The Ordered Assembly of the ÏX174-type Primosome
Gel filtration chromatography was used to isolate both preprimosomal and primosomal complexes formed on single-stranded DNA-binding protein-coated ÏX174 DNA by the combination of PriA, PriB, PriC, DnaT, DnaB, DnaC, and DnaG. The presence and relative amounts of primosomal proteins in these complexe...
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Veröffentlicht in: | The Journal of biological chemistry 1996-06, Vol.271 (26), p.15649 |
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Format: | Artikel |
Sprache: | eng |
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Zusammenfassung: | Gel filtration chromatography was used to isolate both preprimosomal and primosomal complexes formed on single-stranded DNA-binding
protein-coated ÏX174 DNA by the combination of PriA, PriB, PriC, DnaT, DnaB, DnaC, and DnaG. The presence and relative amounts
of primosomal proteins in these complexes were determined by Western blotting. Protein-DNA complexes isolated (i) after assembly
in the presence of 10 μ M ATP, (ii) after preprimosome movement in the presence of 1 m M ATP, (iii) after priming in the presence of the four ribonucleoside triphosphates, or (iv) after complementary strand DNA
replication in the presence of the DNA polymerase III holoenzyme all had the same protein composition; preprimosomes contained
PriA, PriB, PriC, DnaT, and DnaB, whereas primosomes included DnaG. The stable association of DnaG with the protein-DNA complex
could be attributed partially to its ability to remain bound to the primers synthesized. In the absence of PriC, the efficiencies
of priming and replication were reduced by one-third and one-half, respectively, even though PriC was not required for the
formation of stable protein-DNA complexes on a 304-nucleotide-long single strand of DNA containing a primosome assembly site
(Ng, J. Y., and Marians, K. J. (1996) J. Biol. Chem. 271, 15642-15648). We hypothesize that maintenance of the primosome on the replicated DNA may provide a mechanism to allow
primosomes to participate in the resolution of recombination intermediates and intermediates formed during double strand break
repair by permitting the re-establishment of a replication fork. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.271.26.15649 |