PARP1 promotes nucleotide excision repair through DDB2 stabilization and recruitment of ALC1

Pines, Alex; Vrouwe, Mischa G.; Marteijn, Jürgen A.; Typas, Dimitris; Luijsterburg, Martijn S.; Cansoy, Medine; Hensbergen, Paul J.; Deelder, André M.; Groot, Arjan de; Matsumoto, Sakuya; Sugasawa, Kaoru; Thomä, Nicolas H.; Vermeulen, Wim; Vrieling, Harry; Mullenders, Leon H.F.

doi:10.1083/jcb.201112132

Cited by 213 publications

(239 citation statements)

References 58 publications

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“…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 .…”

Section: No Effect Of Parp-1 and Ddb2 Binding On Cpd-photolyase Mediasupporting

confidence: 93%

“…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.…”

mentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

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

Purohit

Robu

Shah

et al. 2016

Sci Rep

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The existing methodologies for studying robust responses of poly (ADP-ribose) polymerase-1 (PARP-1) to DNA damage with strand breaks are often not suitable for examining its subtle responses to altered DNA without strand breaks, such as UV-damaged DNA. Here we describe two novel assays with which we characterized the interaction of PARP-1 with UV-damaged DNA in vivo and in vitro. Using an in situ fractionation technique to selectively remove free PARP-1 while retaining the DNA-bound PARP-1, we demonstrate a direct recruitment of the endogenous or exogenous PARP-1 to the UV-lesion site in vivo after local irradiation. In addition, using the model oligonucleotides with single UV lesion surrounded by multiple restriction enzyme sites, we demonstrate in vitro that DDB2 and PARP-1 can simultaneously bind to UV-damaged DNA and that PARP-1 casts a bilateral asymmetric footprint from −12 to +9 nucleotides on either side of the UV-lesion. These techniques will permit characterization of different roles of PARP-1 in the repair of UV-damaged DNA and also allow the study of normal housekeeping roles of PARP-1 with undamaged DNA.The abundance of poly (ADP-ribose) polymerase-1 (PARP-1) in mammalian cells and its rapid catalytic activation to form polymers of ADP-ribose (PAR) in the presence of various types of DNA damages with or without strand breaks has made it an ideal first responder at the lesion site to influence downstream events 1,2 . Apart from DNA damages, PARP-1 is also recruited to DNA during normal physiological processes such as transcription and chromatin remodeling 3 , which do not involve overt DNA damage but just altered DNA structures. While we know much more about how PARP-1 rapidly recognizes and binds to single or double strand breaks in DNA, we know very little about how PARP-1 interacts with DNA damages or altered DNA structures without strand breaks. 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. Thus, PARP-1 possibly has multiple roles in NER, but we do not yet fully understand its interactions with UV-damaged DNA or other NER proteins due to two major challenges. The first challenge is that unlike for many NER proteins, the abundance of endogenous PARP-1 in the nucleus makes it nearly impossible to visualize its dynamics of recruitment to UV-damaged DNA in situ using conventional immunocytological methods. T...

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Section: No Effect Of Parp-1 and Ddb2 Binding On Cpd-photolyase Mediasupporting

confidence: 93%

mentioning

confidence: 99%

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

Purohit

Robu

Shah

et al. 2016

Sci Rep

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“…Thus we compared early recruitment dynamics of PARP1 and ALC1, both of which are known as fast recruiters with maximum recruitment reached within < 1 min [15–17,29,48,49] (Figure 7). …”

Section: Resultsmentioning

confidence: 99%

Simultaneous dual-channel imaging to quantify interdependent protein recruitment to laser-induced DNA damage sites

Garbrecht

Hornegger

Herbert

et al. 2018

Nucleus

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Fluorescence microscopy in combination with the induction of localized DNA damage using focused light beams has played a major role in the study of protein recruitment kinetics to DNA damage sites in recent years. Currently published methods are dedicated to the study of single fluorophore/single protein kinetics. However, these methods may be limited when studying the relative recruitment dynamics between two or more proteins due to cell-to-cell variability in gene expression and recruitment kinetics, and are not suitable for comparative analysis of fast-recruiting proteins. To tackle these limitations, we have established a time-lapse fluorescence microscopy method based on simultaneous dual-channel acquisition following UV-A-induced local DNA damage coupled with a standardized image and recruitment analysis workflow. Simultaneous acquisition is achieved by spectrally splitting the emitted light into two light paths, which are simultaneously imaged on two halves of the same camera chip. To validate this method, we studied the recruitment of poly(ADP-ribose) polymerase 1 (PARP1), poly (ADP-ribose) glycohydrolase (PARG), proliferating cell nuclear antigen (PCNA) and the chromatin remodeler ALC1. In accordance with the published data based on single fluorophore imaging, simultaneous dual-channel imaging revealed that PARP1 regulates fast recruitment and dissociation of PARG and that in PARP1-depleted cells PARG and PCNA are recruited with comparable kinetics. This approach is particularly advantageous for analyzing the recruitment sequence of fast-recruiting proteins such as PARP1 and ALC1, and revealed that PARP1 is recruited faster than ALC1. Split-view imaging can be incorporated into any laser microirradiation-adapted microscopy setup together with a recruitment-dedicated image analysis package.

