Specific amino acid changes enhance the anti‐recombination activity of the UmuD′C complex

Sommer, Suzanne; Coste, Genevièève; Bailone, Adriana

doi:10.1046/j.1365-2958.2000.01809.x

Cited by 16 publications

(15 citation statements)

References 42 publications

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“…The wild-type plasmid was pGY9738, and the empty vector was pGB2. diated homologous recombination by UmuDЈ 2 C occurs via a physical interaction of Pol V with RecA (10,56,(68)(69)(70). Taken together, these observations suggest that a site of interaction between UmuC and RecA is the location on UmuC where the single-stranded DNA template enters UmuC.…”

Section: Discussionmentioning

confidence: 83%

“…UmuDЈ and UmuC inhibit RecA-mediated homologous recombination when present at elevated levels, such as in the SOS response, which may be important for regulating RecA and its involvement in cellular processes (10,56,68,70,74,76). UmuC variants N32A, N33A, and D34A were expressed from the respective derivatives of plasmid pGY9738 in a ⌬umuDC strain.…”

Section: Resultsmentioning

confidence: 99%

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Characterization of Escherichia coli UmuC Active-Site Loops Identifies Variants That Confer UV Hypersensitivity

2011

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DNA is constantly exposed to chemical and environmental mutagens, causing lesions that can stall replication. In order to deal with DNA damage and other stresses, Escherichia coli utilizes the SOS response, which regulates the expression of at least 57 genes, including umuDC. The gene products of umuDC, UmuC and the cleaved form of UmuD, UmuD, form the specialized E. coli Y-family DNA polymerase UmuD 2 C, or polymerase V (Pol V). Y-family DNA polymerases are characterized by their specialized ability to copy damaged DNA in a process known as translesion synthesis (TLS) and by their low fidelity on undamaged DNA templates. Y-family polymerases exhibit various specificities for different types of DNA damage. Pol V carries out TLS to bypass abasic sites and thymine-thymine dimers resulting from UV radiation. Using alanine-scanning mutagenesis, we probed the roles of two active-site loops composed of residues 31 to 38 and 50 to 54 in Pol V activity by assaying the function of single-alanine variants in UV-induced mutagenesis and for their ability to confer resistance to UV radiation. We find that mutations of the N-terminal residues of loop 1, N32, N33, and D34, confer hypersensitivity to UV radiation and to 4-nitroquinoline-N-oxide and significantly reduce Pol V-dependent UV-induced mutagenesis. Furthermore, mutating residues 32, 33, or 34 diminishes Pol V-dependent inhibition of recombination, suggesting that these mutations may disrupt an interaction of UmuC with RecA, which could also contribute to the UV hypersensitivity of cells expressing these variants.Escherichia coli has five DNA polymerases that replicate DNA under different circumstances (22). The replicative polymerase in E. coli is DNA polymerase III (Pol III), a member of the C family. DNA polymerases IV and V are members of the Y family, which specialize in copying damaged DNA in a process known as translesion synthesis (TLS) (22, 47). Y-family DNA polymerases also copy undamaged DNA in an errorprone manner, possibly subjecting DNA to untargeted mutagenesis and potentially leading to antibiotic resistance or cancer (16,17,22,52).E. coli DNA polymerases IV (DinB) and V (UmuDЈ 2 C) are the products of the dinB and umuDC genes, respectively. Due to their potentially mutagenic nature, these proteins are highly regulated in a specific cellular response to DNA damage and other stresses called the SOS response (22,55). The SOS response is initiated when single-stranded DNA (ssDNA) forms downstream from a lesion in DNA due to the inability of the replicative DNA polymerase to copy damaged DNA. RecA then coats the ssDNA to form a RecA-ssDNA nucleoprotein filament, which is the inducing signal for the SOS response. The RecA-ssDNA filament facilitates the autocleavage of LexA, the repressor of the SOS genes, allowing the expression of at least 57 genes (22,66). While many genes are induced in the SOS response, expression of only the umuDC genes is required for SOS mutagenesis (71).The umuDC genes in E. coli encode UmuD, a polymerase manager protein, and UmuC, t...

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Section: Discussionmentioning

confidence: 83%

Section: Resultsmentioning

confidence: 99%

Characterization of Escherichia coli UmuC Active-Site Loops Identifies Variants That Confer UV Hypersensitivity

2011

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“…This observation suggests that, like DinB (29), the steric gate residue of UmuC specifically facilitates translesion synthesis. The UmuC F10L variant, identified in a screen of for UmuDЈC variants that enhance recombination inhibition, essentially lacks translesion synthesis activity (57,59). The Saccharomyces cerevisiae Pol F34L variant, at the position analogous to UmuC F10, displayed dramatically reduced translesion synthesis activity on a template containing a cis-syn cyclobutane pyrimidine dimer while maintaining DNA synthesis activity (42).…”

