Identification of a domain of Escherichia coli primase required for functional interaction with the DnaB helicase at the replication fork.
Primase plays a key role in governing the sequence of events required on the lagging strand during a cycle of Okazaki fragment synthesis. To begin to probe the protein-protein interactions necessary for primase function at the replication fork, we have used limited trypsinolysis to separate primase into two functional domains, an N-terminal domain of 49 kDa (p49) and a carboxyl-terminal domain of 16 kDa (p16). p49 retained primase activity in replication assays that utilized bacteriophage M13 DNA carrying the bacteriophage G4 origin of DNA replication as the template, but was inactive during general priming or the conversion of phi X174 single-stranded circular (ss(c))-DNA to the replicative form (RF) and could not support lagging-strand DNA synthesis at replication forks reconstituted with the phi X-type primosomal proteins and the DNA polymerase III holoenzyme. On the other hand, p16 inhibited those replication reactions that included the replication fork helicase, DnaB (general priming, phi X174 ss(c)-->RF, and at the replication fork), but had no effect on those that did not (M13Gori ss(c)-->RF). These results demonstrate that p49 defines a domain of primase required for catalytic activity, that p16 defines a domain of primase required for functional interaction with DnaB, and that it is a protein-protein interaction with DnaB that attracts primase to the replication fork.
Tougu
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Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
Peng
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Marians
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KJ
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D004265:DNA Helicases / Q000378:metabolism*; D019915:DNA Primase; D017931:DNA Primers / Q000737:chemistry; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D053488:DnaB Helicases; D004926:Escherichia coli / Q000201:enzymology*; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D012316:RNA Nucleotidyltransferases / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013698:Templates, Genetic
D001426:Bacterial Proteins; D017931:DNA Primers; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D010455:Peptides; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004265:DNA Helicases; D053488:DnaB Helicases