;2(11):e1601605. doi: 10.1126/sciadv.1601605. eCollection 2016 Nov.

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

Author information

1
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.; Department of Biochemistry, Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA.; Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA.; Dan L Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA.; Graduate Program in Integrative Molecular and Biomedical Sciences, Baylor College of Medicine, Houston, TX 77030, USA.
2
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.; Department of Biochemistry, Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA.; Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA.; Dan L Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA.
3
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.; Department of Biochemistry, Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA.; Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA.; Dan L Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA.; Systems, Synthetic, and Physical Biology Program, Rice University, Houston, TX 77030, USA.
4
Department of Molecular Biosciences, University of Texas, Austin, TX 78712, USA.; Institute of Cell and Molecular Biology, University of Texas, Austin, TX 78712, USA.
5
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.; Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA.; Dan L Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA.
6
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.; Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA.; Dan L Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA.; Graduate Program in Integrative Molecular and Biomedical Sciences, Baylor College of Medicine, Houston, TX 77030, USA.
7
Department of Experimental Radiation Oncology, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
8
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.; Dan L Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA.
9
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.; Department of Biochemistry, Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA.; Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA.; Dan L Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA.; Graduate Program in Integrative Molecular and Biomedical Sciences, Baylor College of Medicine, Houston, TX 77030, USA.; Systems, Synthetic, and Physical Biology Program, Rice University, Houston, TX 77030, USA.

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.

KEYWORDS:

BLM; DNA repair; DNA replication; Escherichia coli; RecQ family proteins; RuvC; cancer; holliday junctions; homologous recombination; spontaneous DNA damage

PMID:
28090586
PMCID:
PMC5222578
DOI:
10.1126/sciadv.1601605
[Indexed for MEDLINE]
Free PMC Article

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