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      R-loops and nicks initiate DNA breakage and genome instability in non-growing Escherichia coli

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          Abstract

          Double-stranded DNA ends, often from replication, drive genomic instability, yet their origin in non-replicating cells is unknown. Here we show that transcriptional RNA/DNA hybrids (R-loops) generate DNA ends that underlie stress-induced mutation and amplification. Depleting RNA/DNA hybrids with overproduced RNase HI reduces both genomic changes, indicating RNA/DNA hybrids as intermediates in both. An Mfd requirement and inhibition by translation implicate transcriptional R-loops. R-loops promote instability by generating DNA ends, shown by their dispensability when ends are provided by I- SceI endonuclease. Both R-loops and single-stranded endonuclease TraI are required for end formation, visualized as foci of a fluorescent end-binding protein. The data suggest that R-loops prime replication forks that collapse at single-stranded nicks, producing ends that instigate genomic instability. The results illuminate how DNA ends form in non-replicating cells, identify R-loops as the earliest known mutation/amplification intermediate, and suggest that genomic instability during stress could be targeted to transcribed regions, accelerating adaptation.

          Abstract

          DNA double-strand breaks commonly occur in all replicating cells. Wimberly and colleagues show that in non-replicating cells, aborted transcription/translation forms RNA/DNA hybrid R-loops that prime origin-independent replication, leading to DNA breakage, point mutations and chromosomal rearrangements.

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          Most cited references 46

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          Mechanisms of change in gene copy number.

          Deletions and duplications of chromosomal segments (copy number variants, CNVs) are a major source of variation between individual humans and are an underlying factor in human evolution and in many diseases, including mental illness, developmental disorders and cancer. CNVs form at a faster rate than other types of mutation, and seem to do so by similar mechanisms in bacteria, yeast and humans. Here we review current models of the mechanisms that cause copy number variation. Non-homologous end-joining mechanisms are well known, but recent models focus on perturbation of DNA replication and replication of non-contiguous DNA segments. For example, cellular stress might induce repair of broken replication forks to switch from high-fidelity homologous recombination to non-homologous repair, thus promoting copy number change.
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            Single-strand break repair and genetic disease.

            Hereditary defects in the repair of DNA damage are implicated in a variety of diseases, many of which are typified by neurological dysfunction and/or increased genetic instability and cancer. Of the different types of DNA damage that arise in cells, single-strand breaks (SSBs) are the most common, arising at a frequency of tens of thousands per cell per day from direct attack by intracellular metabolites and from spontaneous DNA decay. Here, the molecular mechanisms and organization of the DNA-repair pathways that remove SSBs are reviewed and the connection between defects in these pathways and hereditary neurodegenerative disease are discussed.
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              A Microhomology-Mediated Break-Induced Replication Model for the Origin of Human Copy Number Variation

              Chromosome structural changes with nonrecurrent endpoints associated with genomic disorders offer windows into the mechanism of origin of copy number variation (CNV). A recent report of nonrecurrent duplications associated with Pelizaeus-Merzbacher disease identified three distinctive characteristics. First, the majority of events can be seen to be complex, showing discontinuous duplications mixed with deletions, inverted duplications, and triplications. Second, junctions at endpoints show microhomology of 2–5 base pairs (bp). Third, endpoints occur near pre-existing low copy repeats (LCRs). Using these observations and evidence from DNA repair in other organisms, we derive a model of microhomology-mediated break-induced replication (MMBIR) for the origin of CNV and, ultimately, of LCRs. We propose that breakage of replication forks in stressed cells that are deficient in homologous recombination induces an aberrant repair process with features of break-induced replication (BIR). Under these circumstances, single-strand 3′ tails from broken replication forks will anneal with microhomology on any single-stranded DNA nearby, priming low-processivity polymerization with multiple template switches generating complex rearrangements, and eventual re-establishment of processive replication.
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                Author and article information

                Journal
                Nat Commun
                Nat Commun
                Nature Communications
                Nature Pub. Group
                2041-1723
                05 July 2013
                : 4
                Affiliations
                [1 ]Department of Molecular and Human Genetics , 1 Baylor Plaza, Houston, Texas 77030, USA
                [2 ]Departments of Biochemistry and Molecular Biology, Molecular Virology and Microbiology and the Dan L Duncan Cancer Center, Baylor College of Medicine , Houston, Texas 77030, USA
                [3 ]Present address: Department of Pathology, Yale University School of Medicine , 333 Cedar Street, New Haven, Connecticut 06510, USA
                Author notes
                Article
                ncomms3115
                10.1038/ncomms3115
                3715873
                23828459
                Copyright © 2013, Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.

                This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivative Works 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/

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