Structure of the Escherichia coli primase/single-strand DNA-binding protein/phage G4oric complex required for primer RNA synthesis.
Escherichia coli primase/SSB/single-stranded phage G4oric is a simple system to study how primase interacts with DNA template to synthesize primer RNA for initiation of DNA replication. By a strategy of deletion analysis and antisense oligonucleotide protection on small single-stranded G4oric fragments, we have identified the DNA sequences required for binding primase and the critical location of single-strand DNA-binding (SSB) protein. Together with the previous data, we have defined the structure of the primase/SSB/G4oric priming complex. Two SSB tetramers bind to the G4oric secondary structure, which dictates the spacing of 3' and 5' bound adjacent SSB tetramers and leaves SSB-free regions on both sides of the stem-loop structure. Two primase molecules then bind separately to specific DNA sequences in the 3' and 5' SSB-free G4oric regions. Binding of the 3' SSB tetramer, upstream of the primer RNA initiation site, is also necessary for priming. The generation of a primase-recognition target by SSB phasing at DNA hairpin structures may be applicable to the binding of initiator proteins in other single-stranded DNA priming systems. Novel techniques used in this study include antisense oligonucleotide protection and RNA synthesis on an SSB-melted, double-stranded DNA template.
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Biochemistry Department, New York University Medical Center, 550 First Avenue, New York 10016, USA.
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D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D019915:DNA Primase / Q000737:chemistry / Q000378:metabolism*; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D046911:Macromolecular Substances; D017915:Microvirus / Q000235:genetics*; D009690:Nucleic Acid Conformation; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D012313:RNA / Q000096:biosynthesis* / Q000235:genetics; D018741:Replication Origin; D017384:Sequence Deletion
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D016376:Oligonucleotides, Antisense; C034477:RNA primers; D012313:RNA; D019915:DNA Primase