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Molecular cloning and DNA sequence analysis of Escherichia coli priA, the gene encoding the primosomal protein
P Nurse1, R J DiGate, K H Zavitz
1Program in Molecular Biology, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.
Insights
Escherichia coli replication factor Y (protein n') gene was cloned and sequenced, revealing its role in chromosomal replication. Overexpression of this gene significantly increased factor Y activity, confirming its function in primosome assembly.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Escherichia coli replication factor Y (protein n') is crucial for primosome assembly in DNA replication.
- Its specific role in E. coli chromosomal replication remains unclear, unlike its known functions in bacteriophage and plasmid DNA replication.
Purpose of the Study:
- To molecularly clone and determine the DNA sequence of the gene encoding E. coli replication factor Y.
- To elucidate the function of replication factor Y in E. coli chromosomal replication.
Main Methods:
- Molecular cloning of the factor Y gene.
- DNA sequencing and analysis of the open reading frame.
- Transient expression using a bacteriophage T7 system.
- Protein expression and activity assays via SDS/polyacrylamide gel electrophoresis and in vitro DNA synthesis.
Main Results:
- An open reading frame encoding an 81.7 kDa polypeptide was identified, matching known amino acid sequences of factor Y.
- Overexpression resulted in a 78 kDa polypeptide that comigrated with authentic factor Y.
- Cell extracts showed a 2000-fold increase in factor Y activity in vitro.
Conclusions:
- The gene encoding factor Y, designated primosome A (priA), maps to 88.5 min on the E. coli chromosome.
- This study confirms the role of factor Y in E. coli chromosomal replication and primosome assembly.
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 phi 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 phi 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).