Volume 271, Number 26, Issue of June 28, 1996 pp. 15642-15648
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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The Ordered Assembly of the X174-type Primosome
I. ISOLATION AND IDENTIFICATION OF INTERMEDIATE PROTEIN-DNA COMPLEXES

(Received for publication, March 7, 1996, and in revised form, April 5, 1996)

Jenny Y. Ng and Kenneth J. Marians ^§

From the  Graduate Program in Molecular Biology, Cornell University Graduate School of Medical Sciences, New York, New York 10021 and the ^§ Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021

The X-type primosome was discovered during the resolution and reconstitution in vitro of the complementary strand DNA replication step of the X174 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 X174 phage DNA, the primosome also contains PriC.

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