Primer-DNA formation during simian virus 40 DNA replication in vitro.
Studies of simian virus 40 (SV40) DNA replication in vitro have identified a small (approximately 30-nucleotide) RNA-DNA hybrid species termed primer-DNA. Initial experiments indicated that T antigen and the polymerase alpha-primase complex are required to form primer-DNA. Proliferating cell nuclear antigen, and presumably proliferating cell nuclear antigen-dependent polymerases, is not needed to form this species. Herein, we present an investigation of the stages at which primer-DNA functions during SV40 DNA replication in vitro. Hybridization studies indicate that primer-DNA is initially formed in the origin region and is subsequently synthesized in regions distal to the origin. At all time points, primer-DNA is synthesized from templates for lagging-strand DNA replication. These studies indicate that primer-DNA functions during both initiation and elongation stages of SV40 DNA synthesis. Results of additional experiments suggesting a precursor-product relationship between formation of primer-DNA and Okazaki fragments are presented.
Denis
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Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111.
Bullock
P A
PA
D000951:Antigens, Neoplasm / Q000378:metabolism; D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism; D001483:Base Sequence; D002474:Cell-Free System; D019915:DNA Primase; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis* / Q000302:isolation & purification; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D018809:Proliferating Cell Nuclear Antigen; D012316:RNA Nucleotidyltransferases / Q000378:metabolism; D015183:Restriction Mapping; D013539:Simian virus 40 / Q000235:genetics*; D013698:Templates, Genetic
D000951:Antigens, Neoplasm; D000952:Antigens, Polyomavirus Transforming; D004279:DNA, Viral; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D018809:Proliferating Cell Nuclear Antigen; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases