A peptide switch regulates DNA polymerase processivity

Saro, Francisco J. López de; Georgescu, Roxana E.; O'Donnell, Mike

doi:10.1073/pnas.2435454100

Cited by 76 publications

(95 citation statements)

References 34 publications

(36 reference statements)

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“…Unlike Y-family polymerases whose structures are adapted to specialist lesion bypass (20), sequence analysis reveals few clues to DnaE2 function. All major DNA polymerase IIIα structural/functional domains (23,24) are readily identified in DnaE2, except for the very C-terminal region which in E. coli has been implicated in the interaction of α with the clamploader subunit, τ (28,29). A strong α-τ interaction enables simultaneous leading and lagging-strand synthesis by the DNA polymerase III holoenzyme (13), and the absence of this region in DnaE2 and all other nonessential dnaE-type α-subunits (Fig.…”

Section: Discussionmentioning

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

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%

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

205

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“…The solutions were removed, and the wells were washed four times with 100 l of buffer C, air-dried, and analyzed using a PhosphorImager. Steady-state Fluorescence Measurements-The E. coli ␤ subunit was labeled at Cys 333 using Oregon Green 488 maleimide (Molecular Probes, Inc., Eugene, OR) to form ␤ OG as described previously (58). Peptide titrations contained ␤ OG at a concentration of 500 nM, 1 M, or 2 M in 60 l of 20 mM Tris-HCl (pH 7.5), 5 mM DTT, 1 mM EDTA, and 50 mM NaCl.…”

Section: Protein-protein Interaction Analysis Using 96-well Microtitermentioning

confidence: 99%

“…This stimulation is species-specific and therefore relies on specific amino acid contacts between the subunit and the DNA polymerase (58,64). may enhance polymerase activity by increasing the affinity of the polymerase for DNA because binds to both DNA and the polymerase.…”

Section: The S Aureus Polc-interaction Is Weak Compared With the E mentioning

confidence: 99%

Conserved Interactions in the Staphylococcus aureus DNA PolC Chromosome Replication Machine

Bruck

Georgescu

O'Donnell

2005

Journal of Biological Chemistry

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The PolC holoenzyme replicase of the Gram-positive Staphylococcus aureus pathogen has been reconstituted from pure subunits. We compared individual S. aureus replicase subunits with subunits from the Gram-negative Escherichia coli polymerase III holoenzyme for activity and interchangeability. The central organizing subunit, , is smaller than its Gram-negative homolog, yet retains the ability to bind single-stranded DNA and contains DNA-stimulated ATPase activity comparable with E. coli . S. aureus also stimulates PolC, although they do not form as stabile a complex as E. coli polymerase III⅐. We demonstrate that the extreme C-terminal residues of PolC bind to and function with ␤ clamps from different bacteria. Hence, this polymerase-clamp interaction is highly conserved. Additionally, the S. aureus ␦ wrench of the clamp loader binds to E. coli ␤. The S. aureus clamp loader is even capable of loading E. coli and Streptococcus pyogenes ␤ clamps onto DNA. Interestingly, S. aureus PolC lacks functionality with heterologous ␤ clamps when they are loaded onto DNA by the S. aureus clamp loader, suggesting that the S. aureus clamp loader may have difficulty ejecting from heterologous clamps. Nevertheless, these overall findings underscore the conservation in structure and function of Gram-positive and Gram-negative replicases despite >1 billion years of evolutionary distance between them.The Escherichia coli chromosomal replicase, DNA polymerase III holoenzyme, is extremely rapid (ϳ1 kb/s) and extends DNA by thousands of nucleotides without dissociating from the DNA template (1-3). These special features require accessory proteins, as the polymerase subunit alone is only weakly active in synthesis and lacks high processivity. The accessory proteins act as a clamp loader ATPase complex and a ring-shaped subunit that functions as a DNA sliding clamp (␤). The ␤ clamp encircles the DNA duplex and binds the polymerase, tethering it to DNA for rapid and processive chain extension. The clamp loader uses energy derived from ATP hydrolysis to pry open the ␤ clamp and to close it around a primed template.The E. coli ␥/ clamp loader consists of five proteins required for clamp loading ( 2 ␥ 1 ␦ 1 ␦Ј 1 ) and also the attached and ancillary subunits (4 -7). In E. coli, the / subunits connect the replicase to single-stranded DNA-binding protein (SSB), 1 but are not required for clamp loading (8 -10). The / subunits also contribute to the stability of the ␥/ complex in vitro (11). In E. coli, the dnaX gene produces two polypeptides, and ␥.(71 kDa) is the full-length product of the gene, whereas ␥ (47 kDa) is a truncated version of , produced by a translational frameshift (12)(13)(14). Both and ␥ are capable of functioning with ␦ and ␦Ј in clamp loading action (15). However, the unique C-terminal 24-kDa C-terminal region of provides extra functions relative to ␥. For example, (but not ␥) binds to the polymerase directly (16). Hence, the presence of multiple subunits within the clamp loader enables it to cross-link two polymerases, ther...

