The active form of DNA polymerase V is UmuD′2C–RecA–ATP

Jiang, Qingfei; Karata, Kiyonobu; Woodgate, Roger; Cox, Michael M.; Goodman, Myron F.

doi:10.1038/nature08178

Cited by 130 publications

(251 citation statements)

References 24 publications

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“…In E. coli, RecA constitutes a key component of the DNA polymerase V "mutasomal complex" comprising UmuD′ 2 C-RecA-ATP (8). Mtb ImuA′ resembles RecA at a structural level, but lacks the characteristic C-terminal domain.…”

Section: Discussionmentioning

confidence: 99%

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Essential roles for imuA ′- and imuB -encoded accessory factors in DnaE2-dependent mutagenesis in Mycobacterium tuberculosis

Warner

Ndwandwe

Abrahams

et al. 2010

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

117

205

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In Mycobacterium tuberculosis (Mtb), damage-induced mutagenesis is dependent on the C-family DNA polymerase, DnaE2. Included with dnaE2 in the Mtb SOS regulon is a putative operon comprising Rv3395c, which encodes a protein of unknown function restricted primarily to actinomycetes, and Rv3394c, which is predicted to encode a Y-family DNA polymerase. These genes were previously identified as components of an imuA-imuB-dnaE2-type mutagenic cassette widespread among bacterial genomes. Here, we confirm that Rv3395c (designated imuA′) and Rv3394c (imuB) are individually essential for induced mutagenesis and damage tolerance. Yeast two-hybrid analyses indicate that ImuB interacts with both ImuA′ and DnaE2, as well as with the β-clamp. Moreover, disruption of the ImuB-β clamp interaction significantly reduces induced mutagenesis and damage tolerance, phenocopying imuA′, imuB, and dnaE2 gene deletion mutants. Despite retaining structural features characteristic of Y-family members, ImuB homologs lack conserved active-site amino acids required for polymerase activity. In contrast, replacement of DnaE2 catalytic residues reproduces the dnaE2 gene deletion phenotype, strongly implying a direct role for the α-subunit in mutagenic lesion bypass. These data implicate differential protein interactions in specialist polymerase function and identify the split imuA′-imuB/dnaE2 cassette as a compelling target for compounds designed to limit mutagenesis in a pathogen increasingly associated with drug resistance. (1), a Cfamily DNA polymerase implicated in error-prone bypass of DNA lesions. Loss of DnaE2 activity renders Mtb hypersensitive to DNA damage and eliminates induced mutagenesis. Moreover, dnaE2 deletion attenuates virulence and reduces the frequency of drug resistance in vivo. Mtb contains two DnaE-type polymerases; the other, DnaE1, provides essential, high-fidelity replicative polymerase function (1). However, the basis for the functional specialization of the DnaE subunits remains unclear (2, 3). Although structural determinants such as active-site architecture contribute significantly to inherent fidelity, it is possible that differential interactions with other DNA metabolic proteins modulate polymerase function.Bacterial genomes containing a DnaE2-type DNA polymerase almost invariably encode a homolog of ImuB (4-6), a putative Yfamily polymerase that is usually present in a LexA-regulated imuA-imuB-dnaE2 gene cassette (5). In Caulobacter crescentus, both ImuB and ImuA are required for induced mutagenesis and damage tolerance (6) whereas plasmid-encoded DnaE2 and ImuB mediate UV-induced mutagenesis in Deinococcus deserti (7). Although distributed widely across the bacterial domain, the imuA-imuB-dnaE2 cassette is not found in organisms possessing umuDC homologs (5). This suggests that the encoded proteins perform an analogous function to DNA polymerase V (8), the Y-family member required for damage-induced mutagenesis in Escherichia coli (9).Mtb contains a putative SOS-inducible operon, Rv3395c-Rv3394c (1, 10), locat...

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Section: Discussionmentioning

confidence: 99%

“…Although distributed widely across the bacterial domain, the imuA-imuB-dnaE2 cassette is not found in organisms possessing umuDC homologs (5). This suggests that the encoded proteins perform an analogous function to DNA polymerase V (8), the Y-family member required for damage-induced mutagenesis in Escherichia coli (9).…”

mentioning

confidence: 95%

Essential roles for imuA ′- and imuB -encoded accessory factors in DnaE2-dependent mutagenesis in Mycobacterium tuberculosis

Warner

Ndwandwe

Abrahams

et al. 2010

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

117

205

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“…Pol III* [(Pol III core) 3 τ 3 δδ′χψ] was reconstituted and purified as described (54). Pol V, RecA, and 30-mer ssDNA were obtained as previously described (12). Pol II containing a His 6 tag was cloned into pET16, expressed, and purified by Ni 2+ affinity chromatography; its activity was the same as that of wt Pol II.…”

Section: Methodsmentioning

confidence: 99%

“…Among these are TLS Pol II (polB) and TLS Pol IV (dinB), which are rapidly (<5 min) up-regulated during the SOS response (7-10). TLS Pol V (UmuD′ 2 C) is induced late (≥45 min) and requires RecA bound to ssDNA (RecA*) for synthetic activity (8,11,12). Pol IV and Pol V belong to the Yfamily Pols (1), whereas Pol II, even though involved in TLS and mutagenesis, is a B-family polymerase (13).…”

mentioning

confidence: 99%

RecA acts as a switch to regulate polymerase occupancy in a moving replication fork

