Nucleotide Excision Repair Is Associated with the Replisome and Its Efficiency Depends on a Direct Interaction between XPA and PCNA

PLOS ONE, Dec 2019

Proliferating cell nuclear antigen (PCNA) is an essential protein for DNA replication, DNA repair, cell cycle regulation, chromatin remodeling, and epigenetics. Many proteins interact with PCNA through the PCNA interacting peptide (PIP)-box or the newly identified AlkB homolog 2 PCNA interacting motif (APIM). The xeroderma pigmentosum group A (XPA) protein, with a central but somewhat elusive role in nucleotide excision repair (NER), contains the APIM sequence suggesting an interaction with PCNA. With an in vivo based approach, using modern techniques in live human cells, we show that APIM in XPA is a functional PCNA interacting motif and that efficient NER of UV lesions is dependent on an intact APIM sequence in XPA. We show that XPA−/− cells complemented with XPA containing a mutated APIM sequence have increased UV sensitivity, reduced repair of cyclobutane pyrimidine dimers and (6–4) photoproducts, and are consequently more arrested in S phase as compared to XPA−/− cells complemented with wild type XPA. Notably, XPA colocalizes with PCNA in replication foci and is loaded on newly synthesized DNA in undamaged cells. In addition, the TFIIH subunit XPD, as well as XPF are loaded on DNA together with XPA, and XPC and XPG colocalize with PCNA in replication foci. Altogether, our results suggest a presence of the NER complex in the vicinity of the replisome and a novel role of NER in post-replicative repair.

A PDF file should load here. If you do not see its contents the file may be temporarily unavailable at the journal website or you do not have a PDF plug-in installed and enabled in your browser.

Alternatively, you can download the file locally and open with any standalone PDF reader:

https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0049199&type=printable

Nucleotide Excision Repair Is Associated with the Replisome and Its Efficiency Depends on a Direct Interaction between XPA and PCNA

Otterlei M (2012) Nucleotide Excision Repair Is Associated with the Replisome and Its Efficiency Depends on a Direct Interaction between XPA and PCNA. PLoS ONE 7(11): e49199. doi:10.1371/journal.pone.0049199 Nucleotide Excision Repair Is Associated with the Replisome and Its Efficiency Depends on a Direct Interaction between XPA and PCNA Karin M. Gilljam 0 Rebekka Mu ller 0 Nina B. Liabakk 0 Marit Otterlei 0 Marco Muzi-Falconi, Universita' di Milano, Italy 0 Department of Cancer Research and Molecular Medicine, Faculty of Medicine, Norwegian University of Science and Technology , Trondheim , Norway Proliferating cell nuclear antigen (PCNA) is an essential protein for DNA replication, DNA repair, cell cycle regulation, chromatin remodeling, and epigenetics. Many proteins interact with PCNA through the PCNA interacting peptide (PIP)-box or the newly identified AlkB homolog 2 PCNA interacting motif (APIM). The xeroderma pigmentosum group A (XPA) protein, with a central but somewhat elusive role in nucleotide excision repair (NER), contains the APIM sequence suggesting an interaction with PCNA. With an in vivo based approach, using modern techniques in live human cells, we show that APIM in XPA is a functional PCNA interacting motif and that efficient NER of UV lesions is dependent on an intact APIM sequence in XPA. We show that XPA2/2 cells complemented with XPA containing a mutated APIM sequence have increased UV sensitivity, reduced repair of cyclobutane pyrimidine dimers and (6-4) photoproducts, and are consequently more arrested in S phase as compared to XPA2/2 cells complemented with wild type XPA. Notably, XPA colocalizes with PCNA in replication foci and is loaded on newly synthesized DNA in undamaged cells. In addition, the TFIIH subunit XPD, as well as XPF are loaded on DNA together with XPA, and XPC and XPG colocalize with PCNA in replication foci. Altogether, our results suggest a presence of the NER complex in the vicinity of the replisome and a novel role of NER in post-replicative repair. - Funding: This work was supported by The National Program for Research in Functional Genomics in Norway (FUGE) from The Research Council of Norway, The Cancer Fund at St. Olavs Hospital, Trondheim, Norway, and The Norwegian Cancer Society and The Liaison Committee between the Central Norway Regional Health Authority (RHA) and the Norwegian University of Science and Technology (NTNU), Norway. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Proper repair of DNA is vital in order to avoid mutations that may cause cancer and other diseases. Cells have therefore evolved numerous pathways to deal with a variety of DNA damage, many of which are associated with the replication machinery [13]. Xeroderma pigmentosum group A (XPA) is a protein in the Nucleotide excision repair (NER) pathway responsible for removal of a wide range of lesions leading to distortions of the DNA helix, most frequently caused by UV radiation (UVR) from the sun. UVR-induced DNA damage inhibits DNA transcription and replication, leading to S phase delay and, if the damage is left unrepaired, may induce DNA double strand breaks (DSBs) [4,5]. However, NER has not been directly coupled to the replicative process. The historically scientific interest in NER has partly been due to the severe clinical phenotype seen in patients with inherited deficiency in this pathway. The NER pathway involves more than 30 proteins and defects in any of the central NER proteins may result in premature aging, neurodegenerative diseases and/or hypersensitivity to UVR. The skin cancer disease xeroderma pigmentosum (XP) is a result of deficiency in any of the seven XPgenes coding for proteins involved in NER. XP patients exhibit more than a 1,000-fold increase in the incidence of sun-induced skin cancer and an increased incidence of internal cancers, primarily in the lung or gastro-intestinal tract [6,7]. Moreover, 30% of XP patients suffer from neurological diseases in addition to the increased incidents of cancer [8]. Solar UV-B and UV-C radiation generate pyrimidine crosslinks, both cyclobutane pyrimidine dimers (CPDs) and 6-4 photo products (6-4 PPs). Particularly the 6-4 PPs are rapidly recognized by NER [9,10]. CPDs, however, are less efficiently recognized by NER, but are easily bypassed by the translesion synthesis (TLS) polymerase POLg [11]. These bypassed CPDs are believed to be repaired by NER prior to next round of replication. DNA damage in the actively transcribed strand is recognized by the stalling of the RNA polymerase in a process called transcription coupled (TC) NER, while damage recognized and repaired independent of transcription is called global genome (GG) NER. After damage recognition, TC-NER and GG-NER have similar mechanisms involving dual incision, removal of a 2530 nucleotid (...truncated)


This is a preview of a remote PDF: https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0049199&type=printable

Karin M. Gilljam, Rebekka Müller, Nina B. Liabakk, Marit Otterlei. Nucleotide Excision Repair Is Associated with the Replisome and Its Efficiency Depends on a Direct Interaction between XPA and PCNA, PLOS ONE, 2012, 11, DOI: 10.1371/journal.pone.0049199