SOS factors involved in translesion synthesis

Napolitano, Rita L.; Lambert, Iain B.; Fuchs, Robert P. P.

doi:10.1073/pnas.94.11.5733

Cited by 56 publications

(75 citation statements)

References 47 publications

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“…A TLS reaction may be viewed as comprising at least two steps: (i) an insertion step, during which a nucleotide is incorporated in the newly synthesized strand across from the lesion. This step generates a key replication intermediate that we refer to as the lesion terminus (LT) (26,27); and (ii) one or several extension step(s) of the LT.…”

Section: Resultsmentioning

confidence: 99%

“…Extension experiments were carried out with a primer͞template substrate that mimics the LT. The 3Ј-end of the primer was located across from the lesion site (primer L0) with a C residue across from the G-AAF adduct, as it was shown both in vivo and in vitro that most of the time G-AAF adducts ''correctly'' direct the insertion of cytosine (26,32). Pol II is able to extend this intermediate generating exclusively bypass product two nucleotides shorter than the one obtained with the lesion-free control template (Fig.…”

Section: Characterization Of the Frameshift Replication Intermediate mentioning

confidence: 99%

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Mechanism of DNA polymerase II-mediated frameshift mutagenesis

Bécherel

Fuchs

2001

Proc. Natl. Acad. Sci. U.S.A.

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Escherichia coli possesses three SOS-inducible DNA polymerases (Pol II, IV, and V) that were recently found to participate in translesion synthesis and mutagenesis. Involvement of these polymerases appears to depend on the nature of the lesion and its local sequence context, as illustrated by the bypass of a single N-2-acetylaminofluorene adduct within the NarI mutation hot spot. Indeed, error-free bypass requires Pol V (umuDC), whereas mutagenic (؊2 frameshift) bypass depends on Pol II (polB). In this paper, we show that purified DNA Pol II is able in vitro to generate the ؊2 frameshift bypass product observed in vivo at the NarI sites. Although the ⌬polB strain is completely defective in this mutation pathway, introduction of the polB gene on a low copy number plasmid restores the ؊2 frameshift pathway. In fact, modification of the relative copy number of polB versus umuDC genes results in a corresponding modification in the use of the frameshift versus error-free translesion pathways, suggesting a direct competition between Pol II and V for the bypass of the same lesion. Whether such a polymerase competition model for translesion synthesis will prove to be generally applicable remains to be confirmed. NarI mutation hot spot ͉ translesion synthesis ͉ slippage mutagenesis ͉ N-2-acetylaminofluorene ͉ umuDC (Pol V) P oint mutations are formed during DNA replication either as genuine replication errors or as a consequence of the presence of damage in the parental DNA. By changing the chemical structure of the bases, damaging agents are often effective blocks to the progression of replicative DNA polymerases. It has now become clear that these blocks are overcome by specialized enzymes (translesional DNA polymerases) that can read through damaged bases in a process known as translesion synthesis (TLS) (1-5). Because of the presence of the lesion, this process is inevitably less accurate than normal replication and will thus trigger increased mutation rates in the newly synthesized strand opposite the damaged base.Recently it was shown that in Escherichia coli, all three SOS-inducible DNA polymerases, Pol II (polB), Pol IV (dinB), and Pol V (umuDC), can be involved in TLS, depending on the nature of the DNA damage and its sequence context (6). We have identified an intriguing situation where the bypass of a given lesion, N-2-acetylaminofluorene (AAF), located within a frameshift mutation hot spot, the NarI site, is mediated by two genetically distinct pathways. Indeed, in that sequence context, the bypass of an AAF guanine adduct requires Pol V for nonslipped elongation, yielding error-free TLS but Pol II for slipped elongation, thus producing Ϫ2 frameshift TLS (6). The nonslipped TLS pathway obeys the rules previously described for base substitution mutagenesis induced by UV light or abasic sites, i.e., Pol V and RecA* dependence (for a recent review, see ref. 7). In contrast, the involvement of DNA Pol II in the slipped Ϫ2 frameshift pathway is more intriguing. Although a series of phenotypes related to DNA repair...

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Section: Resultsmentioning

confidence: 99%

Section: Characterization Of the Frameshift Replication Intermediate mentioning

confidence: 99%

Mechanism of DNA polymerase II-mediated frameshift mutagenesis

Bécherel

Fuchs

2001

Proc. Natl. Acad. Sci. U.S.A.

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“…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).…”

Section: Methodsmentioning

confidence: 99%

“…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). The total number of transformants obtained with both the unmodified and the modified plasmid preparations was the same for both strains (data not shown), thereby eliminating a possible bias in the observations resulting from adduct toxicity.…”

Section: Methodsmentioning

confidence: 99%

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

Baynton

Bresson-Roy

Fuchs

1998

Molecular and Cellular Biology

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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 (REV3 and rev3⌬, respectively) in a mismatch and nucleotide excision repair-defective background (msh2⌬ rad10⌬). 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, 32 P-end-labeled oligonucleotides specific to each strand of the heteroduplex. In both the REV3 and rev3⌬ strains, the two strands of an unmodified heteroduplex plasmid were replicated in ϳ80% 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 rev3⌬ mutant, providing for the first time in vivo biochemical evidence of a requirement for the Rev3 protein in TLS.

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