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      Two Mechanisms Produce Mutation Hotspots at DNA Breaks in Escherichia coli

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      1 , 1 , 1 ,
      Cell Reports
      Cell Press

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          Summary

          Mutation hotspots and showers occur across phylogeny and profoundly influence genome evolution, yet the mechanisms that produce hotspots remain obscure. We report that DNA double-strand breaks (DSBs) provoke mutation hotspots via stress-induced mutation in Escherichia coli. With tet reporters placed 2 kb to 2 Mb (half the genome) away from an I- SceI site, RpoS/DinB-dependent mutations occur maximally within the first 2 kb and decrease logarithmically to ∼60 kb. A weak mutation tail extends to 1 Mb. Hotspotting occurs independently of I-site/ tet-reporter-pair position in the genome, upstream and downstream in the replication path. RecD, which allows RecBCD DSB-exonuclease activity, is required for strong local but not long-distance hotspotting, indicating that double-strand resection and gap-filling synthesis underlie local hotspotting, and newly illuminating DSB resection in vivo. Hotspotting near DSBs opens the possibility that specific genomic regions could be targeted for mutagenesis, and could also promote concerted evolution (coincident mutations) within genes/gene clusters, an important issue in the evolution of protein functions.

          Abstract

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          Highlights

          ► Spontaneous mutation pathway in Escherichia coli causes hotpots at double-strand breaks ► Strong local (2–60 kb) hotspot mechanism double-strand resection and gap-fill ► Weak long-distance (1 Mb) mutagenesis by break-induced replication ► Break-induced replication and length of DNA-end resection in natural repair with sister chromosomes

          Abstract

          Mutation hotspots promote cancer and genome evolution, yet how they occur remains obscure. Rosenberg and colleagues used targeted endonucleolytic cleavages in the Escherichia coli chromosome to show that double-strand breaks cause mutation hotspots. Strong local and weak distant hotspots are caused by two mutation mechanisms that accelerate evolution in stressed cells. Hotspotting at breaks raises the possibility that specific genomic regions can be targeted for mutagenesis and can promote concerted evolution within genes, an important issue in protein evolution.

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          Most cited references45

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          Molecular mechanisms of antibody somatic hypermutation.

          Functional antibody genes are assembled by V-D-J joining and then diversified by somatic hypermutation. This hypermutation results from stepwise incorporation of single nucleotide substitutions into the V gene, underpinning much of antibody diversity and affinity maturation. Hypermutation is triggered by activation-induced deaminase (AID), an enzyme which catalyzes targeted deamination of deoxycytidine residues in DNA. The pathways used for processing the AID-generated U:G lesions determine the variety of base substitutions observed during somatic hypermutation. Thus, DNA replication across the uracil yields transition mutations at C:G pairs, whereas uracil excision by UNG uracil-DNA glycosylase creates abasic sites that can also yield transversions. Recognition of the U:G mismatch by MSH2/MSH6 triggers a mutagenic patch repair in which polymerase eta plays a major role and leads to mutations at A:T pairs. AID-triggered DNA deamination also underpins immunoglobulin variable (IgV) gene conversion, isotype class switching, and some oncogenic translocations in B cell tumors.
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            Is Open Access

            Homologous recombination and its regulation

            Homologous recombination (HR) is critical both for repairing DNA lesions in mitosis and for chromosomal pairing and exchange during meiosis. However, some forms of HR can also lead to undesirable DNA rearrangements. Multiple regulatory mechanisms have evolved to ensure that HR takes place at the right time, place and manner. Several of these impinge on the control of Rad51 nucleofilaments that play a central role in HR. Some factors promote the formation of these structures while others lead to their disassembly or the use of alternative repair pathways. In this article, we review these mechanisms in both mitotic and meiotic environments and in different eukaryotic taxa, with an emphasis on yeast and mammal systems. Since mutations in several proteins that regulate Rad51 nucleofilaments are associated with cancer and cancer-prone syndromes, we discuss how understanding their functions can lead to the development of better tools for cancer diagnosis and therapy.
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              The importance of repairing stalled replication forks.

              The bacterial SOS response to unusual levels of DNA damage has been recognized and studied for several decades. Pathways for re-establishing inactivated replication forks under normal growth conditions have received far less attention. In bacteria growing aerobically in the absence of SOS-inducing conditions, many replication forks encounter DNA damage, leading to inactivation. The pathways for fork reactivation involve the homologous recombination systems, are nonmutagenic, and integrate almost every aspect of DNA metabolism. On a frequency-of-use basis, these pathways represent the main function of bacterial DNA recombination systems, as well as the main function of a number of other enzymatic systems that are associated with replication and site-specific recombination.
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                Author and article information

                Journal
                Cell Rep
                Cell Rep
                Cell Reports
                Cell Press
                2211-1247
                25 October 2012
                25 October 2012
                : 2
                : 4
                : 714-721
                Affiliations
                [1 ]Departments of Molecular and Human Genetics, Biochemistry and Molecular Biology, Molecular Virology and Microbiology, and Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA
                Author notes
                []Corresponding author smr@ 123456bcm.edu
                Article
                CELREP157
                10.1016/j.celrep.2012.08.033
                3607216
                23041320
                8cf8689b-f20a-4103-a061-69544cbcf35a
                © 2012 The Authors
                History
                : 6 July 2012
                : 6 August 2012
                : 30 August 2012
                Categories
                Report

                Cell biology
                Cell biology

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