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R P Fuchs

Publications and source records attributed to R P Fuchs.

At least 37 records · Page 2Linked to original sources
PubMed1999

Distinct roles for Rev1p and Rev7p during translesion synthesis in Saccharomyces cerevisiae.

Translesion synthesis (TLS) in Saccharomyces cerevisiae requires at least Rev1p and polymerase zeta (Pol zeta), a complex of the Rev3 polymerase and its accessory factor Rev7p. Although their precise role(s) are poorly characterized, in vitro studies suggest that each protein contributes to TLS in a manner dependent on the particular lesion and surrounding DNA sequence. In the present study, strand segregation analysis is used to attempt to identify the role(s) of the Rev1 and Rev7 proteins during TLS. This assay uses double-stranded plasmids containing a genetic marker opposite to a replication blocking lesion (N-2-acetylaminofluorene; AAF) to measure TLS quantitatively and qualitatively in vivo. The AAF adduct is localized within a repetitive sequence in a manner that allows the formation of misaligned primer-template replication intermediates. Elongation from a misaligned intermediate fixes a frameshift mutation (slipped TLS), while extension of the correctly aligned lesion terminus yields error-free (non-slipped) TLS. The results indicate that there is a strong requirement for Rev7p during Pol zeta-mediated TLS measured in vivo. Furthermore, Rev1p is needed only for non-slipped TLS; slipped TLS remains efficient in its absence, revealing a previously uncharacterized Rev1p activity similar to Escherichia coli UmuDC function. Specifically, this activity is required for elongation from a correctly aligned lesion terminus.

DNA-Directed DNA Polymerase↗
PubMed1999

Impaired translesion synthesis in xeroderma pigmentosum variant extracts.

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.

Cell Extracts↗
PubMed1999

Mutation spectrum induced by singlet oxygen in Escherichia coli deficient in exonuclease III.

The repair of singlet oxygen (1O2)-induced DNA lesions requires several enzymes of the nucleotide and base excision repair pathways, including exonuclease III and endonuclease IV that are known apurinic/apyrimidinic-endonucleases in Escherichia coli. In order to better understand the relevance of exonuclease III on the repair of these lesions, we investigated the mutagenic events that result from the replication of a 1O2-damaged plasmid in an exonuclease-deficient host (xth). The mutation spectrum in the tRNA supF gene target indicated that the absence of exonuclease III does not change the types of mutations induced by 1O2 (mostly of G:C-->T:A and G:C-->C:G transversions). However, the spectrum shows that the mutations are scattered in the supF gene, which is significatively different from the one obtained in wild-type bacteria. Thus, exonuclease III may act on the repair of 1O2-induced lesions altering the DNA repair sequence specificity.

Base Sequence↗
PubMed1998

Inactivation of DNA proofreading obviates the need for SOS induction in frameshift mutagenesis.

Translesion synthesis at replication-blocking lesions requires the induction of proteins that are controlled by the SOS system in Escherichia coli. Of the proteins identified so far, UmuD', UmuC, and RecA* were shown to facilitate replication across UV-light-induced lesions, yielding both error-free and mutagenic translesion-synthesis products. Similar to UV lesions, N-2-acetylaminofluorene (AAF), a chemical carcinogen that forms covalent adducts at the C8 position of guanine residues, is a strong replication-blocking lesion. Frameshift mutations are induced efficiently by AAF adducts when located within short repetitive sequences in a two-step mechanism; AAF adducts incorporate a cytosine across from the lesion and then form a primer-template misaligned intermediate that, upon elongation, yields frameshift mutations. Recently, we have shown that although elongation from the nonslipped intermediate depends on functional umuDC+ gene products, elongation from the slipped intermediate is umuDC+-independent but requires another, as yet biochemically uncharacterized, SOS function. We now show that in DNA Polymerase III-proofreading mutant strains (dnaQ49 and mutD5 strains), elongation from the slipped intermediate is highly efficient in the absence of SOS induction-in contrast to elongation from the nonslipped intermediate, which still requires UmuDC functions.

2-Acetylaminofluorene↗
PubMed1998

Expansion of CTG repeats from human disease genes is dependent upon replication mechanisms in Escherichia coli: the effect of long patch mismatch repair revisited.

Many human hereditary disease genes have been recently associated with the expansion of CTG/GAC repeats. We have used a plasmid-based assay in Escherichia coli to investigate the instability of a (CTG/GAC) insert containing 64 repeats. Using this assay, expansions were biochemically detected and subsequently quantified. We show that the occurence of expansions within these trinucleotide repeats is dependent upon replicative mechanisms. Expansions of up to 30 repeats and deletions of almost all possible sizes occured regardless of the orientation of the insert relative to the replication origin. In contradiction to a previous report, the mismatch repair pathway was found to strongly stabilize these repeat stretches.