Indiani

Patel

Goodman

et al. 2013

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

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This report discovers a role of Escherichia coli RecA, the cellular recombinase, in directing the action of several DNA polymerases at the replication fork. Bulk chromosome replication is performed by DNA polymerase (Pol) III. However, E. coli contains translesion synthesis (TLS) Pols II, IV, and V that also function with the helicase, primase, and sliding clamp in the replisome. Surprisingly, we find that RecA specifically activates replisomes that contain TLS Pols. In sharp contrast, RecA severely inhibits the Pol III replisome. Given the opposite effects of RecA on Pol III and TLS replisomes, we propose that RecA acts as a switch to regulate the occupancy of polymerases within a moving replisome. Previous studies have shown that all three Pols function at replication forks with DnaB helicase, primase, the β sliding clamp, and the clamp loader (2). Bulk chromosome replication is performed by Pol III, and the mechanism that regulates the access of TLS Pols to the replisome is largely unexplored.Heavy DNA damage in E. coli induces the SOS response, which slows down chromosomal replication (3) and expresses over 40 proteins that aid cell survival and enable genome replication to continue (4, 5). Induction of the SOS response requires formation of ssDNA by replication forks to which the cellular recombinase, RecA, binds. The binding of RecA to ssDNA facilitates RecAmediated self-cleavage of the LexA repressor, thereby activating the SOS response genes (6). Among these are TLS Pol II (polB) and TLS Pol IV (dinB), which are rapidly (<5 min) up-regulated during the SOS response (7-10). TLS Pol V (UmuD′ 2 C) is induced late (≥45 min) and requires RecA bound to ssDNA (RecA*) for synthetic activity (8,11,12). Pol IV and Pol V belong to the Yfamily Pols (1), whereas Pol II, even though involved in TLS and mutagenesis, is a B-family polymerase (13).During normal chromosome duplication, the intracellular levels of Pols II and IV are not sufficient to take over Pol III-based replisomes (2). Upon DNA damage, the induced levels of TLS Pols take over the cellular replicase (2, 14, 15). Our previous studies have shown that Pols II and IV replace Pol III within the replisome without causing fork collapse, as they preserve the DNAbound helicase and sliding clamp to form TLS Pol replisomes (2). Pol II and Pol IV replisomes progress at significantly slower rates than the intrinsic rate of DnaB helicase, and thus regulate the speed of helicase unwinding. Slower fork progression may give the cell additional time to repair DNA lesions using normal repair processes (e.g., nucleotide excision repair), thereby preventing direct encounters of the replication fork with DNA lesions.It has been suggested that polymerase selection at the replication fork is mainly guided by mass action dictated by the concentration of each polymerase and its affinity for the clamp (1,14,16,17). However, the findings of this report demonstrate that RecA adds an additional level of control, acting as a switch that regulates DNA polymerase access to the replicat...

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“…pol V (9) or UmuC (14) has minimal activity in the absence of RecA*. In 2009, we determined that the role of RecA* was to transfer a RecA monomer from the 3Ј-end of the nucleoprotein filament, which, along with a molecule of ATP, "activates" pol V to a mutasome complex, designated as pol V Mut (UmuDЈ 2 C-RecA-ATP) (15).…”

Section: The Origin Of Sos Error-prone Dna Repairmentioning

confidence: 99%

The Discovery of Error-prone DNA Polymerase V and Its Unique Regulation by RecA and ATP

Goodman

2014

Journal of Biological Chemistry

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My career pathway has taken a circuitous route, beginning with a Ph.D. degree in electrical engineering from The Johns Hopkins University, followed by five postdoctoral years in biology at Hopkins and culminating in a faculty position in biological sciences at the University of Southern California. My startup package in 1973 consisted of $2,500, not to be spent all at once, plus an ancient Packard scintillation counter that had a series of rapidly flashing light bulbs to indicate a radioactive readout in counts/minute. My research pathway has been similarly circuitous. The discovery of Escherichia coli DNA polymerase V (pol V) began with an attempt to identify the mutagenic DNA polymerase responsible for copying damaged DNA as part of the well known SOS regulon. Although we succeeded in identifying a DNA polymerase, one that was induced as part of the SOS response, we actually rediscovered DNA polymerase II, albeit in a new role. A decade later, we discovered a new polymerase, pol V, whose activity turned out to be regulated by bound molecules of RecA protein and ATP. This Reflections article describes our research trajectory, includes a review of key features of DNA damage-induced SOS mutagenesis leading us to pol V, and reflects on some of the principal researchers who have made indispensable contributions to our efforts.

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The active form of DNA polymerase V is UmuD′2C–RecA–ATP

Cited by 130 publications

References 24 publications

Essential roles for imuA ′- and imuB -encoded accessory factors in DnaE2-dependent mutagenesis in Mycobacterium tuberculosis

Essential roles for imuA ′- and imuB -encoded accessory factors in DnaE2-dependent mutagenesis in Mycobacterium tuberculosis

RecA acts as a switch to regulate polymerase occupancy in a moving replication fork

The Discovery of Error-prone DNA Polymerase V and Its Unique Regulation by RecA and ATP

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