The Dimeric SOS Mutagenesis Protein UmuD Is Active as a Monomer

Ollivierre, Jaylene N.; Sikora, Jacquelyn L.; Beuning, Penny J.

doi:10.1074/jbc.m110.167254

Cited by 14 publications

(22 citation statements)

References 68 publications

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“…In E. coli , UmuD forms dimers that cleaves intermolecularly (McDonald et al ., ), although recent evidence shows that E. coli UmuD can cleave intramolecularly, albeit only when a specific mutation is engineered into UmuD to prevent homodimerization (Ollivierre et al ., ). However, we found that UmuDAb, unlike UmuD, does not cleave intermolecularly, although UmuDAb contains the conserved asparagine required for UmuD dimerization (Ollivierre et al ., ). In this respect, UmuDAb naturally behaves like a monomer, although its homology to other self‐cleaving serine proteases supports the hypothesis that it may dimerize.…”

Section: Resultsmentioning

confidence: 97%

The Acinetobacter regulatory UmuDAb protein cleaves in response to DNA damage with chimeric LexA/UmuD characteristics

Hare

Adhikari

Lambert

et al. 2012

FEMS Microbiol Lett

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In the DNA damage response of most bacteria, UmuD forms part of the error‐prone (UmuD′2)C polymerase V and is activated for this function by self‐cleavage after DNA damage. However, the umuD homolog (umuDAb) present throughout the Acinetobacter genus encodes an extra N‐terminal region, and in Acinetobacter baylyi, regulates transcription of DNA damage–induced genes. UmuDAb expressed in cells was correspondingly larger (24 kDa) than the Escherichia coli UmuD (15 kDa). DNA damage from mitomycin C or UV exposure caused UmuDAb cleavage in both E. coli wild‐type and ΔumuD cells on a timescale resembling UmuD, but did not require UmuD. Like the self‐cleaving serine proteases LexA and UmuD, UmuDAb required RecA for cleavage. This cleavage produced a UmuDAb′ fragment of a size consistent with the predicted cleavage site of Ala83–Gly84. Site‐directed mutations at Ala83 abolished cleavage, as did mutations at either the Ser119 or Lys156 predicted enzymatic residues. Co‐expression of the cleavage site mutant and an enzymatic mutant did not allow cleavage, demonstrating a strictly intramolecular mechanism of cleavage that more closely resembles the LexA‐type repressors than UmuD. These data show that UmuDAb undergoes a post‐translational, LexA‐like cleavage event after DNA damage, possibly to achieve its regulatory action.

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Section: Resultsmentioning

confidence: 97%

The Acinetobacter regulatory UmuDAb protein cleaves in response to DNA damage with chimeric LexA/UmuD characteristics

Hare

Adhikari

Lambert

et al. 2012

FEMS Microbiol Lett

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“…3C). We previously observed that plasmid-borne umuC can suppress the extreme sensitivity to UV that is characteristic of strains in which both umuC and recJ have been deleted (19,48). RecJ helps to partially degrade DNA at replication forks that are stalled due to lesions to help aid in restart of DNA replication.…”

Section: Resultsmentioning

confidence: 99%

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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“…Recent work suggests that UmuD′ can be active as a monomer [90]. It is not unreasonable to imagine that an UmuD′C/RecA complex lacking distal UmuD′ (darker green in Figure 6) might still retain activity.…”

Section: Resultsmentioning

confidence: 99%

Structural model of the Y-Family DNA polymerase V/RecA mutasome

Chandani¹,

Loechler²

2013

Journal of Molecular Graphics and Modelling

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To synthesize past DNA damaged by chemicals or radiation, cells have lesion bypass DNA polymerases (DNAPs), most of which are in the Y-Family. One class of Y-Family DNAPs includes DNAP η in eukaryotes and DNAP V in bacteria, which have low fidelity when replicating undamaged DNA. In E. coli, DNAP V is carefully regulated to insure it is active for lesion bypass only, and one mode of regulation involves interaction of the polymerase subunit (UmuC) and two regulatory subunits (UmuD′) with a RecA-filament bound to ss-DNA. Taking a docking approach, ~150,000 unique orientations involving UmuC, UmuD′ and RecA were evaluated to generate models, one of which was judged best able to rationalize the following published findings. (1) In the UmuD′2C/RecA-filament model, R64-UmuC interacts with S117-RecA, which is known to be at the UmuC/RecA interface. (2) At the model’s UmuC/RecA interface, UmuC has three basic amino acids (K59/R63/R64) that anchor it to RecA. No other Y-Family DNAP has three basic amino acids clustered in this region, making it a plausible site for UmuC to form its unique interaction with RecA. (3) In the model, residues N32/N33/D34 of UmuC form a second interface with RecA, which is consistent with published findings. (4) Active UmuD′ is generated when 24 amino acids in the N-terminal tail of UmuD are proteolyzed, which occurs when UmuD2C binds the RecA-filament. When UmuD is included in an UmuD2C/RecA-filament model, plausible UmuD/RecA contacts guide the UmuD cleavage site (C24/G25) into the UmuD proteolysis active site (S60/K97). One contact involves E11-UmuD interacting with R243-RecA, where the latter is known to be important for UmuD cleavage. (5) The UmuD2C/RecA-filament model rationalizes published findings that at least some UmuD-to-UmuD′ cleavage occurs intermolecularly. (6) Active DNAP V is known to be the heterotetramer UmuD′2C/RecA, a model of which can be generated by a simple rearrangement of the RecA monomer at the 3′-end of the RecA-filament. The rearranged UmuD′2C/RecA model rationalizes published findings about UmuD′ residues in proximity to RecA. In summary, docking and molecular simulations are used to develop an UmuD′2C/RecA model, whose structure rationalizes much of the known properties of the active form of DNA polymerase V.

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The Dimeric SOS Mutagenesis Protein UmuD Is Active as a Monomer

Cited by 14 publications

References 68 publications

The Acinetobacter regulatory UmuDAb protein cleaves in response to DNA damage with chimeric LexA/UmuD characteristics

The Acinetobacter regulatory UmuDAb protein cleaves in response to DNA damage with chimeric LexA/UmuD characteristics

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

Structural model of the Y-Family DNA polymerase V/RecA mutasome

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