Cell Survival↗
PubMed1998

A single N-2-acetylaminofluorene adduct alters the footprint of T7 (exo-) DNA polymerase bound to a model primer-template junction.

Bovine pancreatic deoxyribonuclease I (DNaseI) has been used to footprint T7 (exo-) DNA polymerase bound to a model primer-template junction. The polymerase was blocked at a specific position either by the omission of dCTP from the reaction mix or by the presence of a N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dGuo-AAF) adduct. This lesion has been shown to be a severe block for several DNA polymerases, both in in vitro primer elongation experiments, and during the in vivo replication of AAF-monomodified single-stranded vectors. The footprints obtained with unmodified primer-template DNA define two protected domains separated by an inter-region that remains sensitive to DNaseI, and several hypersensitive sites located on both strands. Binding of the polymerase to AAF monomodified duplexes results in the same protection pattern as that obtained with the unmodified duplexes. However, the hypersensitive sites either disappear or are dramatically reduced. The results suggest that the AAF lesion alters the correct positioning of the duplex DNA within the polymerase cleft.

2-Acetylaminofluorene↗
PubMed1998

Mutagenic specificity of the food mutagen 2-amino-3-methylimidazo[4,5-f]quinoline in Escherichia coli using the yeast URA3 gene as a target.

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ), a strong mutagen/carcinogen, belongs to a group of heterocyclic amines that are formed (ng/g amounts) during the cooking of protein containing food. The mutational specificity of IQ in Escherichia coli was determined in a forward mutation assay using the yeast URA3 gene as a target. The plasmid pTU-AC, containing the target URA3, was randomly modified in vitro using N-hydroxy-IQ, and subsequently transformed into an E. coli pyrF strain (DB6656). Mutant clones were directly selected by their ability to grow on medium containing 5-fluoro-orotic acid which is toxic to URA3+ clones and thereby selects for URA3- mutants. Single Strand Conformation Polymorphism (SSCP) was used to map the mutation-containing regions of URA3, so that it was necessary to sequence only the relevant, mutation-containing fragment and not the entire gene. At a modification level of 7 IQ-lesions/URA3 gene, the predominant mutations were base substitutions (approximately 70%), followed by complex gene rearrangements (approximately 20%) and frameshifts (approximately 10%). More than 96% of the base substitutions occurred at G:C base pairs and were predominantly G:C-->A:T transitions, followed by G:C-->T:A and G:C-->C:G transversions. Next neighbour analysis revealed that deoxyguanosines situated within the sequence 5'-TGC were more susceptible to mutations induced by IQ. With one exception, all frameshift mutations were -1 deletions at runs of three consecutive dGs. At higher IQ-modification levels, predominantly complex sequence rearrangements were observed.

Base Sequence↗
PubMed1998

Analysis of damage tolerance pathways in Saccharomyces cerevisiae: a requirement for Rev3 DNA polymerase in translesion synthesis.

The replication of double-stranded plasmids containing a single N-2-acetylaminofluorene (AAF) adduct located in a short, heteroduplex sequence was analyzed in Saccharomyces cerevisiae. The strains used were proficient or deficient for the activity of DNA polymerase zeta (REV3 and rev3delta, respectively) in a mismatch and nucleotide excision repair-defective background (msh2delta rad10delta). The plasmid design enabled the determination of the frequency with which translesion synthesis (TLS) and mechanisms avoiding the adduct by using the undamaged, complementary strand (damage avoidance mechanisms) are invoked to complete replication. To this end, a hybridization technique was implemented to probe plasmid DNA isolated from individual yeast transformants by using short, 32P-end-labeled oligonucleotides specific to each strand of the heteroduplex. In both the REV3 and rev3delta strains, the two strands of an unmodified heteroduplex plasmid were replicated in approximately 80% of the transformants, with the remaining 20% having possibly undergone prereplicative MSH2-independent mismatch repair. However, in the presence of the AAF adduct, TLS occurred in only 8% of the REV3 transformants, among which 97% was mostly error free and only 3% resulted in a mutation. All TLS observed in the REV3 strain was abolished in the rev3delta mutant, providing for the first time in vivo biochemical evidence of a requirement for the Rev3 protein in TLS.

2-Acetylaminofluorene↗
PubMed1997

SOS factors involved in translesion synthesis.

