Abstract
Assembly of the Escherichia coli primosome requires six proteins, PriA, PriB, PriC, DnaB, DnaC, and DnaT, acting at a primosome assembly site (pas) on an SSB-coated single-stranded (ss) DNA. Assembly is initiated by interactions of PriA and PriB with ssDNA and the pas. PriC, DnaB, DnaC, and DnaT then act on the PriA-PriB-DNA complex to yield the primosome. In the primosome, the dATPase (ATPase) of PriA becomes hyper-activated. In addition, the assembled primosome appears to block the pas, preventing it from activating additional PriA molecules. Either ATP alone or dATP in combination with GTP is sufficient for primosome assembly, while ATP or GTP provides for its maintenance during isolation. These nucleotide requirements can be reconciled with the need for ATP or dATP for DnaB-DnaC complex formation and hydrolysis of ATP or GTP by DnaB when it binds ssDNA. Such isolated primosomes contain a dATPase, the hallmark of PriA, and a GTPase indicative of DnaB. Further studies indicate that the isolated primosome contains the PriB replication activity in addition to PriA and DnaB.
Highlights
A special hairpin structure on the quantities of each individual protein (14-17h2 sevDNA called a Primosome assembly siteor pas is required eral of these components are more active when isolated from [3,4]
Theprimosome can thenbe recognized by primase to lay the overproducing source than from wild-type cells, probably down RNA primers for complete replication of the template to due to the high degroefeoverexpression and the brevitoyf the the duplex RF by DNA polymerase I11 holoenzyme. ( A s origi- purification [16].2 nally coined, the term“primosome” defineda multiprotein com- With these newly obtained, highly active,homogeneous complex containing a primase sufficient t o generate a primer [5]. ponents, the early stepofs primosome assembly havenow been The protein complex formed prior to the addition of primase investigated.Theinteractions of PriA andPriBwith both was calledapreprimosome.Lack of a stable association of 4x174 and M13 DNA have been studied
The PriA replication activity in these fractions was assayed as described under “Materials and Methods”;the remaining replication components were added in 10 pl and the reaction incubatedfor 10 minat 30 “C to allow DNA synthesis
Summary
Of the Escherichia coli primosome requires tion during which multiple primers must be provided for dissix proteins, PriA, PriB, PriC, DnaB, DnaC, and DnaT, continuous synthesis Either ATP alone or dATP in combination with GTP is sufficient for primosome assembly, while ATP or GTP provides forits maintenance during isolation These nucleotide requirements can be quired for chromosomalreplication [10,11,12]. Identification of these geneshas allowed the conPriB, PriC, DnaB, DnaC, and DnaT assembling a primosome struction of efficient overproducers and the isolation of large on the SSB-coated ssDNA. Ponents, the early stepofs primosome assembly havenow been The protein complex formed prior to the addition of primase investigated.Theinteractions of PriA andPriBwith both was calledapreprimosome.Lack of a stable association of 4x174 and M13 DNA have been studied. The reprimase with the primosome has diminished this distinction. quirements forprimosome assembly and maintenance have To reflect this fact and the varieotfyprimosomal formsin other been characterized and the enzymatic activities iann isolated systems [6, 7], we use the term“primosome” for the multipro- primosome identified. tein complex, knowing that primase must actat this complex, transiently, to synthesize a primer.) This ss to RF
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