Role of poly(ADP-ribose) polymerase-1 in the removal of UV-induced DNA lesions by nucleotide excision repair

Abstract

Among the earliest responses of mammalian cells to DNA damage is catalytic activation of a nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1). Activated PARP-1 forms the polymers of ADPribose (pADPr or PAR) that posttranslationally modify its target proteins, such as PARP-1 and DNA repair-related proteins. Although this metabolism is known to be implicated in other repair pathways, here we show its role in the versatile nucleotide excision repair pathway (NER) that removes a variety of DNA damages including those induced by UV. We show that PARP inhibition or specific depletion of PARP-1 decreases the efficiency of removal of UV-induced DNA damage from human skin fibroblasts or mouse epidermis. Using NER-proficient and -deficient cells and in vitro PARP-1 assays, we show that damaged DNA-binding protein 2 (DDB2), a key lesion recognition protein of the global genomic subpathway of NER (GG-NER), associates with PARP-1 in the vicinity of UV-damaged chromatin, stimulates its catalytic activity, and is modified by pADPr. PARP inhibition abolishes UV-induced interaction of DDB2 with PARP-1 or xeroderma pigmentosum group C (XPC) and also decreases localization of XPC to UV-damaged DNA, which is a key step that leads to downstream events in GG-NER. Thus, PARP-1 collaborates with DDB2 to increase the efficiency of the lesion recognition step of GG-NER.M ammalian cells respond very rapidly to different types of DNA damage by activation of an abundant and ubiquitous nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1). The activated PARP-1 uses NAD + to form polymers of ADP-ribose (pADPr or PAR) that modify PARP-1 itself and selected target proteins, such as histones and DNA repair proteins (1). This posttranslational modification, i.e., PARylation, has been implicated in cellular responses ranging from DNA repair to cell death. Among mammalian DNA repair pathways, PARP-1 has been implicated in base excision repair, homologous recombination, and nonhomologous end-joining pathways (2, 3), but we do not know its role in the most versatile nucleotide excision repair (NER) pathway that removes a wide variety of DNA lesions, including UV-induced thymine dimers (T-T) and other cyclobutane pyrimidine dimers (CPD), as well as 6-4 photoproducts (6-4PP) (4).The core mammalian NER pathway uses more than 30 proteins to recognize the damaged site on DNA, remove 24-to 32-nucleotide-long single-stranded DNA containing the lesion, fill the gap using the nondamaged strand as a template, and finally ligate the nick (4). There are two subpathways of NER: the transcription-coupled NER (TC-NER) removes lesions from the actively transcribed strands of the genes and the global genomic NER (GG-NER) repairs lesions from the entire genome. These two pathways differ in the initial step of lesion recognition: TC-NER is initiated when elongating RNA polymerase II stalls at the lesion, whereas GG-NER is initiated when the lesion is recognized in the chromatin context by DDB2 (XPE), which through its participation in the UV-DDB-E3 ligase complex ub...

Characterization of the interactions of PARP-1 with UV-damaged DNA in vivo and in vitro

“…The N-terminal DNA binding domain of PARP-1 that is known to be recruited to DNA strand breaks 20 was also recruited to UV-lesions without strand breaks, indicating more general property of this domain of PARP-1 to bind to different types of DNA damages. Our model is also consistent with previously reported interactions of DDB2 and PARP-1 at the UV-lesion site 5,7 . We have earlier shown that PARP-1 and DDB2 co-immunoprecipitate at the UV-damaged chromatin in the presence of ethidium bromide, indicating their direct interaction on the same DNA strand 7 .…”

“…We have earlier shown that PARP-1 binds to UV-damaged large oligonucleotide in vitro or to chromatin fragments containing T-T lesions in vivo 11 . We also showed that PARP-1 and DDB2 associate with each other on the chromatin of UV-irradiated cells and that DDB2 stimulates catalytic activity of PARP-1 in the presence of UV-damaged DNA 7 . However, these assays lack the nucleotide level resolution to reveal whether PARP-1 was bound directly to the UV-damaged bases or to any other base in those long pieces of DNA and whether PARP-1 and DDB2 have sufficient space to co-exist around UV-induced DNA lesion.…”

“…We had shown in the cells and in vitro that PARP-1 not only binds to UV-damaged DNA 11 , but also interacts with DDB2 in the vicinity of UV-induced DNA lesions 7 . Using the model oligo described in Fig.…”

“…The key reason is that the existing methodologies that readily identify interactions of PARP-1 with DNA strand breaks are not sufficiently sensitive to study the relatively weaker responses of PARP-1 to DNA damage without strand breaks. The response of PARP-1 to UVC-induced direct photolesions, such as cyclobutane pyrimidine dimers (CPD) that are formed without any DNA strand breaks exemplifies this problem.Recent studies from others and our team have shown the involvement of PARP-1 in the host cell reactivation 4 and specifically in the nucleotide excision repair (NER) of UV-damaged DNA through its interaction with early NER protein DDB2 [5][6][7] . Additional studies have shown that downstream NER proteins XPA 8,9 and XPC 10 are PARylated.…”

“…Recent studies from others and our team have shown the involvement of PARP-1 in the host cell reactivation 4 and specifically in the nucleotide excision repair (NER) of UV-damaged DNA through its interaction with early NER protein DDB2 [5][6][7] . Additional studies have shown that downstream NER proteins XPA 8,9 and XPC 10 are PARylated.…”