Fork-like DNA templates support bypass replication of lesions that block DNA synthesis on single-stranded templates

Abstract

DNA replication is an asymmetric process involving concurrent DNA synthesis on leading and lagging strands. Leading strand synthesis proceeds concomitantly with fork opening, whereas synthesis of the lagging strand essentially takes place on a single-stranded template. The effect of this duality on DNA damage processing by the cellular replication machinery was tested using eukaryotic cell extracts and model DNA substrates containing site-specific DNA adducts formed by the anticancer drug cisplatin or by the carcinogen N-2-acetylaminof luorene. Bypass of both lesions was observed only with fork-like substrates, whereas complete inhibition of DNA synthesis occurred on damaged singlestranded DNA substrates. These results suggest a role for additional accessory factors that permit DNA polymerases to bypass lesions when present in fork-like DNA.The opposing orientation of the two DNA strands in the double helix imposes an asymmetry upon the replication process (1). Since polymerases only synthesize DNA in one direction (5Ј 3 3Ј), one strand (the leading strand) is synthesized continuously in the direction in which the helix unzips, whereas the other (the lagging strand) is synthesized discontinuously in the opposite direction in numerous short pieces known as Okazaki fragments. Therefore, leading strand synthesis proceeds concomitantly with fork opening from a double-stranded template, whereas synthesis of the lagging strand essentially takes place on a single-stranded template. Single-stranded and fork-like DNA templates have been previously suggested as models for lagging and leading strand replication, respectively (2, 3), although current models of DNA replication propose a mechanism in which leading and lagging strand replication are coupled. Genotoxic agents that induce bulky lesions in DNA often hamper progression of the replication fork and result in either nonor miscoding nucleotides. In a process known as translesion synthesis (TLS), the replication machinery will eventually proceed through some lesions with an increased error rate, resulting in the induction of mutations (4 -6). We wished to investigate the effect of the asymmetry of DNA replication on the efficiency of translesion synthesis. For this purpose, we have compared TLS in vitro by eukaryotic cell extracts on forked or single-stranded (ss) DNA templates containing single site-specific adducts formed by a chemotherapeutic agent (cisplatin) or a strong chemical carcinogen (N-2-acetylaminof luorene or AAF). For both adducts, we observed TLS only when the adduct was located in the forked template. MATERIALS AND METHODSMaterials. The DNA damage-inducible CDK inhibitor p21 protein, purified as described (7), was a generous gift from Prof. Bernard Ducommun (Institut de Pharmacologie et de Biologie Structurale, Toulouse, France). Calf thymus DNA polymerases ␣, ␦, and were purified as reported (8). All oligonucleotides were synthesized on a Cyclone Plus DNA synthesizer from MilliGen͞Biosearch and purified on denaturing 20% polyacrylamide gels. [...

Mutator phenotype of BCR – ABL transfected Ba/F3 cell lines and its association with enhanced expression of DNA polymerase β

“…Replicative cell extracts were prepared as previously described (Ho mann et al, 1996). Brie¯y, the control and BCR ± ABL-transfected Ba/F3 cells were washed with icecold PBS, harvested by centrifugation, and the cell pellets were suspended in hypotonic bu er (10 mM Tris-HCl, pH 7.5, 10 mM KCl, 10 mM MgCl 2 , 1 mM DTT) containing protease inhibitors.…”

Analysis of Damage Tolerance Pathways in Saccharomyces cerevisiae: a Requirement for Rev3 DNA Polymerase in Translesion Synthesis

“…AAF-monomodified heteroduplex plasmids were transformed into the above yeast strains and analyzed as described for the unmodified vector. AAF was the lesion of choice, as the chemical adducts it forms in DNA are stable and well characterized and have been shown to be strong blocks to replication (5,14,19,27,31,45). Moreover, AAF-monomodified plasmids have been successfully used as tools to investigate damage tolerance strategies in E. coli (19,31).…”

Deregulated DNA polymerase β strengthens ionizing radiation-induced nucleotidic and chromosomal instabilities

“…The procedure for CHO extracts preparation has been done following the protocol previously described (Ho mann et al, 1996). Brie¯y, the cells were grown as monolayer cultures then washed with ice-cold PBS, scraped o the plates, and harvested by centrifugation.…”