DNA Binding of PriA Protein Requires Cooperation of the N-terminal D-loop/Arrested-fork Binding and C-terminal Helicase Domains

Tanaka, Taku; Mizukoshi, Toshimi; Taniyama, Chika; Kohda, Daisuke; Arai, Ken; Masai, Hisao

doi:10.1074/jbc.m204397200

Cited by 41 publications

(92 citation statements)

References 48 publications

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“…The first is the 3′BD, which has been shown previously to bind the 3′ end of the leading-strand arm of replication fork structures (26). This recognition is thought to help direct PriA replisomal reloading activity to appropriate DNA structures (25)(26)(27)(28). The 3′BD structure from full-length KpPriA is strikingly similar to the previously determined structure of the isolated 3′BD from EcPriA (26) (rmsd = 1.8 Å for 90 common C α atoms).…”

Section: Resultssupporting

confidence: 64%

“…Similar ARLs in RecQ and PcrA DNA helicases bind directly to ssDNA and couple binding to structural changes that stimulate ATPase and helicase activities (30,31). Mutations within this region of EcPriA block binding to D-loop DNA structures (25), which is consistent with the ARL having a direct role in DNA binding in PriA. The second feature is an extended helicase motif V that positions the side chain of Lys543 between the β-phosphate of ADP and the carboxyl group of Asp319 from helicase motif II (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Biochemical studies have identified a two-domain architecture for PriA that includes a DNA-binding domain (DBD; N-terminal ∼200 residues) and a helicase domain (HD; C-terminal ∼530 residues) (18,21,25) (Fig. 1A).…”

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confidence: 99%

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Structural mechanisms of PriA-mediated DNA replication restart

Bhattacharyya

George

Thurmes

et al. 2013

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

100

205

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Collisions between cellular DNA replication machinery (replisomes) and damaged DNA or immovable protein complexes can dissociate replisomes before the completion of replication. This potentially lethal problem is resolved by cellular "replication restart" reactions that recognize the structures of prematurely abandoned replication forks and mediate replisomal reloading. In bacteria, this essential activity is orchestrated by the PriA DNA helicase, which identifies replication forks via structure-specific DNA binding and interactions with fork-associated ssDNA-binding proteins (SSBs). However, the mechanisms by which PriA binds replication fork DNA and coordinates subsequent replication restart reactions have remained unclear due to the dearth of high-resolution structural information available for the protein. Here, we describe the crystal structures of full-length PriA and PriA bound to SSB. The structures reveal a modular arrangement for PriA in which several DNA-binding domains surround its helicase core in a manner that appears to be poised for binding to branched replication fork DNA structures while simultaneously allowing complex formation with SSB. PriA interaction with SSB is shown to modulate SSB/DNA complexes in a manner that exposes a potential replication initiation site. From these observations, a model emerges to explain how PriA links recognition of diverse replication forks to replication restart.X-ray crystal structure | single-molecule FRET

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Section: Resultssupporting

confidence: 64%

Section: Resultsmentioning

confidence: 99%

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Structural mechanisms of PriA-mediated DNA replication restart

Bhattacharyya

George

Thurmes

et al. 2013

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

100

205

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“…Proteins-Wild-type or the LFY mutant (L12G, F16G, Y18G) form of the full-length PriA protein and PriA-(1-105), PriA-(1-181), or PriA- ) N-terminal polypeptides were expressed and purified as previously described (22,24,28). The expression vectors for the C-terminal polypeptide, PriA-(194 -732) and PriA-(109 -181), were constructed by insertion of the PCR fragments amplified from plasmid carrying wild-type PriA sequence (22) using a primer set (sense, 5Ј-gcaactatcatatgggtgagcggttgcgattgaat-3Ј and antisense, 5Ј-ttaggatccttaaccctcaatcggatcaaca-3Ј and sense, 5Ј-gcaactatcatatgcctgcggcgaacgcg-3Ј and antisense, 5Ј-attaggatcctcatggtgtttcacttgc-3Ј, respectively) at the NdeI-BamHI site of pET15b (Novagen).…”

