Enhanced S phase delay and inhibition of replication of an undamaged shuttle vector in UVC-irradiated xeroderma pigmentosum variant

S. K. Bullock, W. K. Kaufmann, M. Cordeiro-Stone
2001 Carcinogenesis  
replication units that did not incur any direct damage. Xeroderma pigmentosum variant (XP-V) cells are defective Velocity sedimentation analyses of radiolabeled nascent in bypass replication of UVC-induced thymine dimers in DNA revealed that UVC-induced inhibition of DNA replication DNA because they lack a novel DNA polymerase (polyresults from inhibition of both replicon initiation and DNA merase η). In this study the effects of UVC on S phase chain elongation (1). Inhibition of DNA chain
more » ... n of DNA chain elongation cells were compared in fibroblasts derived from normal reflects physical blockage of the replication machinery by donors (IDH4) and XP-V patients (CTag) and immortalized template lesions. This passive, cis-acting effect of UVCby expression of the SV40 large T antigen. These transinduced lesions on DNA replication is overcome by postformed fibroblasts did not activate the G 1 checkpoint replication repair (PRR), which includes pathways leading to or inhibit replicon initiation when damaged by UVC or bypass replication of blocking lesions and elimination of γ-rays. The transformed XP-V cells (CTag) retained the daughter strand gaps (2). Thus, PRR promotes damage tolerincreased sensitivity to UVC-induced inhibition of DNA ance and completion of replication of the damaged genome. strand growth previously observed with their diploid Although UVC induces daughter strand gaps in normal cells, counterpart. Cell cycle progression analyses showed that the frequency and half-life of these single-stranded DNA CTag cells displayed a stronger S phase delay than transregions are increased in PRR-defective cells, such as those formed fibroblasts from normal individuals (IDH4) after derived from xeroderma pigmentosum variant (XP-V) patients. treatment with only 2 J/m 2 UVC. Low doses of UVC also XP-V cells have a normal capacity for nucleotide excision caused a lag in CTag cell proliferation. The extent of repair (3-6), but are deficient in bypass replication of UVCreplication of an episomal DNA (pSV011), not previously induced thymine dimers (1,4,(7)(8)(9)(10)(11)(12). This bypass defect is exposed to radiation, was measured after the host cells caused by frameshift mutations in the gene encoding DNA polymerase η (pol η) (13,14). This novel DNA polymerase were irradiated with 1-3 J/m 2 UVC. Replication of pSV011 has been shown to efficiently bypass cis,syn-cyclobutane was barely affected in irradiated IDH4 cells. Plasmid thymine dimers in vitro by incorporating adenines opposite this replication was inhibited by 50% in irradiated CTag cells photoproduct (12,15). The enzymes responsible for mutagenic and this inhibition could not be accounted for by increased bypass of UVC-induced photoproducts in human cells are killing of host cells by UVC. These results suggest that polymerase ζ (16) and hRev1 (17). These proteins, or another even in transformed cells UVC induces DNA damage error-prone DNA polymerase (18,19), must be the ones that responses that are reflected in transient cell cycle arrest, eventually catalyze the bypass of cyclobutane thymine dimers delay in proliferation and inhibition of episomal DNA in XP-V cells, hence their hypermutability to UVC (20,21). replication. These responses are enhanced in CTag cells, The biochemical mechanisms that underlie radiation-induced presumably because of their bypass replication defect. The inhibition of replicon initiation are less clear. It occurs at sites accumulation of replication complexes blocked at thymine away from the primary DNA lesion (a trans effect) in response dimers and extended single-stranded regions in chromoto a signal transduction pathway. Therefore, it is the final somal DNA might sequester replication factors that are result of an active process in which the DNA damage is needed for plasmid and chromosomal replication. Alternarecognized by molecular sensors and the information transtively, aberrant replication structures might activate a mitted to the sites of action by effector molecules. The signal transduction pathway that down-regulates DNA inhibition triggered by ionizing radiation is dependent on the synthesis. ATM (gene mutated in ataxia telangectasia) protein kinase (22-24) and reflects the activity of an intra-S phase checkpoint (22, 24) . UVC-induced DNA lesions also inhibit initiation of Abbreviations: ATM, gene mutated in ataxia telangectasia; BrdU, bromonew replicons in S phase cells (1,25). The primary kinase deoxyuridine; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; initiating this response, however, seems to be the ATM-and MEM, minimum essential medium; PI, PhosphorImager units; pol η, DNA Rad3-related kinase ATR (26,27). Little is known about the polymerase η; PRR, post-replication repair; RPA, replication protein A (singlestranded DNA-binding protein); XP-V, xeroderma pigmentosum variant. effector molecules involved in the S phase checkpoint
doi:10.1093/carcin/22.2.233 pmid:11181443 fatcat:yruda6kuenhbpp2d3wmgxqzdke