title: 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 ofEscherichia coli. We use RuvCDefGFP (RDG) to map genomic footprints of HR at defined DNA breaks inE. coliand 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, theE. coliortholog 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 orthologRAD51and 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-overproducingE. colias a model of the many human tumors with up-regulatedRAD51and provide the first glimpses of important, previously elusive reaction intermediates in DNA replication and repair in single living cells.
authors.affiliation: 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.
authors.forename: Jun
authors.identifier: 0000-0001-7331-0673
authors.initials: J
authors.lastname: Xia
authors.affiliation: 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.
authors.forename: Li-Tzu
authors.initials: LT
authors.lastname: Chen
authors.affiliation: 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.
authors.forename: Qian
authors.initials: Q
authors.lastname: Mei
authors.affiliation: Department of Molecular Biosciences, University of Texas, Austin, TX 78712, USA.; Institute of Cell and Molecular Biology, University of Texas, Austin, TX 78712, USA.
authors.forename: Chien-Hui
authors.identifier: 0000-0001-8753-8556
authors.initials: CH
authors.lastname: Ma
authors.affiliation: 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.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Halliday
authors.affiliation: 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.
authors.forename: Hsin-Yu
authors.initials: HY
authors.lastname: Lin
authors.affiliation: 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.
authors.forename: David
authors.identifier: 0000-0001-9506-8857
authors.initials: D
authors.lastname: Magnan
authors.affiliation: 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.
authors.forename: John P
authors.identifier: 0000-0002-9267-6715
authors.initials: JP
authors.lastname: Pribis
authors.affiliation: 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.
authors.forename: Devon M
authors.identifier: 0000-0002-2480-7269
authors.initials: DM
authors.lastname: Fitzgerald
authors.affiliation: 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.
authors.forename: Holly M
authors.initials: HM
authors.lastname: Hamilton
authors.affiliation: 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.
authors.forename: Megan
authors.initials: M
authors.lastname: Richters
authors.affiliation: 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.
authors.forename: Ralf B
authors.initials: RB
authors.lastname: Nehring
authors.affiliation: Department of Experimental Radiation Oncology, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
authors.forename: Xi
authors.initials: X
authors.lastname: Shen
authors.affiliation: Department of Experimental Radiation Oncology, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
authors.forename: Lei
authors.initials: L
authors.lastname: Li
authors.affiliation: 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.
authors.forename: David
authors.initials: D
authors.lastname: Bates
authors.affiliation: 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.
authors.forename: P J
authors.initials: PJ
authors.lastname: Hastings
authors.affiliation: 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.
authors.forename: Christophe
authors.initials: C
authors.lastname: Herman
authors.affiliation: Department of Molecular Biosciences, University of Texas, Austin, TX 78712, USA.; Institute of Cell and Molecular Biology, University of Texas, Austin, TX 78712, USA.
authors.forename: Makkuni
authors.initials: M
authors.lastname: Jayaram
authors.affiliation: 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.
authors.forename: Susan M
authors.initials: SM
authors.lastname: Rosenberg
mesh_terms: D053904:DNA Breaks, Single-Stranded; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D045566:DNA, Cruciform / Q000235:genetics / Q000378:metabolism; D004273:DNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism; D051135:Rad51 Recombinase / Q000235:genetics / Q000378:metabolism; D053484:RecQ Helicases / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic
chemical_list: D004269:DNA, Bacterial; D045566:DNA, Cruciform; D004273:DNA, Neoplasm; D009363:Neoplasm Proteins; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; C506731:RECQL4 protein, human; C506724:RecQ protein, E coli; D053484:RecQ Helicases
keywords: BLM; DNA repair; DNA replication; Escherichia coli; RecQ family proteins; RuvC; cancer; holliday junctions; homologous recombination; spontaneous DNA damage