Pyrimidine dimers block simian virus 40 replication forks.

Abstract

UV light produces lesions, predominantly pyrimidine dimers, which inhibit DNA replication in mammalian cells. The mechanism of inhibition is controversial: is synthesis of a daughter strand halted at a lesion while the replication fork moves on and reinitiates downstream, or is fork progression itself blocked for some time at the site of a lesion? We directly addressed this question by using electron microscopy to examine the distances of replication forks from the origin in unirradiated and UV-irradiated simian virus 40 chromosomes. If UV lesions block replication fork progression, the forks should be asymmetrically located in a large fraction of the irradiated molecules; if replication forks move rapidly past lesions, the forks should be symmetrically located. A large fraction of the simian virus 40 replication forks in irradiated molecules were asymmetrically located, demonstrating that UV lesions present at the frequency of pyrimidine dimers block replication forks. As a mechanism for this fork blockage, we propose that polymerization of the leading strand makes a significant contribution to the energetics of fork movement, so any lesion in the template for the leading strand which blocks polymerization should also block fork movement.A variety of chemical and physical agents damage DNA within cells, causing inhibition of DNA replication and transcription, alteration of gene expression, mutagenesis, carcinogenesis, or cell death. Inhibition of DNA replication, when it occurs, is crucial to the fate of the cell: although cells may tolerate some mutations, they must replicate their full complement of DNA before dividing. The structures that result from attempting to replicate a damaged DNA template are the substrates for subsequent repair and recovery processes. Understanding the initial interaction between lesions and the replication fork will therefore provide insight into the mechanisms of recovery.UV light is one of the best-studied of DNA-damaging agents. The primary lesion produced by UV irradiation is the cis-syn cyclobutane pyrimidine dimer (31). The mechanism by which UV inhibits eucaryotic DNA replication remains controversial, especially the question of whether dimers inhibit all, most, or no replication forks. The earliest model (16) proposed that a pyrimidine dimer in the template blocked elongation of a daughter strand without affecting the progression (unwinding and movement) of the replication fork, which reinitiated synthesis about 1,000 nucleotides downstream, leaving a long single-stranded gap that could later be filled de novo (Fig. 1B). This resembles a situation in Escherichia coli, except that in E. coli the gaps are filled by recombination (23,24). An alternative hypothesis (7) is that lesions block the movement of the replication fork itself, as in Fig. 1C. With increasing awareness of the semidiscontinuous nature of DNA replication in mammalian cells (Fig. 1A), the latter model was modified to suggest that lesions in the template for the continuously synthesized (leading) strand block...

Stability of the human polymerase δ holoenzyme and its implications in lagging strand DNA synthesis

“…Such events would leave behind an ssDNA gap opposite the offending lesion. Indeed, when SV40-transformed human cells were irradiated with UV, ssDNA gaps were observed in the replicated portions distal to the replication forks within SV40 DNA molecules and the size of the gaps (50-300 nt) agreed with the expectation for incomplete synthesis of Okazaki fragments (42,43). In similar fashion, on SV40-based plasmids containing a single unique site CPD in the lagging strand template, synthesis of the Okazaki fragment containing the CPD lesion was selectively inhibited compared with the flanking fragments.…”

“…For this model system, all aspects of replicating the double-stranded SV40 DNA except for helicase-assisted unwinding are carried out by the replication machinery of the human host cell. In the absence of DNA damage, small internal ssDNA gaps (50-300 nt) indicative of premature release and recycling of pol δ were not observed within the replicated regions of SV40 DNA molecules (42,43). Altogether, this suggests that lagging strand synthesis within a human cell may not require maximum processivity/efficiency and that aborted Okazaki fragments are rebound in the event pol δ dissociates prematurely (Fig.…”

Regulation of Cellular and SV40 Virus Origins of Replication by Chk1-dependent Intrinsic and UVC Radiation-induced Checkpoints

“…The inhibitory effects of UVC radiation on cellular DNA replication are rapid, occurring maximally over a period of 1 h followed by recovery of DNA replication a few hours later (3,7). For comparison, we analyzed the time-dependent inhibitory effects on SV40 DNA replication induced by 25 J/m 2 UVC radiation.…”

“…The elongation arrest observed at high doses of UVC radiation also was thought to occur in cis when lesions formed a physical block to replication fork progression (7). However, more recent experiments indicate that the inhibitory effects of ionizing radiation (8 -10) and methylation damage (11) on episomal DNA replication can occur in trans.…”

UV‐Induced Changes in Cell Cycle and Gene Expression within Rabbit Lens Epithelial Cells

“…I t is known that inhibitors of DNA synthesis decrease levels of histone transcripts (DeLisle et al, 1983). W-induced DNA damage inhibits DNA synthesis by blocking DNA fork displacement (Berger and Edenberg, 1986) and reduces the initiation of replication (Cleaver et al, 1983;Painter, 1985 Hiller, R., Giacometti, L. and Yuen, K. (1977). Sunlight and cataract: an epidemiologic investigation.…”