RecA acts in trans to allow replication of damaged DNA by DNA polymerase V

Schlacher, Katharina; Cox, Michael M.; Woodgate, Roger; Goodman, Myron F.

doi:10.1038/nature05042

Cited by 94 publications

(116 citation statements)

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“…In addition to the roles of RecA in the induction of the SOS response and the cleavage of UmuD, RecA also plays a role in the activation of Pol V for TLS (23,31,58,60,61). While the exact mechanism of Pol V stimulation by RecA remains to be determined, it is understood that RecA is required for Pol V mutagenesis.…”

mentioning

confidence: 99%

“…The ␤ clamp substantially increases processivity in both Pol III and Pol IV in E. coli but increases the processivity of Pol V only 3-to 5-fold (25,35,42,80). The ␤ clamp interacts directly with UmuC utilizing a canonical (4,6,20,41,73) ␤-binding motif, an interaction that is critical for UmuC to participate in TLS (4,6,73).In addition to the roles of RecA in the induction of the SOS response and the cleavage of UmuD, RecA also plays a role in the activation of Pol V for TLS (23,31,58,60,61). While the exact mechanism of Pol V stimulation by RecA remains to be determined, it is understood that RecA is required for Pol V mutagenesis.…”

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

2011

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“…UmuD 2 interacts preferentially with the ␤-processivity subunit of Pol III, whereas UmuDЈ 2 favors the ␣-catalytic subunit (8). RecA:ssDNA interacts with UmuD 2 to promote cleavage to UmuDЈ 2 (3), whereas UmuDЈ 2 C requires trans RecA:ssDNA for efficient TLS (9). UmuD 2 may be degraded by either Lon (10) or ClpXP proteases (11), whereas UmuDЈ 2 must first exchange into the UmuDЈD heterodimer to be degraded by ClpXP (12).…”

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confidence: 99%

“…Both proteins have been shown to interact with UmuC (5), DinB (the Y family DNA Pol IV) (7), and three subunits of the replicative DNA Pol III (8). Additionally, both interact with RecA:ssDNA nucleoprotein filaments (3,9).…”

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Regulation of Escherichia coli SOS mutagenesis by dimeric intrinsically disordered umuD gene products

Simon

Sousa²,

Mohana‐Borges³

et al. 2008

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

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Products of the umuD gene in Escherichia coli play key roles in coordinating the switch from accurate DNA repair to mutagenic translesion DNA synthesis (TLS) during the SOS response to DNA damage. Homodimeric UmuD 2 is up-regulated 10-fold immediately after damage, after which slow autocleavage removes the Nterminal 24 amino acids of each UmuD. The remaining fragment, UmuD 2, is required for mutagenic TLS. The small proteins UmuD2 and UmuD 2 make a large number of specific protein-protein contacts, including three of the five known E. coli DNA polymerases, parts of the replication machinery, and RecA recombinase. We show that, despite forming stable homodimers, UmuD 2 and UmuD 2 have circular dichroism (CD) spectra with almost no ␣-helix or ␤-sheet signal at physiological concentrations in vitro. High protein concentrations, osmolytic crowding agents, and specific interactions with a partner protein can produce CD spectra that resemble the expected ␤-sheet signature. A lack of secondary structure in vitro is characteristic of intrinsically disordered proteins (IDPs), many of which act as regulators. A stable homodimer that lacks significant secondary structure is unusual but not unprecedented. Furthermore, previous single-cysteine cross-linking studies of UmuD 2 and UmuD 2 show that they have a nonrandom structure at physiologically relevant concentrations in vitro. Our results offer insights into structural characteristics of relatively poorly understood IDPs and provide a model for how the umuD gene products can regulate diverse aspects of the bacterial SOS response.natively unfolded ͉ SOS response ͉ unstructured ͉ denatured ͉ DNA repair

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“…2C), rendering a delay of PolV function compared to other members of the SOS regulon (14). Furthermore, the catalytic activation of PolV occurs in trans by RecAˆlaments, i.e., byˆlla-ment present on separate ssDNA molecules from the strand on which PolV becomes active (19). These exquisite and multiple steps regulating the activation of PolV appear to restrict error-prone TLS to the right place and right time when otherwise overwhelming DNA damage needs to be bypassed.…”

Section: Dna Damage Bypass In Prokaryotes; Sos' Signals Rescue Cells mentioning

confidence: 99%

Roadworks of DNA Damage Bypass during and after Replication

Daigaku

2012

Genes and Environment

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Duplication of the genome must be faithfully carried out in proliferating cells. DNA replication is the stage in which DNA damage becomes truly dangerous and potentially causes cell death or genomic instability. DNA damage bypass mechanisms have evolved as the`last minute' processes to protect the quality of genome replication from these risks. Damage bypass provides a highly ‰exible mechanism to tolerate various types of DNA damage during replication. Recent studies have highlighted that bypass mechanisms can be uncoupled from global genome replication: i.e., the time of action of DNA damage bypass is notˆxed at a particular point during genome replication. Although DNA damage bypass mechanisms are conserved throughout organisms, their regulation is diŠerent between prokaryotes and eukaryotes. On one hand, the bypass mechanisms of prokaryotes are mainly dependent on upregulation of transcripts under the SOS regulon. On the other hand, in eukaryotes, DNA damage bypass is activated by ubiquitylation of the replication sliding clamp, PCNA. This review starts with our understanding of the basis of lesion-bypassing mechanisms in the bacterial system, advances to recent views of the molecular mechanisms underlying eukaryotic DNA damage bypass and speciˆcally focuses on how the bypassing mechanisms provide temporally and structurally ‰exible functions.

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RecA acts in trans to allow replication of damaged DNA by DNA polymerase V

Cited by 94 publications

References 29 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

Regulation of Escherichia coli SOS mutagenesis by dimeric intrinsically disordered umuD gene products

Roadworks of DNA Damage Bypass during and after Replication

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