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Nucleotide excision repair-initiating proteins bind to oxidative DNA lesions in vivo.

ABSTRACT: Base excision repair (BER) is the main repair pathway to eliminate abundant oxidative DNA lesions such as 8-oxo-7,8-dihydroguanine. Recent data suggest that the key transcription-coupled nucleotide excision repair factor (TC-NER) Cockayne syndrome group B (CSB) and the global genome NER-initiating factor XPC are implicated in the protection of cells against oxidative DNA damages. Our novel live-cell imaging approach revealed a strong and very rapid recruitment of XPC and CSB to sites of oxidative DNA lesions in living cells. The absence of detectable accumulation of downstream NER factors at the site of local oxidative DNA damage provide the first in vivo indication of the involvement of CSB and XPC in the repair of oxidative DNA lesions independent of the remainder of the NER reaction. Interestingly, CSB exhibited different and transcription-dependent kinetics in the two compartments studied (nucleolus and nucleoplasm), suggesting a direct transcription-dependent involvement of CSB in the repair of oxidative lesions associated with different RNA polymerases but not involving other NER proteins.

SUBMITTER: Menoni H

PROVIDER: S-EPMC3529521 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Nucleotide excision repair-initiating proteins bind to oxidative DNA lesions in vivo.

Menoni Hervé H Hoeijmakers Jan H J JH Vermeulen Wim W

The Journal of cell biology 20121217 7

Base excision repair (BER) is the main repair pathway to eliminate abundant oxidative DNA lesions such as 8-oxo-7,8-dihydroguanine. Recent data suggest that the key transcription-coupled nucleotide excision repair factor (TC-NER) Cockayne syndrome group B (CSB) and the global genome NER-initiating factor XPC are implicated in the protection of cells against oxidative DNA damages. Our novel live-cell imaging approach revealed a strong and very rapid recruitment of XPC and CSB to sites of oxidativ ...[more]

PMID: 23253478

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Project description:Nucleotide excision repair (NER) is the most versatile DNA repair pathway that removes a wide variety of DNA lesions caused by different types of physical and chemical agents, such as ultraviolet radiation (UV), environmental carcinogen benzo[a]pyrene and anti-cancer drug carboplatin. The mammalian NER utilizes more than 30 proteins, in a multi-step process that begins with the lesion recognition within seconds of DNA damage to completion of repair after few hours to several days. The core proteins and their biochemical reactions are known from in vitro DNA repair assays using purified proteins, but challenge was to understand the dynamics of their rapid recruitment and departure from the lesion site and their coordination with other proteins and post-translational modifications to execute the sequential steps of repair. Here, we provide a brief overview of various techniques developed by different groups over last 20 years to overcome these challenges. However, more work is needed for a comprehensive knowledge of all aspects of mammalian NER. With this aim, here we provide detailed protocols of three simple yet innovative methods developed by many teams that range from local UVC irradiation to in situ extraction and sub-cellular fractionation that will permit study of endogenous as well as exogenous NER proteins in any cellular model. These methods do not require unique reagents or specialized instruments, and will allow many more laboratories to explore this repair pathway in different models. These techniques would reveal intracellular movement of these proteins to the DNA lesion site, their interactions with other proteins during repair and the effect of post-translational modifications on their functions. We also describe how these methods led us to identify hitherto unexpected role of poly(ADP-ribose) polymerase-1 (PARP1) in NER. Collectively these three simple techniques can provide an initial assessment of the functions of known and unknown proteins in the core or auxiliary events associated with mammalian NER. The results from these techniques could serve as a solid foundation and a justification for more detailed studies in NER using specialized reagents and more sophisticated tools. They can also be suitably modified to study other cellular processes beyond DNA repair.

Dataset Information

Nucleotide excision repair-initiating proteins bind to oxidative DNA lesions in vivo.

Publications

Nucleotide excision repair-initiating proteins bind to oxidative DNA lesions in vivo.

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