|Gene:||priA||Accession Numbers: EG10763 (EcoCyc), b3935, ECK3927|
Synonyms: srgA, replication factor Y
Component of: primosome (summary available)
The role of PriA in the cell appears to be the restart of stalled replication forks. PriA, along with DNA polymerase II, is required to restart DNA synthesis immediately following UV exposure [Rangarajan02]. Based on genetic interactions, there seem to be two replication restart pathways that require PriA, one using RecG, the other using RuvABC [Meddows04, Gregg02].
PriA binds to the 3' terminus of nascent DNA at stalled replication forks in vitro, leading to assembly of the full primosome at the binding site [Mizukoshi03]. PriA preferentially binds to stalled forks with D loops, which are intermediates in recombination-based recovery from a stall [Liu99, McGlynn97]. Larger single-stranded gaps at the stall site favor PriC-mediated restart rather than PriA involvement [Heller05]. PriA binding is most effective when the lagging strand is duplex and the leading strand is single stranded [Jones01]. D loop binding leads to assembly of the full primosome at the stall site [Liu99a].
In many plasmids and phages, PriA and the primosome function in initiation of normal DNA replication. In this context, PriA binds to the primosome assembly site (PAS), a double-hairpin DNA structure that acts as the origin of replication for replicative DNA synthesis [Greenbaum85, Soeller82, Zipursky81, Zipursky80, Allen93, Jones99]. Following PAS binding, PriA translocates 3' to 5' along ssDNA in an ATP-dependent fashion, after which it can move onto and unwind duplex DNA, a process that also uses ATP [Lee90, Lee87]. This PriA-mediated unwinding moves just as fast as unwinding catalyzed by the normal replicative helicase, DnaB, and is stimulated by interaction with single-strand binding protein (SSB) via the SSB carboxy-terminus [Mok87, Cadman04]. PriA helicase activity is blocked by Tus bound at ter replication termination sites [Hiasa92, Lee92a].
PriA binding is the first step in assembly of the primosome, a multiprotein complex that restarts stalled replication forks in E. coli and initiates replication in various plasmids and phages [Ng96]. The primosomal protein PriB helps PriA bind DnaT, as well as stimulating the processivity and helicase activity of PriA [Liu96, Cadman05]. PriA activity doubles in the completed primosome [Allen93]. Notably, PriA that lacks its helicase and translocation activities can still catalyze primosome assembly, and this alone appears to be sufficient to correct defects in homologous recombination and double-strand-break repair that are normally present in priA null mutants [Zavitz92, Kogoma96].
PriA binds ssDNA as a monomer via a single DNA-binding site, binding to as few as 8 nucleotides [Jezewska00, Jezewska00a]. This binding occurs in the PriA amino-terminal domain, though the helicase domain works together with it synergistically to allow high-affinity binding of D loops at replication forks [Tanaka02, Chen04]. Helicase activity is not required for this binding [Tanaka03]. The kinetics of PriA binding to ssDNA have been examined in detail [Galletto04].
The amino-terminal domain of PriA has been crystallized [Sasaki06].
PriA is required for inducible and constitutive stable DNA replication in the absence of protein synthesis [Masai94].
priA null mutants are unable to generate phiX174 phage or maintain plasmids that use an E. coli origin of replication. They also have growth defects and a filamentous phenotype [Lee91]. The SOS response is induced in these mutants [Nurse91]. Loss of PriA helicase activity suppresses the deleterious effects of the loss of RecG recombinational helicase function [AlDeib96].
