Frequency and Fidelity of Translesion Synthesis of Site-specific N-2-Acetylaminofluorene Adducts during DNA Replication in a Human Cell Extract

Psoralens are mutagenic compounds of vegetable origin that are used as photosensitizing agents in the treatment of various skin diseases, blood cell cancer, and autoimmune disorders. To study the mechanism of mutagenicity of psoralens in humans, we examined the efficiency and fidelity of simian virus 40 origindependent replication in a human cell extract of M13mp2 DNA randomly treated with the psoralen derivative 4-hydroxymethyl-4,5,8-trimethyl psoralen plus UVA irradiation. Replication of DNA treated with variable amounts of 4-hydroxymethyl-4,5,8-trimethyl psoralen and a fixed UVA fluence was inhibited in a concentration-dependent manner. However, covalently closed monomer-length circular replication products were observed. Product analysis by renaturing agarose gel electrophoresis after cross-linking with 250-to 280-nm UV light indicated that approximately 1 of 9 psoralen monoadducts was bypassed during in vitro replication. Introduction of product DNA into Escherichia coli to score replication errors in the lacZ␣ reporter gene demonstrated that replication of the damaged DNA was more mutagenic than was replication of undamaged DNA. Sequence analysis of lacZ mutants revealed that damage-dependent replication errors were predominantly T ⅐ A3C ⅐ G transitions, transversions at C ⅐ G base pairs, and deletions of single A ⅐ T base pairs, the last occurring most frequently in homopolymeric runs. A comparison of error specificities with two substrates having the replication origin asymmetrically placed on opposite sides of the mutational target suggests that the lagging-strand replication apparatus is less accurate than the leading-strand replication apparatus for psoralen monoadduct-dependent deletion errors. A model is proposed based on the preferential loopout of the monoadducted base from the strand that templates retrograde discontinuous synthesis.DNA replication in eukaryotic cells is a complex process requiring a variety of proteins to synthesize the leading and lagging strands in an asymmetric yet coordinated manner (14). Because of this asymmetry, DNA adducts that escape repair may have different potentials for blocking replication or promoting replication infidelity, depending on which strand the adduct is located on. For example, in mammalian cells deficient in excision repair, mutations were predominantly found on one of the two strands of the hprt gene (6,19,20,50,51), leading to the suggestion that the fidelity of translesion bypass is more error prone on the leading strand (20, 51).To study the processes that determine the fidelity of DNA replication, we have been using the SV40 replication system as a model for human chromosomal replication (for reviews, see references 29 and 37). Using substrates containing the SV40 origin of replication, we (27,30,31,41) and others (11) have shown that the replication apparatus in mammalian cell extracts is quite accurate when replicating undamaged DNA, with an error rate ranging from less than or equal to 6.2 ϫ 10 Ϫ6 to 1 ϫ 10 Ϫ7 error per nucleotide. These observ...

Section: Discussioncontrasting

confidence: 48%

Section: Discussionmentioning

confidence: 99%

Strand Specificity of Mutagenic Bypass Replication of DNA Containing Psoralen Monoadducts in a Human Cell Extract

Thomas

Svoboda

Vos

et al. 1996

“…Complete replication past site-specific UV-induced lesions (6,29) or AAF adducts (30,31) in double-stranded DNA carrying the simian virus 40 origin of replication has been observed in HeLa cell extracts, in the presence of T antigen. Recently, using a similar approach, three groups (8,9,28) demonstrated that in contrast to normal cell extracts, extracts from XPV cells were completely (8) or partially (9, 28) deficient in the bypass of a single cis-syn thymine dimer.…”

Section: Discussionmentioning

confidence: 99%

“…AAF adducts at the C8 position of guanine (dGua-C8-AAF) are severe blocks to in vitro DNA synthesis by purified prokaryotic and eukaryotic DNA polymerases (2,21,22). TLS past AAF adducts in both double-stranded (30,31) and forked single-stranded (13) DNA templates has been observed to some extent in cell extracts. This suggests that extracts accurately mimic at least some of the mechanisms that rescue a blocked replication fork in vivo.…”

