Abstract
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.
Highlights
The nucleotide excision repair (NER) is the most versatile DNA repair pathway that eliminates 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 three simple methods described above do not require specialized reagents and equipment and can be readily performed in most laboratories. These methods will permit analyses of the fate and functions of various endogenous or exogenous proteins during NER. These functions range from recruitment and persistence or departure from the lesion, their interactions with other partners and role of post-translational modifications (PTM) in these processes
If one uses exogenous tagged NER proteins, note that PTM may be affected by the presence of tag, as observed for DDB2 and Xeroderma pigmentosum C (XPC) (Puumalainen et al, 2014; Matsumoto et al, 2015), which will require validation with untagged or endogenous proteins
Summary
The nucleotide excision repair (NER) is the most versatile DNA repair pathway that eliminates 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. To stimulate more studies in NER, there is a need for widespread accessibility to techniques that will allow many more laboratories to explore this repair pathway in different models In this context, some of the leading techniques in the field, such as live cell microscopy imaging using fluorescent tagged proteins (Vermeulen, 2011; Ticli and Prosperi, 2019) or UVC laser with quartz optics to cause damage in defined sub-nuclear zones (Dinant et al, 2007) have produced excellent data and will continue to be useful in future. The cellular fractionation protocol provides an enriched nucleoplasmic or chromatin-bound protein fraction that can be useful for many downstream applications, such as immunoprecipitation and proteomics to identify the components of repair complexes or partners of the target proteins 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 the efficiency of mammalian NER.
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