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.
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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)