Section: Discussionmentioning

confidence: 99%

Steric Gate Variants of UmuC Confer UV Hypersensitivity onEscherichia coli

et al. 2009

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Y family DNA polymerases are specialized for replication of damaged DNA and represent a major contribution to cellular resistance to DNA lesions. Although the Y family polymerase active sites have fewer contacts with their DNA substrates than replicative DNA polymerases, Y family polymerases appear to exhibit specificity for certain lesions. Thus, mutation of the steric gate residue of Escherichia coli DinB resulted in the specific loss of lesion bypass activity. We constructed variants of E. coli UmuC with mutations of the steric gate residue Y11 and of residue F10 and determined that strains harboring these variants are hypersensitive to UV light. Moreover, these UmuC variants are dominant negative with respect to sensitivity to UV light. The UV hypersensitivity and the dominant negative phenotype are partially suppressed by additional mutations in the known motifs in UmuC responsible for binding to the ␤ processivity clamp, suggesting that the UmuC steric gate variant exerts its effects via access to the replication fork. Strains expressing the UmuC Y11A variant also exhibit decreased UV mutagenesis. Strikingly, disruption of the dnaQ gene encoding the replicative DNA polymerase proofreading subunit suppressed the dominant negative phenotype of a UmuC steric gate variant. This could be due to a recruitment function of the proofreading subunit or involvement of the proofreading subunit in a futile cycle of base insertion/excision with the UmuC steric gate variant.

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“…These mutant recA alleles result from amino acid substitutions that are located on the surface of the RecA protein close to the head-tail interface between RecA monomers. Subsequently, umu mutant alleles that restore recombination inhibition have been isolated, further illustrating the interplay between Pol V and the RecA filament (26). To maintain a permanent contact between an advancing Pol V molecule and the tip of the RecA filament, we suggest that the RecA filament ''slides back'' in a 3Ј-Ͼ5Ј direction, RecA monomer dissociating from the 5Ј-end of the filament in an ATP-catalyzed reaction (17).…”

Section: Pol V Forms a Stable Initiation Complex On Interaction With Thementioning

confidence: 95%

Biochemical basis for the essential genetic requirements of RecA and the β-clamp in Pol V activation

Fujii

Fuchs

2009

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

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In Escherichia coli, it is genetically well established that the ␤-clamp and RecA are essential cofactors that endow DNA polymerase (Pol) V with lesion bypass activity. However, the biochemical basis for these requirements is still largely unknown. Because the process of translesion synthesis (TLS) requires that the specialized DNA polymerase synthesize in a single binding event a TLS patch that is long enough to resist external proofreading, it is critical to monitor Pol V burst synthesis. Here, we dissect the distinct roles that RecA and the ␤-clamp perform during the Pol V activation process using physiologically relevant long single-stranded template DNA, similar to those used in genetic assays. Our data show that the ␤-clamp endows the complex between Pol V and the template DNA with increased stability. Also, the RecA filament formed in cis on the single-stranded DNA produced downstream from the lesion stretches the template DNA to allow smooth elongation of the nascent strand by Pol V. The concurrent action of both cofactors is required for achieving productive TLS events. The present article presents an integrated view of TLS under physiologically relevant conditions in E. coli that may represent a paradigm for lesion bypass in other organisms. When a replication apparatus encounters a replicationblocking lesion in one of the template strands, the replicative helicase keeps opening the parental duplex, replication continues on the undamaged strand, and a gap is formed in the damaged chromatid opposite the lesion (1, 2). Functional uncoupling of the leading and lagging strand DNA polymerases has been observed both in vivo (3) and in vitro (4, 5). The singlestranded gaps formed opposite the lesions are converted into the molecular signal for the SOS response on formation of a single-stranded ssDNA-RecA filament. Translesion DNA synthesis (TLS) that is largely mediated by DNA polymerase (Pol) V (encoded by the umuDC operon) requires RecA (6-10) and the ␤-clamp, the replication processivity factor (11). In vivo, RecA filament formation is a complex reaction that involves the recombination mediator proteins RecFOR to counteract the high propensity of ssDNA-binding (SSB) protein to bind ssDNA (12). Indeed, RecFOR proteins are essential for efficient Pol V-mediated TLS and mutagenesis both in vitro and in vivo (13). Naked regions of ssDNA are unlikely to persist in vivo even for short time periods as shown during lagging-strand replication (14). Because Pol V-mediated TLS is induced late during the SOS response, the ssDNA gaps generated downstream the replication-blocking lesions are initially covered by SSB and gradually replaced by RecA in a RecFOR mediated reaction path. It is, thus, sensible to include these proteins in Pol V-mediated TLS reconstitution experiments as we showed previously (13). The mechanism by which RecA activates Pol V has been a matter of debate over the years (15), and we will briefly discuss it in the light of a recently published article (16).TLS entails at least two DNA polymerase swi...

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Specific amino acid changes enhance the anti‐recombination activity of the UmuD′C complex

Cited by 16 publications

References 42 publications

Characterization of Escherichia coli UmuC Active-Site Loops Identifies Variants That Confer UV Hypersensitivity

Characterization of Escherichia coli UmuC Active-Site Loops Identifies Variants That Confer UV Hypersensitivity

Steric Gate Variants of UmuC Confer UV Hypersensitivity onEscherichia coli

Biochemical basis for the essential genetic requirements of RecA and the β-clamp in Pol V activation

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