Molecular cloning and DNA sequence analysis of Escherichia coli priA, the gene encoding the primosomal protein replication factor Y Journal Article
| Authors: | Nurse, P.; DiGate, R. J.; Zavitz, K. H.; Marians, K. J. |
| Article Title: | Molecular cloning and DNA sequence analysis of Escherichia coli priA, the gene encoding the primosomal protein replication factor Y |
| Abstract: | Escherichia coli replication factor Y (protein n') functions in the assembly of a mobile multiprotein replication-priming complex called the primosome. Although the role of factor Y in primosome assembly during replication in vitro of bacteriophage ∅X174 and plasmid pBR322 DNA is clear, its role in E. coli chromosomal replication is not. To address this issue, the gene for factor Y has been cloned molecularly and its DNA sequence has been determined. The cloned fragment of DNA contained an open reading frame capable of encoding a polypeptide of 81.7 kDa. This open reading frame contains amino acid sequences identical to 13 N-terminal amino acids of purified factor Y, as well as to a 10-amino acid internal sequence (from a cyanogen bromide fragment) as determined by gas-phase microsequencing. Expression of the polypeptide encoded by this open reading frame using a bacteriophage T7 transient expression system resulted in the accumulation of a polypeptide with an apparent molecular mass of 78 kDa that comigrated with bona fide factor Y during SDS/polyacrylamide gel electrophoresis. Soluble extracts made from cells overexpressing the product of the putative factor Y open reading frame showed a 2000-fold increase in factor Y activity during bacteriophage ∅X174 complementary-strand DNA synthesis in vitro when compared to control extracts. The gene encoding factor Y, which maps to 88.5 min on the E. coli chromosome, has been designated primosome A (priA). |
| Keywords: | unclassified drug; nonhuman; dna replication; molecular cloning; cloning, molecular; bacterial proteins; amino acid sequence; molecular sequence data; kinetics; escherichia coli; base sequence; plasmids; dna sequence; molecular weight; dna, bacterial; dna helicase; chromosomes, bacterial; chromosome mapping; primosome; replication factor; blotting, southern; priority journal; article; support, u.s. gov't, p.h.s.; genes, structural, bacterial; replication factor y |
| Journal Title: | Proceedings of the National Academy of Sciences of the United States of America |
| Volume: | 87 |
| Issue: | 12 |
| ISSN: | 0027-8424 |
| Publisher: | National Academy of Sciences |
| Date Published: | 1990-06-01 |
| Start Page: | 4615 |
| End Page: | 4619 |
| Language: | English |
| DOI: | 10.1073/pnas.87.12.4615 |
| PUBMED: | 2162049 |
| PROVIDER: | scopus |
| PMCID: | PMC54167 |
| DOI/URL: | |
| Notes: | Article -- Export Date: 27 January 2020 -- Source: Scopus |
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