Roles of poly(ADP-ribose) polymerase-1 in mammalian transcription-coupled nucleotide excision repair of DNA damage

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Roles of PARP-1 in mammalian transcription-coupled-NER (TCR) of DNA damage Background: Nucleotide excision repair (NER) is the most versatile DNA repair pathway that removes a wide variety of lesions caused by diverse agents. There are two sub-pathways of NER: the transcription-coupled NER (TCR) rapidly removes lesions from the transcribed strand of the active genes, whereas the global genomic NER (GGR) slowly removes the lesions from rest of the genome. In this NSERC-supported program, we have previously identified two key roles of the mammalian nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP1) in GGR. Here, we will focus on its role in TCR. Preliminary Results and Hypotheses: TCR is initiated when the transcribing RNA polymerase II (RNAPII) stalls at a DNA lesion and recruits the Cockayne syndrome B (CSB) protein to begin a multistep process to repair the damage and restart the transcription. Using various cellular TCR models, we have now observed that: (i) Impaired PARP1 sensitizes cells to DNA damage that is repaired by TCR; (ii) PARP1 interacts with RNAPII both before and after it encounters DNA damage, but it interacts with CSB only at the DNA damage; and (iii) In PARP1-impaired cells, the interaction of CSB with RNAPII, a required step to launch TCR, is significantly suppressed. Therefore, our first hypothesis is that PARP1 present with RNAPII during normal transcription elongation, switches to a repair role when RNAPII stalls at the lesion site to facilitate efficient initiation of TCR. Our second hypothesis is that PARP1 plays a role in transcription regulation before and after TCR to facilitate lesion detection and transcription restart, respectively. To test the hypothesis, the immediate goals are to: 1) Elucidate the role of PARP1 with the elongating RNAPII and CSB in the initiation of TCR; 2) Examine the role of PARP1 in the de novo transcription after DNA damage that plays a role in the lesion detection and in the restart of transcription after TCR. Methods and Approaches: We have established several cellular and DNA damage models to study the TCR or GGR. The impact of PARP1 function will be examined using PARP1-gene knockout or knockdown, enzyme-dead PARP1 and PARP-inhibitors. The transcription and TCR will be monitored by Click-IT techniques for TCR-specific RNA recovery synthesis and unscheduled DNA synthesis. We will use ChIP-Seq and BrU-Seq techniques to identify global impact of PARP1 in TCR and transcription of all genes. The long-term objective is to determine if the intervention of PARP1 in TCR is universally observed in other DNA repair pathways that remove different types of transcription-blocking lesions on the transcribed strand of genes. Impact: Our research program aimed at identification of novel roles of PARP1 in transcription and TCR will not only advance our text-book level knowledge of these important processes but also allow better comprehension of the diseases associated with failure of TCR.