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“…Previous reports showed that CEES induced DNA mono adducts are predominantly repaired by BER and NER pathways (Jowsey et al, 2009;Matijasevic et al, 2001). PARylation plays an active role in both repair pathway, potentially by being activated through DNA strand breaks introduced by endonucleases as intermediates during the repair processes (Fischer et al, 2014;Pines et al, 2013Pines et al, , 2012Robert et al, 2013). Thus, even though mustards do not induce DNA strand breaks directly, strand breaks can occur in response to mustard treatment by enzymatic processes that actively introduce them in the course of repair processes, thereby triggering PARP activation.…”

Section: Application Of the Optimized Cees Treatment Prototocol For Tmentioning

confidence: 99%

Immunochemical analysis of poly(ADP-ribosyl)ation in HaCaT keratinocytes induced by the mono-alkylating agent 2-chloroethyl ethyl sulfide (CEES): Impact of experimental conditions

et al. 2016

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Biological effects of CEES treated cells are significantly influenced by treatment protocol. Optimized protocol for the treatment of HaCaT cells with CEES. CEES induces a dose and time dependent PARylation response in HaCaT cells. CEES induced PAR formation is predominantly due to the activation of PARP1. Keywords:Poly(ADP-ribose) Poly(ADP-ribose) polymerases Sulfur mustard CEES HaCaT keratinocytes Solvent A B S T R A C T Sulfur mustard (SM) is a bifunctional alkylating agent with a long history of use as a chemical weapon. Although its last military use is dated for the eighties of the last century, a potential use in terroristic attacks against civilians remains a significant threat. Thus, improving medical therapy of mustard exposed individuals is still of particular interest. PARP inhibitors were recently brought into the focus as a potential countermeasure for mustard induced pathologies, supported by the availability of efficient compounds successfully tested in cancer therapy.PARP activation after SM treatment was reported in several cell types and tissues under various conditions; however, a detailed characterization of this phenomenon is still missing. This study provides the basis for such studies by developing and optimizing experimental conditions to investigate poly(ADP ribosyl)ation (PARylation) in HaCaT keratinocytes upon treatment with the monofunctional alkylating agent 2 chloroethyl ethyl sulfide ("half mustard", CEES). By using an immunofluorescence based approach, we show that optimization of experimental conditions with regards to the type of solvent, dilution factors and treatment procedure is essential to obtain a homogenous PAR staining in HaCaT cell cultures. Furthermore, we demonstrate that different CEES treatment protocols significantly influence the cytotoxicity profiles of treated cells. Using an optimized treatment protocol, our data reveals that CEES induces a dose and time dependent dynamic PARylation response in HaCaT cells that could be completely blocked by treating cells with the clinically relevant pharmacological PARP inhibitor ABT888 (also known as veliparib). Finally, siRNA experiments show that CEES induced PAR formation is predominantly due to the activation of PARP1. In conclusion, this study provides a detailed analysis of the CEES induced PARylation response in HaCaT keratinocytes, which forms an experimental basis to study the molecular mechanism of PARP1 activation and its functional consequences after mustard treatment in general.

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PARP1 promotes nucleotide excision repair through DDB2 stabilization and recruitment of ALC1

Abstract: PARP1-mediated poly(ADP-ribosyl)ation of DDB2 prolongs its occupation on UV-damaged chromatin and promotes the recruitment of the chromatin remodeler ALC1.

Cited by 213 publications

References 58 publications

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

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

Simultaneous dual-channel imaging to quantify interdependent protein recruitment to laser-induced DNA damage sites

Immunochemical analysis of poly(ADP-ribosyl)ation in HaCaT keratinocytes induced by the mono-alkylating agent 2-chloroethyl ethyl sulfide (CEES): Impact of experimental conditions

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