Multiple roles of poly(ADP-ribose) polymerase-1 in mammalian nucleotide excision repair

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Multiple Roles of PARP-1 in Mammalian NER Background: Nucleotide excision repair (NER) is the most versatile DNA repair pathway that removes a wide variety of DNA lesions including those induced by ultraviolet radiations (UV). Despite extensive studies with lower organisms on NER pathways, we are still far from understanding the full complement of proteins and processes involved in efficiently executing mammalian NER. Our research program will identify and characterize different roles of poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme of higher organisms that is absent in bacteria or yeast, in mammalian NER. Our earlier studies have shown that PARP-1 is catalytically activated to form polymers of ADP-ribose in response to UV-irradiation of cells and this process plays a role in the repair of UV-induced DNA damage from viral reporter genes or from the genome of mammalian cells. More recent studies from others and our team identified the first direct mechanism of intervention of PARP-1 in mammalian NER via its interaction with and modification of UV-DNA damage binding protein 2 (DDB2), a key lesion recognition protein of NER. Recently, we also developed two novel techniques to study recruitment of PARP-1 to UV-induced DNA lesion site in vivo and identified the bilateral asymmetric footprint of PARP-1 around UV-induced DNA lesion that would permit it to collaborate with other NER proteins at the lesion site. Based on our recent data, we hypothesize that PARP-1 has multiple additional roles in NER, specifically through its interaction with two NER proteins, namely Xeroderma pigmentosum C (XPC) and Cockayne syndrome B (CSB), each of which play a key role in controlling the initial events in the global genomic and transcription-coupled sub-pathways of NER (GG-NER and TC-NER), respectively. We also hypothesize that PARP-1 activation by UV-damaged DNA and PARylation affects ubiquitination of various NER proteins, which facilitates chromatin remodelling and movement of NER proteins to efficiently conduct the repair events. To test our hypotheses, three immediate goals of our program are: 1) To identify and characterize novel role of PARP-1 in the GG-NER via its interaction with and control of the function of XPC, which starts the process of repair by GG-NER; 2) To determine whether PARP-1 influences the function of CSB, which is the earliest protein recruited by the stalled RNA polymerase II at the damaged site for initiating the process of TC-NER; 3) To examine whether PARP-1 and PARylation control the functions of different E3 ubiquitin ligase complexes involved in ubiquitination of NER proteins, which facilitate GG-NER and TC-NER. NER pathway serves as an excellent complex model to examine multiple roles of this versatile and abundant yet enigmatic protein in mammalian cells, and our long-term goal is to apply this knowledge to understand various other functions of PARP-1 in mammalian cells.