Nucleotide excision repair is associated with the replisome and its efficiency depends on a direct interaction between XPA and PCNA

PLoS One. 2012;7(11):e49199. doi: 10.1371/journal.pone.0049199. Epub 2012 Nov 13.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • DNA Repair* / radiation effects
  • DNA Replication / radiation effects
  • DNA-Directed DNA Polymerase / metabolism*
  • HEK293 Cells
  • HeLa Cells
  • Histones / metabolism
  • Humans
  • Models, Biological
  • Molecular Sequence Data
  • Multienzyme Complexes / metabolism*
  • Mutant Proteins / metabolism
  • Proliferating Cell Nuclear Antigen / metabolism*
  • Protein Binding / radiation effects
  • Protein Transport / radiation effects
  • Structure-Activity Relationship
  • Ultraviolet Rays
  • Xeroderma Pigmentosum Group A Protein / chemistry
  • Xeroderma Pigmentosum Group A Protein / metabolism*

Substances

  • H2AX protein, human
  • Histones
  • Multienzyme Complexes
  • Mutant Proteins
  • Proliferating Cell Nuclear Antigen
  • XPA protein, human
  • Xeroderma Pigmentosum Group A Protein
  • DNA synthesome
  • DNA-Directed DNA Polymerase

Grant support

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. Olav's 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.