Section: Resultsmentioning

confidence: 99%

Impaired Translesion Synthesis in Xeroderma Pigmentosum Variant Extracts

Cordonnier¹,

Lehmann

Fuchs³

1999

Xeroderma pigmentosum variant (XPV) cells are characterized by a cellular defect in the ability to synthesize intact daughter DNA strands on damaged templates. Molecular mechanisms that facilitate replication fork progression on damaged DNA in normal cells are not well defined. In this study, we used single-stranded plasmid molecules containing a single N-2-acetylaminofluorene (AAF) adduct to analyze translesion synthesis (TLS) catalyzed by extracts of either normal or XPV primary skin fibroblasts. In one of the substrates, the single AAF adduct was located at the 3 end of a run of three guanines that was previously shown to induce deletion of one G by a slippage mechanism. Primer extension reactions performed by normal cellular extracts from four different individuals produced the same distinct pattern of TLS, with over 80% of the products resulting from the elongation of a slipped intermediate and the remaining 20% resulting from a nonslipped intermediate. In contrast, with cellular extracts from five different XPV patients, the TLS reaction was strongly reduced, yielding only low amounts of TLS via the nonslipped intermediate. With our second substrate, in which the AAF adduct was located at the first G in the run, thus preventing slippage from occurring, we confirmed that normal extracts were able to perform TLS 10-fold more efficiently than XPV extracts. These data demonstrate unequivocally that the defect in XPV cells resides in translesion synthesis independently of the slippage process.Xeroderma pigmentosum (XP) is an autosomal recessive disorder characterized by a genetic predisposition to sunlightinduced skin cancer. Fibroblasts derived from patients with XP are extremely sensitive to the mutagenic effect of UV irradiation (19). The majority of XP patients are deficient in nucleotide excision repair, and there have been dramatic advances in our understanding of the molecular defects in these patients. In contrast, there has been little progress in our understanding of the molecular defect in the XP variant (XPV) group, which comprises a substantial minority (approximately 20%) of XP patients. They have normal levels of nucleotide excision repair and normal sensitivity to the lethal effects of UV irradiation (5) but a marked defect in the ability to synthesize intact daughter DNA strands during DNA replication after some (4,17,20,26) but not all (4, 7) types of carcinogenic damage. The cellular defect in DNA replication in UV-irradiated XPV cells was discovered as long ago as 1975 (17), but because of the normal sensitivity to killing by UV light, the XPV gene has been refractory to cloning. The precise nature of the molecular defect in this important class of XP patients remains one of the major unsolved problems in this area.The UV hypermutability of XPV cells could be due to an abnormal error-prone mechanism of replication. This hypothesis is supported by results showing that mutation spectra in UV-irradiated XPV cells are distinct from those observed in normal cells (32,33). In XPV cells, the UV-ind...

“…Psoralen monoadducts (37) and acetylaminofluorene adducts (36) are bypassed with lower fidelity when the adducts are in the lagging-strand template than when they are in the leading-strand template. Furthermore, acetylaminofluorene adducts are much stronger blocks to replication when they are situated in the leading-strand template (40).…”

Section: Discussionmentioning

confidence: 99%

Abnormal, Error-Prone Bypass of Photoproducts by Xeroderma Pigmentosum Variant Cell Extracts Results in Extreme Strand Bias for the Kinds of Mutations Induced by UV Light

McGregor

Wang

Maher

et al. 1999

Xeroderma pigmentosum (XP) is a rare genetic disease characterized by a greatly increased susceptibility to sunlight-induced skin cancer. Cells from the majority of patients are defective in nucleotide excision repair. However, cells from one set of patients, XP variants, exhibit normal repair but are abnormally slow in replicating DNA containing UV photoproducts. The frequency of UV radiation-induced mutations in the XP variant cells is significantly higher than that in normal human cells. Furthermore, the kinds of UV-induced mutations differ very significantly from normal. Instead of transitions, mainly C3T, 30% of the base substitutions consist of C3A transversions, all arising from photoproducts located in one strand. Mutations involving cytosine in the other strand are almost all C3T transitions. Forty-five percent of the substitutions involve thymine, and the majority are transversions. To test the hypothesis that the UV hypermutability and the abnormal spectrum of mutations result from abnormal bypass of photoproducts in DNA, we compared extracts from XP variant cells with those from HeLa cells and a fibroblast cell strain, MSU-1.2, for the ability to replicate a UV-irradiated form I M13 phage. The M13 template contains a simian virus 40 origin of replication located directly to the left or to the right of the target gene, lacZ␣, so that the template for the leading and lagging strands of DNA replication is defined. Reduction of replication to ϳ37% of the control value required only 1 photoproduct per template for XP variant cell extracts, but ϳ2.2 photoproducts for HeLa or MSU-1.2 cell extracts. The frequency of mutants induced was four times higher with XP variant cell extracts than with HeLa or MSU-1.2 cell extracts. With XP variant cell extracts, the proportion of C3A transversions reached as high as 43% with either M13 template and arose from photoproducts located in the template for leading-strand synthesis; with HeLa or MSU-1.2 cell extracts, this value was only 5%, and these arose from photoproducts in either strand. With the XP variant extracts, 26% of the substitutions involved thymine, and virtually all were T3A transversions. Sequence analysis of the coding region of the catalytic subunit of DNA polymerase delta in XP variant cell lines revealed two polymorphisms, but these do not account for the reduced bypass fidelity. Our data indicate that the UV hypermutability of XP variant cells results from reduced bypass fidelity and that unlike for normal cells, bypass of photoproducts involving cytosine in the template for the leading strand differs significantly from that of photoproducts in the lagging strand.Xeroderma pigmentosum (XP) patients are genetically predisposed to sunlight-induced skin cancer, and the cells from a majority of such patients are defective in some aspect of nucleotide excision repair (NER) (28). However, the cells from one set of XP patients, termed XP variants, have normal rates of global and gene-specific NER of UV-induced photoproducts (7, 38, 43) but are significantly ...