|Map Position: [4,122,635 <- 4,124,833] (88.86 centisomes, 320°)||Length: 2199 bp / 732 aa|
Molecular Weight of Polypeptide: 81.655 kD (from nucleotide sequence), 78 kD (experimental) [Nurse90 ]
Unification Links: ASAP:ABE-0012860 , CGSC:27611 , DIP:DIP-10562N , EchoBASE:EB0756 , EcoGene:EG10763 , EcoliWiki:b3935 , Mint:MINT-1274231 , ModBase:P17888 , OU-Microarray:b3935 , PortEco:priA , PR:PRO_000023591 , Protein Model Portal:P17888 , RefSeq:NP_418370 , RegulonDB:EG10763 , SMR:P17888 , String:511145.b3935 , UniProt:P17888
Relationship Links: InterPro:IN-FAMILY:IPR001650 , InterPro:IN-FAMILY:IPR005259 , InterPro:IN-FAMILY:IPR011545 , InterPro:IN-FAMILY:IPR014001 , InterPro:IN-FAMILY:IPR027417 , PDB:Structure:2D7E , PDB:Structure:2D7G , PDB:Structure:2D7H , PDB:Structure:2DWL , PDB:Structure:2DWM , PDB:Structure:2DWN , Pfam:IN-FAMILY:PF00270 , Pfam:IN-FAMILY:PF00271 , Prosite:IN-FAMILY:PS51192 , Prosite:IN-FAMILY:PS51194 , Smart:IN-FAMILY:SM00487 , Smart:IN-FAMILY:SM00490
In Paralogous Gene Group: 563 (2 members)
|Biological Process:||GO:0006260 - DNA replication
[UniProtGOA11, GOA01, Rangarajan02]
GO:0006261 - DNA-dependent DNA replication [Liu99]
GO:0006268 - DNA unwinding involved in DNA replication [Lee87]
GO:0006270 - DNA replication initiation [Masai94]
GO:0006276 - plasmid maintenance [Lee91]
GO:0006302 - double-strand break repair [Kogoma96]
GO:0006310 - DNA recombination [Kogoma96]
GO:0010332 - response to gamma radiation [Kogoma96]
GO:0046677 - response to antibiotic [Kogoma96]
GO:0006269 - DNA replication, synthesis of RNA primer [UniProtGOA11]
|Molecular Function:||GO:0004386 - helicase activity
GO:0005515 - protein binding [Kozlov10, Lopper07]
GO:0043140 - ATP-dependent 3'-5' DNA helicase activity [Lee90]
GO:0000166 - nucleotide binding [UniProtGOA11]
GO:0003676 - nucleic acid binding [GOA01]
GO:0003677 - DNA binding [UniProtGOA11, GOA01]
GO:0005524 - ATP binding [UniProtGOA11, GOA01]
GO:0016787 - hydrolase activity [UniProtGOA11]
GO:0046872 - metal ion binding [UniProtGOA11]
|Cellular Component:||GO:0005829 - cytosol
GO:1990077 - primosome complex [UniProtGOA11]
|MultiFun Terms:||information transfer → DNA related → DNA replication|
|Growth Medium||Growth?||T (°C)||O2||pH||Osm/L||Growth Observations|
|LB Lennox||Yes||37||Aerobic||7||Yes [Baba06, Comment 1]|
|M9 medium with 1% glycerol||Yes||37||Aerobic||7.2||0.35||Yes [Joyce06, Comment 2]|
|MOPS medium with 0.4% glucose||Yes||37||Aerobic||7.2||0.22||Yes [Baba06, Comment 1]|
Subunit of: primosome
Subunit composition of
primosome = [(DnaB)6][(DnaT)3][(PriB)2][PriA][PriC][DnaG]
replicative DNA helicase = (DnaB)6 (extended summary available)
primosomal protein DnaT = (DnaT)3 (extended summary available)
primosomal protein DnaT = DnaT
primosomal replication protein N = (PriB)2 (extended summary available)
primosome factor N' = PriA (extended summary available)
primosomal replication protein N'' = PriC (extended summary available)
DNA primase = DnaG (extended summary available)
The primosome is a six-protein complex that appears to be involved in restart of stalled replication forks, as well as in replication initiation in certain phages and plasmids. See the individual subunit entries for additional information on the function of the primosome.
The primosome undergoes ordered assembly beginning with PriA binding to DNA. Following this, PriB binds to PriA, then DnaT binds. After this, DnaC loads DnaB in an ATP-dependent manner. DnaG associates with the complex and synthesizes an RNA primer [Ng96]. Despite its absence from this model of ordered assembly, PriC is also found in isolated intact primosomes [Ng96a]. Note that the primosome components have many functions in the cell that do not require the full primosome.
|Conserved-Region||211 -> 377|
|Nucleotide-Phosphate-Binding-Region||224 -> 231|
|Protein-Segment||320 -> 323|
|Zn-Finger-Region||436 -> 448|
|Conserved-Region||447 -> 638|
|Zn-Finger-Region||463 -> 479|
10/20/97 Gene b3935 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10763; confirmed by SwissProt match.