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“…Second, the tetranucleotide synthesized by T7 DNA primase leaves only a nick between Okazaki fragments, at which point T7 DNA polymerase dissociates from the DNA. The 5′-attached tRNA may require strand-displacement synthesis, thus interfering with the coordination mechanism (22). Despite those shortcomings, tRNA may serve as a backup source of primers and might even be used for priming at the chromosomal origin of replication.…”

Section: Discussionmentioning

confidence: 99%

Gene 5.5 protein of bacteriophage T7 in complex with Escherichia coli nucleoid protein H-NS and transfer RNA masks transfer RNA priming in T7 DNA replication

Zhu

Lee

Tan

et al. 2012

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

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DNA primases provide oligoribonucleotides for DNA polymerase to initiate lagging strand synthesis. A deficiency in the primase of bacteriophage T7 to synthesize primers can be overcome by genetic alterations that decrease the expression of T7 gene 5.5, suggesting an alternative mechanism to prime DNA synthesis. The product of gene 5.5 (gp5.5) forms a stable complex with the Escherichia coli histone-like protein H-NS and transfer RNAs (tRNAs). The 3′-terminal sequence (5′-ACCA-3′) of tRNAs is identical to that of a functional primer synthesized by T7 primase. Mutations in T7 that suppress the inability of primase reduce the amount of gp5.5 and thus increase the pool of tRNA to serve as primers. Alterations in T7 gene 3 facilitate tRNA priming by reducing its endonuclease activity that cleaves at the tRNA-DNA junction. The tRNA bound to gp5.5 recruits H-NS. H-NS alone inhibits reactions involved in DNA replication, but the binding to gp5.5-tRNA complex abolishes this inhibition.A 3′-hydroxyl end of an RNA or DNA strand positioned at the catalytic site of DNA polymerase functions as a primer to initiate synthesis of DNA. In most instances, primers are short oligoribonucleotides synthesized by enzymes designated DNA primases (1). DNA primases catalyze the template-directed synthesis of short oligoribonucleotides and stabilize the newly synthesized primer on the template and hand it off to DNA polymerase for initiation of DNA synthesis (2, 3).Bacteriophage T7 has provided a model system for the study of DNA replication. Only three T7 proteins-gene 5 DNA polymerase (gp5), gene 4 primase-helicase (gp4), and gene 2.5 ssDNAbinding protein (gp2.5)-together with host Escherichia coli thioredoxin, are sufficient for leading and lagging strands synthesis in a reconstituted replication system (4). The genes encoding these proteins, together with genes 2 (E. coli RNA polymerase inhibitor), 3 (endonuclease), 3.5 (lysozyme), and 6 (exonuclease) constitute the DNA metabolism class II genes. More than a dozen additional genes within the class II group remain uncharacterized (5).The primase activity of T7 is located in the N-terminal half of the 63-kDa gp4, whereas the C-terminal half of gene 4 encodes the DNA helicase (4). Like other prokaryotic homologs, the primase domain recognizes a specific sequence (1, 6) 5′-(G/T)(G/T) GTC-3′ in the ssDNA template to catalyze the template-directed synthesis of functional tetraribonucleotides (pppACCA, pppACCC, and pppACAC) that the lagging strand DNA polymerase can use as primers to initiate the synthesis of Okazaki fragments. Gene 4 encodes two colinear proteins, the full-length 63-kDa gp4 and a short 56-kDa form in equal amount in vivo from an internal start codon and ribosome-binding site (4). This 56-kDa gp4 has full helicase activity but lacks the N-terminal zinc-binding domain (ZBD), an indispensable motif for primer synthesis (7). Despite its inability to synthesize template-dependent tetraribonucleotides, the 56-kDa gp4 is capable of stabilizing certain preformed oligonucleotides on ...

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A peptide switch regulates DNA polymerase processivity

Cited by 76 publications

References 34 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

Conserved Interactions in the Staphylococcus aureus DNA PolC Chromosome Replication Machine

Gene 5.5 protein of bacteriophage T7 in complex with Escherichia coli nucleoid protein H-NS and transfer RNA masks transfer RNA priming in T7 DNA replication

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