The ordered assembly of the phiX174-type primosome. I. Isolation and identification of intermediate protein-DNA complexes.
The phiX-type primosome was discovered during the resolution and reconstitution in vitro of the complementary strand DNA replication step of the phiX174 viral life cycle. This multienzyme bidirectional helicase-primase complex can provide the DNA unwinding and Okazaki fragment-priming functions at the replication fork and has been implicated in cellular DNA replication, repair, and recombination. We have used gel mobility shift assays and enhanced chemiluminescence Western analysis to isolate and identify the pathway of primosome assembly at a primosome assembly site (PAS) on a 300-nucleotide-long single-stranded DNA fragment. The first three steps do not require ATP and are as follows: (i) PriA recognition and binding to the PAS, (ii) stabilization of the PriA-PAS complex by the addition of PriB, and (iii) formation of a PriA-PriB-DnaT-PAS complex. Subsequent formation of the preprimosome involves the ATP-dependent transfer of DnaB from a DnaB-DnaC complex to the PriA-PriB-DnaT-PAS complex. The final preprimosomal complex contains PriA, PriB, DnaT, and DnaB but not DnaC. A transient interaction between the preprimosome and DnaG generates the five-protein primosome. As described in an accompanying article (Ng, J. Y., and Marians, K. J. (1996) J. Biol. Chem. 271, 15649-15655), when assembled on intact phiX174 phage DNA, the primosome also contains PriC.
Ng
J Y
JY
Graduate Program in Molecular Biology, Cornell University Graduate School of Medical Sciences, New York, New York 10021, USA.
Marians
K J
KJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D010584:Bacteriophage phi X 174 / Q000235:genetics* / Q000648:ultrastructure; D004265:DNA Helicases; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D003852:Deoxyribonucleoproteins / Q000737:chemistry*; D053488:DnaB Helicases; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D051716:Replication Protein A
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D003852:Deoxyribonucleoproteins; C071702:DnaC protein, E coli; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; C086004:PriB protein, E coli; D051716:Replication Protein A; D000255:Adenosine Triphosphate; D004265:DNA Helicases; D053488:DnaB Helicases