Differential replication of a single, UV-induced lesion in the leading or lagging strand by a human cell extract: fork uncoupling or gap formation.

Abstract

We have constructed simian virus 40 minireplicons containing uniquely placed cis,syn-thymine dimers (T< >T) for the analysis of leading-and lagging-strand bypass replication. Assaying for replication in a human cellfree extract through the analysis of full-size labeled product molecules and restriction fragments spanning the T< >T site resulted in the following findings: (i) Despite these investigations, a basic understanding of replication of damaged DNA in normal human cells is lacking (2).In vitro studies using primed single-stranded DNA templates and purified DNA polymerases (9-13) have demonstrated the existence of both stalled replication intermediates and translesion synthesis. These studies involved DNA synthesis on primed templates rather than semiconservative coordinated replication of leading and lagging strands at a bona fideThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. replication fork. In vitro replication of SV40-based minireplicons carrying randomly distributed, UVC-induced Pyr<>Pyr has been analyzed in human cell extracts (14,15). A UV fluence-dependent inhibition of covalently closed circular DNA (form I DNA) synthesis was observed, which confirmed the blocking effect of such lesions on DNA replication. The presence of a UV endonuclease V from phage T4 (T4 UV endo)-sensitive product implied that replication of Pyr<>Pyr occurred in such a system. A detailed examination of the molecular mechanism of UV damage processing has become possible with the development of site-specific lesioncontaining DNA (11-13). Thus, we now report sites of blockage and rates of bypass of individual lesions in the leading and lagging strands.The most prevalent DNA modification resulting from UVC irradiation is the cis,syn-thymine dimer (T<>T), which has been a paradigm DNA lesion (16). We have prepared plasmids containing the SV40 origin of replication together with uniquely placed T<>T and have analyzed their in vitro replication in a human cell-free extract. We have developed these assays to study the mechanism of replication of DNA damaged in either the leading or lagging strands of a human replication fork. Specifically, we have determined the site of blockage of strand elongation on T<>T-containing DNA templates, compared the bypass of T<>T in the leading and lagging strands, and analyzed the potential uncoupling of coordinated leading-and lagging-strand synthesis at T<>T-blocked replication forks. MATERIALS AND METHODS

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

“…However, human pol δ cannot replicate past them, suggesting that disassembly of the pol δ holoenzyme on encountering such lesions is predominantly driven by an abrupt decrease in k pol . Indeed, blockage of strand elongation by human pol δ was observed directly 3′ to these lesions rather than upstream (8,44,45,50). The results presented in Fig.…”

“…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. Furthermore, strand elongation past and beyond the lesion was observed in only a fraction of the analyzed molecules (50,61). Altogether, these observations suggest that the lagging strand template is reprimed 5′ to the damage, and pol δ is recycled to nascent primers upstream of the damage, leaving behind a ssDNA gap extending from the CPD lesion to the 5′ terminus of the downstream Okazaki fragment.…”

Minding the gap: The underground functions of BRCA1 and BRCA2 at stalled replication forks

“…Studies in mammalian cells of replicating UV-irradiated plasmids have revealed DNA polymerase stalling on both the leading and lagging strands and showed the existence of ssDNA gaps on the lagging strand, presumably caused by interruption of Okazaki fragment synthesis [41][42][43][44]. This suggests that DSGs also arise during mammalian DNA replication, although it is not yet clear whether they can form on the leading strand.…”

Genetic evidence for a role of Saccharomyces cerevisiae Mph1 in recombinational DNA repair under replicative stress

“…In particular, a polymeraseblocking lesion on the lagging strand template should not arrest replication, as it proceeds with synthesis of the next Okazaki fragment. The result is a lagging strand with a gap comprising the lesion (Svoboda and Vos, 1995) to be repaired at a later time, after gap filling, by either TLS or HR. Although a similar mechanism has been proposed, when the leading strand polymerase stops at a lesion on the template (Heller and Marians, 2006;Lopes et al, 2006), an alternative possibility is decoupling of the leading and lagging strand polymerase with the lagging strand being further elongated for another 1 or 2 kb (Cordeiro-Stone et al, 1999;Pagés and Fuchs, 2003).…”