Bound Lac repressor protein differentially inhibits the unwinding reactions catalyzed by DNA helicases.
A partial duplex DNA substrate containing the Lac repressor binding site, within the duplex region, was constructed to examine the effect of bound Lac repressor on the unwinding reaction catalyzed by several DNA helicases. The substrate contained 90 base pairs of double-stranded DNA and, in the absence of Lac repressor, was effectively unwound by each of the seven helicases tested. The unwinding reactions catalyzed by Escherichia coli Rep protein, bacteriophage T4 Dda protein and E. coli DNA helicase I were not inhibited by the presence of bound Lac repressor. Both SV40 T antigen and E. coli helicase II were partially inhibited by bound repressor at the highest repressor concentrations tested. The helicase reactions catalyzed by E. coli DnaB protein and helicase IV were substantially inhibited by the presence of bound protein. When the length of the duplex region was increased to 323 base pairs the inhibition spectrum caused by bound Lac repressor on the unwinding reactions catalyzed by DnaB protein, helicase I and helicase II was essentially the same as that observed using the shorter partial duplex molecule. Inhibition of the unwinding reaction was due to the presence of bound Lac repressor as evidenced by the substantially weaker inhibition of helicase IV by Lac repressor in the presence of IPTG. In addition, we have shown that Rep protein displaces the bound repressor protein during the course of an unwinding reaction.
Yancey-Wrona
J E
JE
Department of Biology, University of North Carolina, Chapel Hill 27599.
Matson
S W
SW
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors; D000818:Animals; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases / Q000037:antagonists & inhibitors*; D004256:DNA Polymerase I / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins; D003850:Deoxyribonuclease I; D053488:DnaB Helicases; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D007700:Kinetics; D009690:Nucleic Acid Conformation; D012097:Repressor Proteins / Q000378:metabolism*; D015183:Restriction Mapping; D012474:Salmon; D013379:Substrate Specificity; D013604:T-Phages / Q000378:metabolism; D014764:Viral Proteins
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; C506744:rep protein, E coli; D004247:DNA; D004256:DNA Polymerase I; D004262:DNA Restriction Enzymes; D003850:Deoxyribonuclease I; D000251:Adenosine Triphosphatases; C027093:TraI protein, E coli; C506730:dnaB protein, E coli; C059716:helD protein, E coli; D004265:DNA Helicases; D053488:DnaB Helicases; C077401:dda DNA helicase protein, Bacteriophage T4