Mutations are permanent DNA sequence changes that can be induced when replication occurs on a damaged DNA template. In Escherichia coli, the process of translesion synthesis past a lesion that hinders replication requires the induction of SOS-controlled gene products, among which are those of the umuDC operon. To study translesion synthesis in vivo, we have constructed single-stranded vectors containing single 2-acetylaminofluorene adducts located within -1 and -2 frameshift mutation hot spots formed by short repetitive sequences. These adducts strongly hinder DNA replication as only 2-5% of the molecules give rise to progeny under non-SOS-induced conditions. Induction of the SOS response lead to a 10-fold increase in survival. Adducts present within repetitive sequences trigger the formation of misaligned primer/template replication intermediates which, upon elongation, will result in the fixation of frameshift errors (mutagenic translesion synthesis). Surprisingly we find that elongation from the nonslipped intermediate depends upon functional umuDC+ gene products, whereas elongation from the slipped intermediate is umuDC+ independent but requires another, as yet biochemically uncharacterized, SOS function. These data are discussed in terms of the different steps involved during translesion synthesis through a replication-blocking lesion.

2-Acetylaminofluorene↗
PubMed1997

Sequence-dependent modulation of nucleotide excision repair: the efficiency of the incision reaction is inversely correlated with the stability of the pre-incision UvrB-DNA complex.

The UvrABC excinuclease is involved in the nucleotide excision repair (NER) pathway. Sequence-dependent differences in repair efficiency have been reported for many different lesions, and it is often suggested that sites with poor repair contribute to the occurrence of mutation hot spots. However, guanine bases modified by N-2-acetylaminofluorence (AAF) within the NarI site (5'-G1G2CG3CC-3') are incised by the UvrABC excinuclease with different efficiencies in a pattern not correlated with the potency of mutation induction. To gain insight into the mechanism of sequence-dependent modulation of NER, we analyzed the formation, the structure and the stability of UvrB-DNA pre-incision complexes formed at all three positions of the AAF-modified NarI site. We show that the efficiency of release of UvrA2 from specific UvrA2B-DNA complexes is sequence-dependent and that the efficiency of incision is inversely related to the stability of the pre-incision complex. We propose that the pre-incision complex, [UvrB-DNA], when formed upon dissociation of UvrA2, undergoes a conformational change (isomerization step) giving rise to an unstable but incision-competent complex that we call [UvrB-DNA]'. The [UvrB-DNA] complex is stable and unable to form an incision-competent complex with UvrC. As the release of UvrA2, this isomerization step is sequence-dependent. Both steps contribute to modulate NER efficiency.

2-Acetylaminofluorene↗
PubMed1997

Adducts formed by the food mutagen 2-amino-3-methylimidazo(4,5-f) quinoline induce frameshift mutations at hot spots through an SOS-independent pathway.

The potency of 2-amino-3-methylimidazo(4,5-f)quinoline (IQ) adducts to induce -2, -1 and +1 frameshift mutations has been determined on specific target DNA sequences, namely short runs of alternating GpC sequences and short runs of guanines. The genetic control of the mutational processes has been analyzed using different Escherichia coli mutants, affected either in the control or in the mutagenesis pathway of the SOS system. We have shown that IQ adducts induce very efficiently both -1 and -2 frameshift mutations in E. coli. Both types of deletion mutations are induced in bacteria without the need of SOS induction, indicating that no LexA-controlled functions, in particular the UmuDC proteins, are required for mutation fixation. We have also shown that the frequency of IQ-induced -2 frameshift mutations in alternating GC sequences increases with the length of the repetition. The efficiency of IQ adducts to induce -1 and -2 frameshift mutations is similar to that of N-2-acetylaminofluorene (AAF) adducts. Both chemicals are potent carcinogens which form covalent adducts at the C8 position of guanines. We suggest that in both cases the adduct-induced DNA structure allows the replication complex to perform a mutagenic bypass of the lesion by a slippage mechanism. However, in contrast to AAF-induced frameshift mutagenesis, IQ-induced frameshift mutagenesis is SOS-independent.

DNA Adducts↗
PubMed1997

Leading versus lagging strand mutagenesis induced by 7,8-dihydro-8-oxo-2'-deoxyguanosine in Escherichia coli.

