Interaction of Escherichia coli primase with a phage G4ori(c)-E. coli SSB complex.
We earlier reported that Escherichia coli single-stranded DNA-binding protein (SSB) bound in a fixed position to the stem-loop structure of the origin of complementary DNA strand synthesis in phage G4 (G4ori(c)), leaving stem-loop I and the adjacent 5' CTG 3', the primer RNA initiation site, as an SSB-free region (W. Sun and G. N. Godson, J. Biol. Chem. 268:8026-8039, 1993). Using a small 278-nucleotide (nt) G4ori(c) single-stranded DNA fragment that supported primer RNA synthesis, we now demonstrate by gel shift that E. coli primase can stably interact with the SSB-G4ori(c) complex. This stable interaction requires Mg2+ for specificity. At 8 mM Mg2+, primase binds to an SSB-coated 278-nt G4ori(c) fragment but not to an SSB-coated control 285-nt LacZ ss-DNA fragment. In the absence of Mg2+, primase binds to both SSB-coated fragments and gives a gel shift. T4 gene 32 protein cannot substitute for E. coli SSB in this reaction. Stable interaction of primase with naked G4ori(c). single-stranded DNA was not observed. DNase I and micrococcal nuclease footprinting, of both 5' and 3' 32P-labeled DNA, demonstrated that primase interacts with two regions of G4ori(c): one covering stem-loop I and the 3' sequence flanking stem-loop I which contains the pRNA initiation site and another located on the 5' sequence flanking stem-loop III.
Sun
W
W
Biochemistry Department, New York University Medical Center, New York, New York 10016, USA.
Godson
G N
GN
D001483:Base Sequence; D003090:Coliphages / Q000235:genetics / Q000378:metabolism*; D018983:DNA Footprinting; D019915:DNA Primase; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000096:biosynthesis; D012316:RNA Nucleotidyltransferases / Q000378:metabolism*; D018741:Replication Origin
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; C034477:RNA primers; D012313:RNA; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D008274:Magnesium