Cellular strategies for accommodating replication-hindering adducts in DNA: control by the SOS response in Escherichia coli.

Koffel-Schwartz, Nicole; Coin, Frédéric; Veaute, Xavier; Fuchs, Robert P. P.

doi:10.1073/pnas.93.15.7805

Cited by 61 publications

(95 citation statements)

References 51 publications

(62 reference statements)

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“…This construction, introduced into SOS-induced bacteria by electroporation, confers a LacZ Ϫ phenotype that can be reverted to LacZ ϩ by a Ϫ2 frameshift mutation (22,23). SOS induction was achieved by prior UV irradiation at a dose of 50 J͞m 2 , as previously described (24). The fraction of Ϫ2 frameshift TLS events is determined as the fraction of blue colonies among all transformants on indicator plates containing carbenicillin, 5-bromo-4-chloro-3-indolyl ␤-Dgalactoside, and isopropyl ␤-D-thiogalactoside.…”

Section: Methodsmentioning

confidence: 99%

“…Colonies resulting from either error-free TLS or damage avoidance events form white colonies on these plates. Among the white colonies, the fraction of colonies resulting from error-free TLS is determined by using the colony hybridization assay developed previously (22,24). The majority of colonies represent colonies where plasmid replication occurred via damage avoidance (22,24,25).…”

Section: Methodsmentioning

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: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Mechanism of DNA polymerase II-mediated frameshift mutagenesis

Bécherel

Fuchs

2001

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

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“…As a consequence, we developed a methodology that allows TLS to be studied within cells (E. coli, yeast, mammalian cells) (Figure 4) (Baynton et al, 1998;Koffel-Schwartz et al, 1996). Analysis of translesion synthesis in vivo is made possible by the use of double stranded plasmids containing a single adduct located within a short heteroduplex sequence.…”

Section: How To Analyse Tls and Mutagenesis In Vivo?mentioning

confidence: 99%

“…A colony that hybridizes with the target and marker strand probes is scored as a TLS colony (Figure 4, bottom left). Our assay also allows discriminating between error-free and mutagenic TLS events either phenotypically or by sequencing (Baynton et al, 1998;Koffel-Schwartz et al, 1996). A colony that hybridizes only with the marker strand specific probe result from Damage Avoidance pathways, such as daughter strand gap repair by recombination or DNA polymerase template switch (Figure 4, bottom, right).…”

Section: How To Analyse Tls and Mutagenesis In Vivo?mentioning

confidence: 99%

How DNA lesions are turned into mutations within cells?

2002

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Genomes of all living organisms are constantly injured by endogenous and exogenous agents that modify the chemical integrity of DNA and in turn challenge its informational content. Despite the efficient action of numerous repair systems that remove lesions in DNA in an error-free manner, some lesions, that escape these repair mechanisms, are present when DNA is being replicated. Although replicative DNA polymerases are usually unable to copy past such lesions, it was recently discovered that cells are equipped with specialized DNA polymerases that will assist the replicative polymerase during the process of Translesion Synthesis (TLS). These TLS polymerases exhibit relaxed fidelity that allows them to copy past lesions in DNA with an inherent risk of generating mutations at high frequency. We present recent aspects related to the genetics and biochemistry of TLS and highlight some of the remaining hot topics of this field.

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“…In contrast to error-free repair, damage-inducible TLS generates a significant mutational load (17). Most TLS depends on the damage inducible UmuD 2 Ј and UmuC proteins, which heterotrimerize to form E. coli pol V (UmuD 2 Ј C; refs.…”

Section: Coping With Dna Damage In E Colimentioning

confidence: 99%

Roles of DNA polymerases V and II in SOS-induced error-prone and error-free repair in Escherichia coli

Pham

Rangarajan

Woodgate

et al. 2001

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

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DNA polymerase V, composed of a heterotrimer of the DNA damage-inducible UmuC and UmuD 2 proteins, working in conjunction with RecA, single-stranded DNA (ssDNA)-binding protein (SSB), ␤ sliding clamp, and ␥ clamp loading complex, are responsible for most SOS lesion-targeted mutations in Escherichia coli, by catalyzing translesion synthesis (TLS). DNA polymerase II, the product of the damage-inducible polB (dinA ) gene plays a pivotal role in replication-restart, a process that bypasses DNA damage in an error-free manner. Replication-restart takes place almost immediately after the DNA is damaged (Ϸ2 min post-UV irradiation), whereas TLS occurs after pol V is induced Ϸ50 min later. We discuss recent data for pol V-catalyzed TLS and pol II-catalyzed replicationrestart. Specific roles during TLS for pol V and each of its accessory factors have been recently determined. Although the precise molecular mechanism of pol II-dependent replication-restart remains to be elucidated, it has recently been shown to operate in conjunction with RecFOR and PriA proteins.T wo seemingly unconnected questions arising during the early and mid 1970s were to decipher the biochemical basis of SOS mutagenesis in Escherichia coli, often referred to as SOS errorprone repair (1), and to determine a cellular role for E. coli DNA polymerase II. A tentative link between the two was established when it was determined that pol II was induced as part of the LexA-regulon (2). pol II was subsequently shown to be encoded by the DNA damage-inducible polB (dinA) gene (3-5). A ⌬polB strain shows no measurable UV sensitivity, and SOS-induced mutagenesis occurs at normal levels (6, 7). However, a ⌬polB ⌬umuDC double mutant strain is more sensitive to killing by UV light than either of the single mutant strains, implying that the two SOS-induced polymerases might play compensatory roles in vivo (8).The ability of pols II and V to complement each other does not mean that these activities are functionally redundant, and indeed they are not. pol V is able to copy UV-damaged DNA in a process referred to as error-prone translesion synthesis (TLS). TLS generates mutations targeted specifically to DNA template damage sites (9-12). In contrast, pol II copies chromosomal DNA during error-free replication-restart (8). Although both polymerases are induced by DNA damage, they appear to function on widely disparate time frames-pol II-catalyzed replication-restart occurs 2 min post-UV irradiation whereas pol V-catalyzed TLS begins roughly 50 min later (8). In this paper, we discuss current models for the roles of pol V in TLS and pol II in replication-restart.Coping with DNA Damage in E. coli There are over 40 genes induced on DNA damage in E. coli that have been identified recently by using microarray chip technology (13), of which at least 31 are known to be negatively regulated at the transcriptional level by the LexA protein (14). Many of these genes encode proteins required to repair DNA damage (15). The overriding importance of DNA repair is apparent from the ob...

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Cellular strategies for accommodating replication-hindering adducts in DNA: control by the SOS response in Escherichia coli.

Cited by 61 publications

References 51 publications

Mechanism of DNA polymerase II-mediated frameshift mutagenesis

Mechanism of DNA polymerase II-mediated frameshift mutagenesis

How DNA lesions are turned into mutations within cells?

Roles of DNA polymerases V and II in SOS-induced error-prone and error-free repair in Escherichia coli

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