We have previously shown that a single N-2-acetylaminofluorene (AAF) adduct bound to the C-8 position of a guanine residue located within plasmids containing the unidirectional ColE1 origin of replication induces a 20-fold higher mutation frequency when the adduct is located in the lagging strand as compared to the leading strand. In this study, single 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) lesions have been introduced in the leading and lagging strand orientation within the same sequence context as for the AAF adducts. The induced frequency of guanine to thymine transversions has been measured, using a specific PCR-based quantitative assay, in strains deficient in the repair of the oxidative lesion. The potential involvement of the UvrABC excision repair system in the removal of 8-oxodG has also been investigated and ruled out. Concerning the mutation frequency asymmetry, in contrast to AAF adducts, 8-oxodG adducts induce the same mutation frequency, irrespective of their location in the leading or lagging strands. This striking difference between 8-oxodG and dGuo-C8-AAF adducts is discussed in terms of their differential capacity to block DNA replication.

2-Acetylaminofluorene↗
PubMed1997

Frameshift mutagenesis induced in Escherichia coli after in vitro treatment of double-stranded DNA with methylene blue plus white light: evidence for the involvement of lesion(s) other than 8-oxo-7,8-dihydro-2'-deoxyguanosine.

By means of specific mutation assays, we show here that in vitro treatment of double-stranded plasmid DNA with methylene blue and white light efficiently promotes frameshift mutagenesis in Escherichia coli. The assays detect either -1 or -2 frameshift mutations within previously characterized hot spot sequences for frameshift mutagenesis induced by the chemical carcinogen N-2-acetylaminofluorene, namely, short runs of contiguous guanines and alternating GpC sequences, respectively. The SOS and umuDC dependences of these mutagenic processes have been investigated. Both -1 and -2 frameshift mutagenesis are increased when the host SOS functions are induced. However, and although functional UmuDC proteins are required for maximal mutation induction, the inducibility of both -1 and -2 frameshift mutagenesis is partially independent upon the integrity of the umuDC operon. In addition, results obtained using plasmids with a site specifically located 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dGuo) residue show that this lesion, the major methylene blue plus light induced lesion characterized so far, is inefficient in promoting frameshift mutagenesis. Together, these results led us to conclude that methylene blue plus light treatment of DNA induces, at relatively high rates, lesion(s) other than 8-oxo-dGuo, that efficiently promote(s) frameshift mutagenesis in E. coli.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1997

New strategy for the construction of single-stranded plasmids with single mutagenic lesions.

Single-stranded DNA vectors containing single adducts offer a unique opportunity to study the biochemistry and genetics of trans lesion synthesis, a process during which a DNA polymerase synthesizes across a lesion. We describe a new and general strategy to produce high-quality single-stranded plasmids containing a single adduct within a predetermined sequence context starting with a short oligonucleotide containing the lesion of interest. These vectors are isolated from the corresponding double-stranded constructs by selective enzymatic degradation in vitro of the nonadducted uracil-containing strand. Efficient and complete removal of this strand was achieved using uracil DNA glycosilase to generate AP sites followed by the action of the AP endonuclease associated with exonuclease III and the robust 3'-->5' exonuclease activity associated with T7 DNA polymerase. We show the utility of these constructs for the study of trans lesion synthesis in vitro and in vivo in the case of the highly carcinogenic N-2-acetylaminofluorene adducts located within frameshift mutation hot spots. The possibility to construct both single-stranded and double-stranded plasmids, with the same origin of replication (i.e., ColE1), will allow a direct comparison between single-stranded and double-stranded DNA replication in site-specific mutagenesis studies.

DNA Adducts↗
PubMed1997

Two distinct models account for short and long deletions within sequence repeats in Escherichia coli.

In Escherichia coli, (GpC)n sequences cloned into plasmid DNA molecules are deletion-prone with the occurrence of both short (<2 bp) and long (>2 bp) deletion events. These repetitive tracts can be stabilized by interrupting the strict monotony of the repetition with a variant dinucleotide sequence. The stabilization of short deletion events that is mediated by the variant sequence is completely lost in E. coli mismatch repair-deficient strains. In contrast, this repair pathway has no influence on the frequency of occurrence of long deletion events, even in sequences containing the variant repeat. These results lead us to propose two distinct models to account for short and long deletions within repetitive sequences in E. coli. Furthermore, this study reveals that the deletions occur preferentially at the end of the repeat sequence that is distal with respect to the origin of replication.

DNA Repair↗
PubMed1996

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

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-acetylaminofluorene. Bypass of both lesions was observed only with fork-like substrates, whereas complete inhibition of DNA synthesis occurred on damaged single-stranded DNA substrates. These results suggest a role for additional accessory factors that permit DNA polymerases to bypass lesions when present in fork-like DNA.

2-Acetylaminofluorene↗