The ordered assembly of the φX174-type primosome: I. Isolation and identification of intermediate protein-DNA complexes Journal Article
| Authors: | Ng, J. Y.; Marians, K. J. |
| Article Title: | The ordered assembly of the φX174-type primosome: I. Isolation and identification of intermediate protein-DNA complexes |
| Abstract: | The φX-type primosome was discovered during the resolution and reconstitution in vitro of the complementary strand DNA replication step of the φX174 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 φX174 phage DNA, the primosome also contains PriC. |
| Keywords: | dna binding protein; genetics; dna-binding proteins; nonhuman; dna replication; metabolism; dna repair; protein assembly; bacterial protein; chemistry; bacterial proteins; genetic recombination; molecular recognition; escherichia coli; dna, viral; helicase; adenosine triphosphate; single stranded dna; dna, single-stranded; ultrastructure; dna helicases; replication protein a; escherichia coli proteins; macromolecule; macromolecular substances; dna denaturation; virus dna; escherichia coli protein; molecular stability; dna protein complex; deoxyribonucleoprotein; dnab helicases; bacteriophage phi x 174; priority journal; article; dnab helicase; dnac protein, e coli; prib protein, e coli; deoxyribonucleoproteins |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 271 |
| Issue: | 26 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 1996-06-28 |
| Start Page: | 15642 |
| End Page: | 15648 |
| Language: | English |
| DOI: | 10.1074/jbc.271.26.15642 |
| PUBMED: | 8663104 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Article -- Export Date: 22 November 2017 -- Source: Scopus |
