A universal protein–protein interaction motif in the eubacterial DNA replication and repair systems

Dalrymple, Brian P.; Kongsuwan, Kritaya; Wijffels, Gene; Dixon, Nicholas E.; Jennings, Peter Andrew

doi:10.1073/pnas.191384398

Cited by 312 publications

(390 citation statements)

References 42 publications

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“…The three SOS-inducible DNA polymerases (II, IV and V) contain a short peptide (consensus motif: QLxLF) that was identified in a yeast two-hybrid screen to mediate their interaction with the b-clamp (Dalrymple et al, 2001). As these three polymerases are known to be involved in lesion bypass, we wanted to analyse the functional role of Figure 6 Competing bypass pathways: a single AAF adduct may be bypassed in an error-free manner by Pol V or yield a -2 frameshift product when Pol II is involved Fuchs et al, 2001;Napolitano et al, 2000).…”

Section: The Emerging Complexity Of Tls Pathways In Vivomentioning

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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Section: The Emerging Complexity Of Tls Pathways In Vivomentioning

confidence: 99%

How DNA lesions are turned into mutations within cells?

2002

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“…Rv3394c (ImuB) contains a predicted β-clamp-binding motif, which designates the protein as a DinB3-type Y-family polymerase (11). Notably, neither DnaE2 nor ImuA′ possesses an identifiable clamp-binding motif (1,12). The β-clamp modulates the recruitment to the replication machinery of proteins involved in bacterial DNA replication and repair (13), binding replicative and specialist lesion bypass polymerases simultaneously to enable rapid interchange (14,15).…”

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confidence: 99%

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.

120

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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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“…Pol III consists of ␣, the catalytic polymerase subunit associated with the ⑀ 3Ј 3 5Ј exonuclease, and (22). Pol III gains its special replicative properties by its ability to associate with ␤ and through interactions enabled by sequences located in the carboxyl-terminal third of the ␣ subunit (23)(24)(25)(26).…”

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confidence: 99%

DNA Polymerase III Holoenzyme from Thermus thermophilus Identification, Expression, Purification of Components, and Use to Reconstitute a Processive Replicase

Bullard¹,

Williams²,

Acker³

et al. 2002

Journal of Biological Chemistry

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DNA replication in bacteria is performed by a specialized multicomponent replicase, the DNA polymerase III holoenzyme, that consist of three essential components: a polymerase, the ␤ sliding clamp processivity factor, and the DnaX complex clamp-loader. We report here the assembly of the minimal functional holoenzyme from Thermus thermophilus (Tth), an extreme thermophile. The minimal holoenzyme consists of ␣ (pol III catalytic subunit), ␤ (sliding clamp processivity factor), and the essential DnaX ( /␥), ␦ and ␦ components of the DnaX complex. We show with purified recombinant proteins that these five components are required for rapid and processive DNA synthesis on long single-stranded DNA templates. Subunit interactions known to occur in DNA polymerase III holoenzyme from mesophilic bacteria including ␦-␦ interaction, ␦␦ -/␥ complex formation, and ␣-interaction, also occur within the Tth enzyme. As in mesophilic holoenzymes, in the presence of a primed DNA template, these subunits assemble into a stable initiation complex in an ATP-dependent manner. However, in contrast to replicative polymerases from mesophilic bacteria, Tth holoenzyme is efficient only at temperatures above 50°C, both with regard to initiation complex formation and processive DNA synthesis. The minimal Tth DNA polymerase III holoenzyme displays an elongation rate of 350 bp/s at 72°C and a processivity of greater than 8.6 kilobases, the length of the template that is fully replicated after a single association event.DNA replication in all biological systems is performed by specialized multiprotein replicases (1, 2). Cellular replicases consist of three major subassemblies: a sliding clamp processivity factor, a clamp loader, and a specialized polymerase. Replicases, especially bacterial replicases, are rapid and processive consistent with the requirement for them to synthesize a several megabase genome from a single origin in less than one hour.In the prototypic Escherichia coli replication system, a key determinant of processive DNA synthesis is the interaction between the ␤ processivity factor and pol III 1 (3, 4). The dimeric ␤ subunit is a bracelet-shaped molecule that clamps around DNA permitting it to rapidly slide along duplex DNA without dissociating (5). ␤ binds to the pol III ␣ subunit through protein-protein contacts preventing the polymerase from dissociating from the template, ensuring high processivity. Efficient loading of the ␤ subunit onto DNA requires ATP-dependent opening and closing of the clamp by the DnaX complex. The DnaX complex contains the essential DnaX, ␦ and ␦Ј subunits plus two ancillary proteins, and (6 -9). The dnaX gene encodes two proteins, ␥ and , by programmed ribosomal frameshifting (10 -15). Both and the shorter ␥ product share ATPbinding domain I, domain II, and domain III that is responsible for DnaX oligomerization, -binding, and binding of ␦-␦Ј (16 -19). contains two unique domains. domain IV forms a link with the DnaB helicase and domain V binds pol III (17,20,21). Pol III consists of ␣, the catalyti...

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A universal protein–protein interaction motif in the eubacterial DNA replication and repair systems

Cited by 312 publications

References 42 publications

How DNA lesions are turned into mutations within cells?

How DNA lesions are turned into mutations within cells?

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

DNA Polymerase III Holoenzyme from Thermus thermophilus Identification, Expression, Purification of Components, and Use to Reconstitute a Processive Replicase

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