Florence Le Page

RETRACTED: Transcription-Coupled Repair of 8-oxoGuanine

Analysis of transcription-coupled repair (TCR) of oxidative lesions here reveals strand-specific removal of 8-oxo-guanine (8-oxoG) and thymine glycol both in normal human cells and xeroderma pigmentosum (XP) cells defective in nucleotide excision repair. In contrast, Cockayne syndrome (CS) cells including CS-B, XP-B/CS, XP-D/CS, and XP-G/CS not only lack TCR but cannot remove 8-oxoG in a transcribed sequence, despite its proficient repair when not transcribed. The XP-G/CS defect uniquely slows lesion removal in nontranscribed sequences. Defective TCR leads to a mutation frequency at 8-oxoG of 30%-40% compared to the normal 1%-4%. Surprisingly, unrepaired 8-oxoG blocks transcription by RNA polymerase II. These data imply that TCR is required for polymerase release to allow repair and that CS results from defects in TCR of oxidative lesions.

Role of XRCC1 in the Coordination and Stimulation of Oxidative DNA Damage Repair Initiated by the DNA Glycosylase hOGG1

XRCC1 participates in DNA single strand break and base excision repair (BER) to preserve genetic stability in mammalian cells. XRCC1 participation in these pathways is mediated by its interactions with several of the acting enzymes. Here, we report that XRCC1 interacts physically and functionally with hOGG1, the human DNA glycosylase that initiates the repair by BER of the mutagenic oxidized base 8-oxoguanine. This interaction leads to a 2- to 3-fold stimulation of the DNA glycosylase activity of hOGG1. XRCC1 stimulates the formation of the hOGG1 Schiff-base DNA intermediate without interfering with the endonuclease activity of APE1, the second enzyme in the pathway. On the contrary, the stimulation in the appearance of the incision product seems to reflect the addition of the effects of XRCC1 on the two first enzymes of the pathway. The data presented support a model by which XRCC1 will pass on the DNA intermediate from hOGG1 to the endonuclease APE1. This results in an acceleration of the overall repair process of oxidized purines to yield an APE1-cleaved abasic site, which can be used as a substrate by DNA polymerase β. More importantly, the results unveil a highly coordinated mechanism by which XRCC1, through its multiple protein-protein interactions, extends its orchestrating role from the base excision step to the resealing of the repaired DNA strand.

A global DNA repair mechanism involving the Cockayne syndrome B (CSB) gene product can prevent the in vivo accumulation of endogenous oxidative DNA base damage

The Cockayne syndrome B (CSB) gene product is involved in the repair of various types of base modifications in actively transcribed DNA sequences. To investigate its significance for the repair of endogenous oxidative DNA damage, homozygous csb−/−/ogg1−/− double knockout mice were generated. These combine the deficiency of CSB with that of OGG1, a gene coding for the mammalian repair glycosylase that initiates the base excision repair of 7,8-dihydro-8-oxoguanine (8-oxoG). Compared to ogg1−/− mice, csb−/−/ogg1−/− mice were found to accumulate with age severalfold higher levels of oxidited purine modifications in hepatocytes, splenocytes and kidney cells. In contrast, the basal (steady-state) levels of oxidative DNA modifications in cells from csb−/− mice were not different from those in wild-type mice and did not increase with age. The analysis of the repair rates of additional oxidative DNA base modifications induced by photosensitization in immortalized embryonic fibroblasts was in accordance with these findings: compared to wild-type cells, the global repair was only slightly affected in csb−/− cells, more compromised in ogg1−/− cells, but virtually absent in csb−/−/ogg1−/− cells. An inhibition of transcription by α-amanitin did not block the Csb-dependent repair in ogg1−/− fibroblasts. The influence of Csb on the global repair of 8-oxoG was not detectable in assays with total protein extracts and in a shuttle vector system. The data indicate a role for Csb in the removal of 8-oxoG from the overall genome that is independent of both Ogg1-mediated base excision repair and regular transcription.

Repair and mutagenesis survey of 8-hydroxyguanine in bacteria and human cells

8-Hydroxyguanine is one of the major products formed by the reactive oxygen species which are generated in living cells as a consequence of either the normal metabolic pathways or an exogeneous chemical or physical stress. The production of the oxidative damage is described and the different repair pathways of the oxidative lesions are analyzed from bacteria to human cells. Analysis of repair in human cells harboring different deficiencies in the nucleotide excision repair mechanism such as xeroderma pigmentosum cells from different complementation groups and cells from Cockaynes syndrome patients allows us to emphasize the possibility of the intervention of this repair mechanism on the elimination of oxidative damages. Finally, a repair model of oxidative lesions is proposed.

Repair of 8-oxoguanine and oggl-incised apurinic sites in a CHO cell line

The repair mechanisms involved in the removal of 8-oxo-7,8-dihydroguanine (8-oxoG) in damaged DNA have been investigated using cell-free extracts or purified proteins. However, in vivo repair assays are required to further dissect mechanisms involved in the repair of 8-oxoG in the cellular context. In this study, we analyzed the removal of 8-oxoG from plasmids that contain a single 8-oxoG.C base pair in a sequence that can be transcribed (TS) or nontranscribed (NTS) in a chinese hamster ovary (CHO) cell line. The results show that 8-oxoG located in a TS is removed faster than in a NTS, indicating transcription-coupled repair (TCR) of 8-oxoG in rodent cells. The results also show that CHO cells efficiently repair DNA molecules that contain an Ogg1-incised AP site, which is the first intermediate in the course of base excision repair of 8-oxoG.

Transcription-coupled repair of 8-oxoguanine in human cells.

Publisher Summary Because there are no specific genotoxic agents able to produce only 8-oxoguanine (8-oxoG or GO) in cellular DNA and because the antibodies raised against this lesion are not specific enough, a shuttle vector is used to assay allowing to construct a plasmid DNA containing only one GO lesion at a given position. The assay is the only one at this time to allow the study of the repair and mutagenic potency of unique oxidative damage in human cells. This technology, which is equally applicable for other types of oxidative base damage, has been used in the past mostly for DNA lesions repaired by nucleotide excision repair (NER). Results obtained with shuttle vectors in repair and mutagenesis analyses are highly reproducible and when used in NER studies have always been validated by genomic DNA data when available. The results using a shuttle vector containing a single 8-oxoG showed for the first time the existence of transcription-coupled repair of this lesion in human cells. The observed deficiency in this pathway in Cockayne syndrome (CS) and xeroderma pigmentosum (XP)/CS cells correlates with progressive neurological disorders observed in the patients. It is plausible that in nondividing brain cells the accumulation of unrepaired oxidative lesions on transcribed strands leads to apoptosis and therefore to progressive neurological deterioration.