Interactions of bacteriophage T7 DNA primase/helicase protein with single-stranded and double-stranded DNAs.
Protein-DNA interactions of bacteriophage T7 DNA primase/helicase protein 4A' with small synthetic oligodeoxynucleotides were investigated using a 20-base-paired hairpin duplex, and 10-, 30-, and 60-base-long single-stranded DNA. The effect of nucleotide cofactors on DNA binding was examined using membrane binding assays which showed that 4A' binds DNA optimally only in the presence of MgdTMP-PCP, the nonhydrolyzable analog of dTTP. About 20% of single-stranded DNA binding was observed in the presence of MgdTDP, but none was detectable in the absence of nucleotides. Native polyacrylamide gel electrophoresis showed that the DNAs bind predominantly to the hexameric form of 4A'. Larger oligomers of 4A' can bind DNA, but no DNA binding was observed to species smaller than the hexamer. Quantitative equilibrium binding studies at increasing 4A' concentrations and at increasing DNA concentrations showed tight binding of one 10-mer or 30-mer per hexamer. The 4A' hexamer can bind a second strand of DNA, but with a 50-fold weaker affinity than the first strand. The 60-mer showed tight binding to two 4A' hexamers, suggesting that a hexamer may interact with only 30-40 bases of single-stranded DNA. This was corroborated by nuclease protection experiments where the smallest length of DNA protected by 4A' or 4B protein was found to be about 30 bases. Equilibrium binding studies and competitive DNA binding data are consistent with a weaker affinity of 4A' for the duplex DNA. Only 20-25% of duplex DNA binding was observed at increasing 4A' protein in the presence of MgdTMP-PCP. About four duplex DNAs can bind each 4A' hexamer at increasing DNA concentrations, but their weaker binding was evident from their facile dissociation from 4A' in the presence of competing single-stranded DNA.
Hingorani
M M
MM
Department of Biochemistry, Ohio State University, Columbus 43210.
Patel
S S
SS
D017123:Bacteriophage T7 / Q000378:metabolism*; D001483:Base Sequence; D001667:Binding, Competitive; D004247:DNA / Q000378:metabolism*; D019915:DNA Primase; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003852:Deoxyribonucleoproteins / Q000737:chemistry; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D012316:RNA Nucleotidyltransferases / Q000378:metabolism*
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D003852:Deoxyribonucleoproteins; D009711:Nucleotides; D009838:Oligodeoxyribonucleotides; D004247:DNA; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases