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      Alternative-NHEJ Is a Mechanistically Distinct Pathway of Mammalian Chromosome Break Repair

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          Abstract

          Characterizing the functional overlap and mutagenic potential of different pathways of chromosomal double-strand break (DSB) repair is important to understand how mutations arise during cancer development and treatment. To this end, we have compared the role of individual factors in three different pathways of mammalian DSB repair: alternative-nonhomologous end joining (alt-NHEJ), single-strand annealing (SSA), and homology directed repair (HDR/GC). Considering early steps of repair, we found that the DSB end-processing factors KU and CtIP affect all three pathways similarly, in that repair is suppressed by KU and promoted by CtIP. In contrast, both KU and CtIP appear dispensable for the absolute level of total-NHEJ between two tandem I-SceI–induced DSBs. During later steps of repair, we find that while the annealing and processing factors RAD52 and ERCC1 are important to promote SSA, both HDR/GC and alt-NHEJ are significantly less dependent upon these factors. As well, while disruption of RAD51 causes a decrease in HDR/GC and an increase in SSA, inhibition of this factor did not affect alt-NHEJ. These results suggest that the regulation of DSB end-processing via KU/CtIP is a common step during alt-NHEJ, SSA, and HDR/GC. However, at later steps of repair, alt-NHEJ is a mechanistically distinct pathway of DSB repair, and thus may play a unique role in mutagenesis during cancer development and therapy.

          Author Summary

          Changes to the sequence of DNA, or mutations, can disrupt cellular growth control genes, which can lead to cancer development. Such mutations likely arise from damage to DNA that is repaired in a way that fails to restore the original sequence. One type of DNA damage is a chromosomal double-strand break. We have developed assays to measure how these breaks are repaired, and also how such repair can lead to mutations. In particular, we present an assay to measure a pathway of repair that results in deletion mutations, often with evidence of short homologous sequences at the repair junctions (alt-NHEJ). We have compared the genetic requirements of this repair pathway in relation to other pathways of repair that use extensive homology. We find that factors KU and CtIP appear to affect the initial stages of repair of each of these pathways, regardless of the length of homology. However, these pathways appear to diverge at later steps, as relates to the role of the repair factors RAD52, ERCC1, and RAD51. Given that mutations observed in some cancer cells are consistent with alt-NHEJ repair, these mechanistic descriptions provide models for how such mutations could arise in cancer.

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          Most cited references43

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          DNA double-strand breaks: signaling, repair and the cancer connection.

          To ensure the high-fidelity transmission of genetic information, cells have evolved mechanisms to monitor genome integrity. Cells respond to DNA damage by activating a complex DNA-damage-response pathway that includes cell-cycle arrest, the transcriptional and post-transcriptional activation of a subset of genes including those associated with DNA repair, and, under some circumstances, the triggering of programmed cell death. An inability to respond properly to, or to repair, DNA damage leads to genetic instability, which in turn may enhance the rate of cancer development. Indeed, it is becoming increasingly clear that deficiencies in DNA-damage signaling and repair pathways are fundamental to the etiology of most, if not all, human cancers. Here we describe recent progress in our understanding of how cells detect and signal the presence and repair of one particularly important form of DNA damage induced by ionizing radiation-the DNA double-strand break (DSB). Moreover, we discuss how tumor suppressor proteins such as p53, ATM, Brca1 and Brca2 have been linked to such pathways, and how accumulating evidence is connecting deficiencies in cellular responses to DNA DSBs with tumorigenesis.
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            Human CtIP promotes DNA end resection.

            In the S and G2 phases of the cell cycle, DNA double-strand breaks (DSBs) are processed into single-stranded DNA, triggering ATR-dependent checkpoint signalling and DSB repair by homologous recombination. Previous work has implicated the MRE11 complex in such DSB-processing events. Here, we show that the human CtIP (RBBP8) protein confers resistance to DSB-inducing agents and is recruited to DSBs exclusively in the S and G2 cell-cycle phases. Moreover, we reveal that CtIP is required for DSB resection, and thereby for recruitment of replication protein A (RPA) and the protein kinase ATR to DSBs, and for the ensuing ATR activation. Furthermore, we establish that CtIP physically and functionally interacts with the MRE11 complex, and that both CtIP and MRE11 are required for efficient homologous recombination. Finally, we reveal that CtIP has sequence homology with Sae2, which is involved in MRE11-dependent DSB processing in yeast. These findings establish evolutionarily conserved roles for CtIP-like proteins in controlling DSB resection, checkpoint signalling and homologous recombination.
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              XRCC3 promotes homology-directed repair of DNA damage in mammalian cells.

              Homology-directed repair of DNA damage has recently emerged as a major mechanism for the maintenance of genomic integrity in mammalian cells. The highly conserved strand transferase, Rad51, is expected to be critical for this process. XRCC3 possesses a limited sequence similarity to Rad51 and interacts with it. Using a novel fluorescence-based assay, we demonstrate here that error-free homology-directed repair of DNA double-strand breaks is decreased 25-fold in an XRCC3-deficient hamster cell line and can be restored to wild-type levels through XRCC3 expression. These results establish that XRCC3-mediated homologous recombination can reverse DNA damage that would otherwise be mutagenic or lethal.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Genet
                plos
                plosgen
                PLoS Genetics
                Public Library of Science (San Francisco, USA )
                1553-7390
                1553-7404
                June 2008
                June 2008
                27 June 2008
                : 4
                : 6
                : e1000110
                Affiliations
                [1 ]Department of Radiation Biology, Beckman Research Institute of the City of Hope, Duarte, California, United States of America
                [2 ]City of Hope Graduate School of Biological Sciences, Duarte, California, United States of America
                Brandeis University, United States of America
                Author notes

                Conceived and designed the experiments: NB JS. Performed the experiments: NB AC NH JS. Analyzed the data: NB JS. Contributed reagents/materials/analysis tools: NB AC NH JS. Wrote the paper: NB AC JS.

                Article
                07-PLGE-RA-0994R2
                10.1371/journal.pgen.1000110
                2430616
                18584027
                d8c5fba6-4b41-4d2b-a03d-6aebf2c5161f
                Bennardo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 31 October 2007
                : 28 May 2008
                Page count
                Pages: 10
                Categories
                Research Article
                Cell Biology/Cell Growth and Division
                Genetics and Genomics/Chromosome Biology
                Molecular Biology/DNA Repair

                Genetics
                Genetics

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