8,450 research outputs found

    Protein dynamics mammalian genome maintenance

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    Protein dynamics mammalian genome maintenance

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    Nucleotide Excision Repair in S. cerevisiae

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    Mechanisms and Dynamics of Oxidative DNA Damage Repair in Nucleosomes

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    DNA provides the blueprint for cell function and growth, as well as ensuring continuity from one cell generation to the next. In order to compact, protect, and regulate this vital information, DNA is packaged by histone proteins into nucleosomes, which are the fundamental subunits of chromatin. Reactive oxygen species, generated by both endogenous and exogenous agents, can react with DNA, altering base chemistry and generating DNA strand breaks. Left unrepaired, these oxidation products can result in mutations and/or cell death. The Base Excision Repair (BER) pathway exists to deal with damaged bases and single-stranded DNA breaks. However, the packaging of DNA into chromatin provides roadblocks to repair. Damaged DNA bases may be buried within nucleosomes, where they are inaccessible to repair enzymes and other DNA binding proteins. Previous in vitro studies by our lab have demonstrated that BER enzymes can function within this challenging environment, albeit in a reduced capacity. Exposure to ionizing radiation often results in multiple, clustered oxidative lesions. Near-simultaneous BER of two lesions located on opposing strands within a single helical turn of DNA of one another creates multiple DNA single-strand break intermediates. This, in turn, may create a potentially lethal double-strand break (DSB) that can no longer be repaired by BER. To determine if chromatin offers protection from this phenomenon, we incubated DNA glycosylases with nucleosomes containing clustered damages in an attempt to generate DSBs. We discovered that nucleosomes offer substantial protection from inadvertent DSB formation. Steric hindrance by the histone core in the nucleosome was a major factor in restricting DSB formation. As well, lesions positioned very close to one another were refractory to processing, with one lesion blocking or disrupting access to the second site. The nucleosome itself appears to remain intact during DSB formation, and in some cases, no DNA is released from the histones. Taken together, these results suggest that in vivo, DSBs generated by BER occur primarily in regions of the genome associated with elevated rates of nucleosome turnover or remodeling, and in the short linker DNA segments that lie between adjacent nucleosomes. DNA ligase IIIα (LigIIIα) catalyzes the final step in BER. In order to facilitate repair, DNA ligase must completely encircle the DNA helix. Thus, DNA ligase must at least transiently disrupt histone-DNA contacts. To determine how LigIIIα functions in nucleosomes, given this restraint, we incubated the enzyme with nick-containing nucleosomes. We found that a nick located further within the nucleosome was ligated at a lower rate than one located closer to the edge. This indicated that LigIIIα must wait for DNA to spontaneously, transiently unwrap from the histone octamer to expose the nick for recognition. Remarkably, the disruption that must occur for ligation is both limited and transient: the nucleosome remains resistant to enzymatic digest before and during ligation, and reforms completely once LigIIIα dissociates

    ATP-dependent chromatin remodeling in the DNA-damage response

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    The integrity of DNA is continuously challenged by metabolism-derived and environmental genotoxic agents that cause a variety of DNA lesions, including base alterations and breaks. DNA damage interferes with vital processes such as transcription and replication, and if not repaired properly, can ultimately lead to premature aging and cancer. Multiple DNA pathways signaling for DNA repair and DNA damage collectively safeguard the integrity of DNA. Chromatin plays a pivotal role in regulating DNA-associated processes, and is itself subject to regulation by the DNA-damage response. Chromatin influences access to DNA, and often serves as a docking or signaling site for repair and signaling proteins. Its structure can be adapted by post-translational histone modifications and nucleosome remodeling, catalyzed by the activity of ATP-dependent chromatin-remodeling complexes. In recent years, accumulating evidence has suggested that ATP-dependent chromatin-remodeling complexes play important, although poorly characterized, roles in facilitating the effectiveness of the DNA-damage response. In this review, we summarize the current knowledge on the involvement of ATP-dependent chromatin remodeling in three major DNA repair pathways: nucleotide excision repair, homologous recombination, and non-homologous end-joining. This shows that a surprisingly large number of different remodeling complexes display pleiotropic functions during different stages of the DNA-damage response. Moreover, several complexes seem to have multiple functions, and are implicated in various mechanistically distinct repair pathways

    Chromatin Remodeling in the UV-induced DNA Damage Response

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