Role of nucleotide excision repair factors in genome maintenance in human cells under oxidative stress

Abstract

The role of nucleotide excision repair (NER) in the maintenance of DNA integrity under oxidative assault has yet to be elucidated. A defective NER can result in Xeroderma Pigmentosa (XP) or Cockayne Syndrome (CS), both autosomal recessive diseases, presenting with increased cancer risk and segmental progeria. Although the NER is characterized to be involved in repairing UV-induced damage, it is difficult to attribute all the symptoms of XP and CS to UV-damage. Oxidative stress is thus likely to be an important factor. Other DNA repair proteins including a component of the NER pathway, XPF, have been reported to be involved in telomere dynamics. As the importance of the NER pathway in removing oxidative stress-induced DNA lesions is still unclear, we sought to understand the role of NER in oxidative stress-induced damage protection and telomere-mediated chromosome integrity. In our study, we utilized primary cells derived from patients suffering from XP (XP-A and XP-D) and CS Type II (CS-B), as well as transformed lymphoblastoid<br>cells from XP-A and XP-D patients.<br><br>The XPA protein verifies DNA damage sites, an event integral for the recruitment of downstream factors such as XPD which is a helicase domain-containing protein involved in both the NER and basal transcription. CSB, which displaces stalled RNA polymerase II, is involved in restoring UV-inhibited transcription and basal transcription. Dysfunction of any of these proteins impedes the progression of the NER.<br><br>Following induction of oxidative stress by either sodium arsenite (NaAsO2) or hydrogen peroxide (H2O2), we performed assays related to survival, genome stability and growth kinetics. NER-deficient primary fibroblasts retained higher viability but displayed cell cycle dysfunction and increased DNA damage following exposure to H2O2. Single cell gel electrophoresis assay showed that both fibroblasts and lymphoblastoids deficient in NER were more susceptible to H2O2-induced DNA damage and retained more damage following recovery. Cells lacking functional NER also displayed an increased number of chromosomal aberrations. Mutant fibroblasts displayed decreased population doubling rate, increased telomere attrition rate and early emergence of senescent characteristics under chronic exposure to low level oxidative stress. Our results show that NER dysfunction increases mutagenesis rate following oxidative stress, suggesting that oxidative stress is a major contributor to the manifestations of XP and CS phenotype; XP and CS symptoms cannot be explained simply by the inability to completely remove UVinduced DNA damage. A dysfunctional NER increases tolerance to oxidative stress while increasing the susceptibility to DNA damage, contributing to cancer risk and premature ageing characteristics in XP and CS patients. Our findings have implications in the mechanisms of DNA repair in oxidative stress, mutagenesis, carcinogenesis and ageing.