Effects of the bacteriophage T4 gene 41 and gene 32 proteins on RNA primer synthesis: coupling of leading- and lagging-strand DNA synthesis at a replication fork.
We have demonstrated previously that the template sequences 5'-GTT-3' and 5'-GCT-3' serve as necessary and sufficient signals for the initiation of new DNA chains that start with pentaribonucleotide primers of sequence pppApCpNpNpN or pppGpCpNpNpN, respectively. Normally, the complete T4 primosome, consisting of the T4 gene 41 (DNA helicase) and gene 61 (primase) proteins, is required to produce RNA primers. However, a high concentration of the 61 protein alone can prime DNA chain starts from the GCT sites [Cha, T.-A., & Alberts, B. M. (1986) J. Biol. Chem. 261, 7001-7010]. We show here that the 61 protein can catalyze a single-stranded DNA template-dependent reaction in which the dimers pppApC and pppGpC are the major products and much longer oligomers of various lengths are minor ones. Further addition of the 41 protein is needed to form a primosome that catalyzes efficient synthesis of the physiologically relevant pentaribonucleotides that are responsible for the de novo DNA chain starts on the lagging strand of a replication fork. The helicase activity of the 41 protein is necessary and sufficient to ensure a high rate and processivity of DNA synthesis on the leading strand [Cha, T.-A., & Alberts, B. M. (1989) J. Biol. Chem. 264, 12220-12225]. Coupling an RNA primase to this helicase in the primosome therefore coordinates the leading- and lagging-strand DNA syntheses at a DNA replication fork. Our experiments reveal that the addition of the T4 helix-destabilizing protein (the gene 32 protein) is required to confine the synthesis of RNA primers to those sites where they are used to start an Okazaki fragment, causing many potential priming sites to be passed by the primosome without triggering primer synthesis.
Cha
T A
TA
Department of Biochemistry and Biophysics, University of California, San Francisco 94143.
Alberts
B M
BM
D001483:Base Sequence; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D019915:DNA Primase; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005814:Genes, Viral; D008961:Models, Structural; D008969:Molecular Sequence Data; D009843:Oligoribonucleotides / Q000138:chemical synthesis; D012316:RNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D012293:Rifampin / Q000494:pharmacology; D013604:T-Phages / Q000235:genetics* / Q000378:metabolism; D013698:Templates, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D015678:Viral Structural Proteins / Q000235:genetics
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D009843:Oligoribonucleotides; D012367:RNA, Viral; D014764:Viral Proteins; D015678:Viral Structural Proteins; C063983:gene 41 protein, Enterobacteria phage T4; C060035:gp32 protein, Enterobacteria phage T4; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D012321:DNA-Directed RNA Polymerases; D004265:DNA Helicases; D012293:Rifampin