Baba06: Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006). "Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection." Mol Syst Biol 2;2006.0008. PMID: 16738554
DiazMejia09: Diaz-Mejia JJ, Babu M, Emili A (2009). "Computational and experimental approaches to chart the Escherichia coli cell-envelope-associated proteome and interactome." FEMS Microbiol Rev 33(1);66-97. PMID: 19054114
Galletto04: Galletto R, Jezewska MJ, Bujalowski W (2004). "Multistep sequential mechanism of Escherichia coli helicase PriA protein-ssDNA interactions. Kinetics and energetics of the active ssDNA-searching site of the enzyme." Biochemistry 43(34);11002-16. PMID: 15323559
Gregg02: Gregg AV, McGlynn P, Jaktaji RP, Lloyd RG (2002). "Direct rescue of stalled DNA replication forks via the combined action of PriA and RecG helicase activities." Mol Cell 9(2);241-51. PMID: 11864599
Heller05: Heller RC, Marians KJ (2005). "The disposition of nascent strands at stalled replication forks dictates the pathway of replisome loading during restart." Mol Cell 17(5);733-43. PMID: 15749022
Hiasa92: Hiasa H, Marians KJ (1992). "Differential inhibition of the DNA translocation and DNA unwinding activities of DNA helicases by the Escherichia coli Tus protein." J Biol Chem 267(16);11379-85. PMID: 1317865
Jezewska00a: Jezewska MJ, Rajendran S, Bujalowski W (2000). "Escherichia coli replicative helicase PriA protein-single-stranded DNA complex. Stoichiometries, free energy of binding, and cooperativities." J Biol Chem 275(36);27865-73. PMID: 10875934
Jones01: Jones JM, Nakai H (2001). "Escherichia coli PriA helicase: fork binding orients the helicase to unwind the lagging strand side of arrested replication forks." J Mol Biol 312(5);935-47. PMID: 11580240
Joyce06: Joyce AR, Reed JL, White A, Edwards R, Osterman A, Baba T, Mori H, Lesely SA, Palsson BO, Agarwalla S (2006). "Experimental and computational assessment of conditionally essential genes in Escherichia coli." J Bacteriol 188(23);8259-71. PMID: 17012394
Kogoma96: Kogoma T, Cadwell GW, Barnard KG, Asai T (1996). "The DNA replication priming protein, PriA, is required for homologous recombination and double-strand break repair." J Bacteriol 178(5);1258-64. PMID: 8631700
Kozlov10: Kozlov AG, Jezewska MJ, Bujalowski W, Lohman TM (2010). "Binding specificity of Escherichia coli single-stranded DNA binding protein for the chi subunit of DNA pol III holoenzyme and PriA helicase." Biochemistry 49(17);3555-66. PMID: 20329707
Lee90: Lee MS, Marians KJ (1990). "Differential ATP requirements distinguish the DNA translocation and DNA unwinding activities of the Escherichia coli PRI A protein." J Biol Chem 265(28);17078-83. PMID: 2170365
Liu96: Liu J, Nurse P, Marians KJ (1996). "The ordered assembly of the phiX174-type primosome. III. PriB facilitates complex formation between PriA and DnaT." J Biol Chem 271(26);15656-61. PMID: 8663106
Mizukoshi03: Mizukoshi T, Tanaka T, Arai K, Kohda D, Masai H (2003). "A critical role of the 3' terminus of nascent DNA chains in recognition of stalled replication forks." J Biol Chem 278(43);42234-9. PMID: 12917421
Ng96a: Ng JY, Marians KJ (1996). "The ordered assembly of the phiX174-type primosome. II. Preservation of primosome composition from assembly through replication." J Biol Chem 271(26);15649-55. PMID: 8663105
Nurse90: Nurse P, DiGate RJ, Zavitz KH, Marians KJ (1990). "Molecular cloning and DNA sequence analysis of Escherichia coli priA, the gene encoding the primosomal protein replication factor Y." Proc Natl Acad Sci U S A 87(12);4615-9. PMID: 2162049
Rangarajan02: Rangarajan S, Woodgate R, Goodman MF (2002). "Replication restart in UV-irradiated Escherichia coli involving pols II, III, V, PriA, RecA and RecFOR proteins." Mol Microbiol 43(3);617-28. PMID: 11929519
Sasaki06: Sasaki K, Ose T, Tanaka T, Mizukoshi T, Ishigaki T, Maenaka K, Masai H, Kohda D (2006). "Crystallization and preliminary crystallographic analysis of the N-terminal domain of PriA from Escherichia coli." Biochim Biophys Acta 1764(1);157-60. PMID: 16226927
Soeller82: Soeller WC, Marians KJ (1982). "Deletion mutants defining the Escherichia coli replication factor Y effector site sequences in pBR322 DNA." Proc Natl Acad Sci U S A 79(23);7253-7. PMID: 6130524
Tanaka02: Tanaka T, Mizukoshi T, Taniyama C, Kohda D, Arai K, Masai H (2002). "DNA binding of PriA protein requires cooperation of the N-terminal D-loop/arrested-fork binding and C-terminal helicase domains." J Biol Chem 277(41);38062-71. PMID: 12151393
Tanaka03: Tanaka T, Taniyama C, Arai K, Masai H (2003). "ATPase/helicase motif mutants of Escherichia coli PriA protein essential for recombination-dependent DNA replication." Genes Cells 8(3);251-61. PMID: 12622722
Wickner75: Wickner S, Hurwitz J (1975). "Association of phiX174 DNA-dependent ATPase activity with an Escherichia coli protein, replication factor Y, required for in vitro synthesis of phiX174 DNA." Proc Natl Acad Sci U S A 72(9);3342-6. PMID: 127175
Zavitz92: Zavitz KH, Marians KJ (1992). "ATPase-deficient mutants of the Escherichia coli DNA replication protein PriA are capable of catalyzing the assembly of active primosomes." J Biol Chem 267(10);6933-40. PMID: 1313026
Zipursky80: Zipursky SL, Marians KJ (1980). "Identification of two Escherichia coli factor Y effector sites near the origins of replication of the plasmids (ColE1 and pBR322." Proc Natl Acad Sci U S A 77(11);6521-5. PMID: 6109282
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