Section: Methodsmentioning

confidence: 99%

“…PriA is composed of two domains, namely the N-terminal DNA binding domain and the C-terminal DEXH-type helicase domain (21)(22)(23). The former contains a 3Ј terminus binding pocket that specifically recognizes and binds to a 3Ј-end of DNA.…”

mentioning

confidence: 99%

Escherichia coli PriA Protein, Two Modes of DNA Binding and Activation of ATP Hydrolysis

Tanaka

Mizukoshi

Sasaki

et al. 2007

Journal of Biological Chemistry

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Escherichia coli PriA protein plays crucial roles in processing of arrested replication forks. PriA serves as a sensor/stabilizer for an arrested replication fork and eventually promotes restart of DNA replication through assembly of a primosome. PriA carries a 3 terminus binding pocket required for its high affinity binding to a specific arrested fork as well as for its biological functions. We show here that PriA binds to DNA in a manner either dependent on or independent of 3 terminus recognition. The former mode of binding requires the 3 terminus binding pocket present at the N-terminal half of the 181-residue DNA binding domain and exhibits specific bipartite interaction on the template DNA. The latter mode is independent of the pocket function, but requires the C-terminal half of the same domain. ATP hydrolysis activity of PriA can be stimulated in vitro by either of the two binding modes. We propose architecture of PriA bound to various arrested replication fork structures and discuss its implication in helicase activation and ATP hydrolysis.

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Bacterial Primosome

Tanaka¹,

Masai²

2010

Encyclopedia of Life Sciences

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DNA ( deoxyribonucleic acid ) replication requires operation of a molecular machinery which efficiently synthesises of nucleotide chains on both strands. This process requires not only the enzymes synthesising DNA ( DNA polymerases ) but also those providing primer RNAs ( ribonucleic acid ) and continuously melting the duplex DNA. The primosome refers to a protein complex capable of processive unwinding of duplex DNA and primer RNA synthesis on the lagging strand at a replication fork. The prepriming proteins, DNA helicase and primase are sequentially assembled on the template DNA to generate primosome. Once assembled, it, in conjunction with DNA polymerases , facilitates DNA chain elongation. The assembly of bacterial primosome is triggered by an ‘initiator’ protein including DnaA and PriA , which recognise the site of assembly. Primosome is reassembled in replication restart process at stalled or processed replication forks, triggered by PriA . Thus, primosome constitutes an essential component for active replication fork machinery. Key concepts: Replication fork is the site of DNA replication where two replicating single‐stranded DNA separates. Primer RNA is a short stretch of RNA, the 3′‐terminus of which is utilised by DNA polymerases for DNA elongation. Primosome is a name given to the protein complex capable of duplex DNA unwinding and primer RNA synthesis at the replication fork. oriC is the replication ori gin of c hromosome. The initiation site of bacterial chromosomal DNA replication under a normal growth condition. DnaA is the initiator protein for bacterial chromosomal replication, which binds to oriC to assemble a primosome. PriA is a conserved replication factor which triggers assembly of the so‐called ϕX174‐type primosome, which is assembled at a stalled replication fork for replication restart. Stalled replication fork, the replication fork the movement of which is blocked by internal and external ‘replication stress’ including DNA damages and depletion of nucleotide precursors. Replication restart is a process of reassembly of primosome at a stalled replication fork to resume DNA chain elongation. Recombination intermediate is the intermediate structure (e.g. D‐loop structure) of homologous recombination reaction. DNA helicase is a protein capable of unwinding a duplex DNA at a replication fork by using the energy derived from the hydrolysis of nucleotides. Prepriming proteins are proteins required for the stage preceding the association of the primase (an enzyme synthesising primer RNAs) during the assembly of a primosome.

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DNA Binding of PriA Protein Requires Cooperation of the N-terminal D-loop/Arrested-fork Binding and C-terminal Helicase Domains

Cited by 41 publications

References 48 publications

Structural mechanisms of PriA-mediated DNA replication restart

Structural mechanisms of PriA-mediated DNA replication restart

Escherichia coli PriA Protein, Two Modes of DNA Binding and Activation of ATP Hydrolysis

Bacterial Primosome

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