An ATR- and Chk1-Dependent S Checkpoint Inhibits Replicon Initiation following UVC-Induced DNA Damage

Abstract

Inhibition of replicon initiation is a stereotypic DNA damage response mediated through S checkpointmechanisms not yet fully understood. Studies were undertaken to elucidate the function of checkpoint proteins in the inhibition of replicon initiation following irradiation with 254 nm UV light (UVC) of diploid human fibroblasts immortalized by the ectopic expression of telomerase. Velocity sedimentation analysis of nascent DNA molecules revealed a 50% inhibition of replicon initiation when normal human fibroblasts were treated with a low dose of UVC (1 J/m 2 ). Ataxia telangiectasia (AT), Nijmegen breakage syndrome (NBS), and AT-like disorder fibroblasts, which lack an S checkpoint response when exposed to ionizing radiation, responded normally when exposed to UVC and inhibited replicon initiation. Pretreatment of normal and AT fibroblasts with caffeine or UCN-01, inhibitors of ATR (AT mutated and Rad3 related) and Chk1, respectively, abolished the S checkpoint response to UVC. Moreover, overexpression of kinase-inactive ATR in U2OS cells severely attenuated UVC-induced Chk1 phosphorylation and reversed the UVC-induced inhibition of replicon initiation, as did overexpression of kinase-inactive Chk1. Taken together, these data suggest that the UVC-induced S checkpoint response of inhibition of replicon initiation is mediated by ATR signaling through Chk-1 and is independent of ATM, Nbs1, and Mre11.Accurate replication and segregation of the human genome depends on interactions between cell cycle checkpoints and pathways of DNA repair. Cell cycle checkpoints are biochemical surveillance pathways that slow or arrest progression through the cell cycle, pending completion of essential events and/or repair of DNA damage. DNA damage checkpoints minimize the probability of replicating and segregating damaged DNA and therefore reduce the frequencies of mutations and chromosomal aberrations that are induced by genotoxic stress.

Suppression of replication fork progression in low-dose-specific p53-dependent S-phase DNA damage checkpoint

“…Therefore, the lowdose specificity and p53 dependency of the suppression of replication fork progression may well have been overlooked. In fact, 1.5 Gy gamma-radiation was reported to induce suppression of replication fork progression in p53 proficient normal human fibroblasts (Heffernan et al, 2002). Furthermore, (Painter and Young, 1980) found radiation-induced suppression of chain elongation in p53 proficient E-11 normal human diploid cells whereas the same authors found no such suppression in p53 mutated cell line as discussed above (Painter and Young, 1975).…”

Caffeine and human DNA metabolism: the magic and the mystery

“…The original mutant fibroblast strains were obtained from the NIGMS Human Genetic Cell Repository (GM02052A, AT) and the American Type Culture Collection (CRL1162, XP-V, strain XP4BE). Immortalized cell lines from these strains of human fibroblasts were obtained by ectopic expression of human telomerase (hTERT), as previously described [25]. The derivative cell lines described below are also immortalized.…”

“…These were performed essentially as described [25]. Briefly, logarithmically growing cells were seeded at 10 6 per 100 mm dish and incubated for 40 h. Cultures were treated as specified in the figure legends, harvested by trypsinization, washed once in PBS, and resuspended in lysis buffer (10 mM sodium phosphate buffer, pH 7.2, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 1% NP40, supplemented with 10 mM 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), 10 mM β-glycerophosphate, 10 mM sodium orthovanadate, and 10 μg/ml of leupeptin and aprotinin).…”

“…The effects of caffeine are especially notable in XP variant cells [24], which lack DNA pol η and must rely upon other more error-prone mechanisms of replication through UVCinduced photoproducts. Thus, while caffeine is now known to reverse an ATR-dependent S checkpoint that slows the rate of replicon initiation in UVC-treated cells [25], it may have an additional effect on pol η-independent bypass of cyclobutane pyrimidine dimers, which could involve translesion synthesis by other DNA polymerases or recombinational repair of daughter-strand gaps.…”

Mapping of an origin of DNA replication in the promoter of fragile X gene FMR1

“…The human cells used in these studies were NHF1-hTERT, a cell line derived from normal neonatal foreskin fibroblasts (Boyer et al, 1991) and immortalized by ectopic expression of the catalytic subunit of telomerase (Heffernan et al, 2002); GM1604-hTERT, a male fetal lung fibroblast cell line also immortalized by telomerase expression (Ouellette et al, 2000); and CRL-1502 cells (ATCC), a strain of female fetal lung fibroblasts. The hTERT-immortalized cell lines were grown in minimal essential medium (Invitrogen, Carlsbad, CA) containing 2X the concentration of MEM non-essential amino acids (Invitrogen).…”