Cooperative interactions of nucleotide ligands are linked to oligomerization and DNA binding in bacteriophage T7 gene 4 helicases.
The equilibrium nucleotide binding and oligomerization of bacteriophage T7 gene 4 helicases have been investigated using thymidine 5'-triphosphate (dTTP), deoxythymidine 5'-(beta, gamma-methylenetriphosphate)(dTMP-PCP), thymidine 5'-diphosphate (dTDP), adenosine 5'-triphosphate (ATP), and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S). In the presence of nucleotide ligands, T7 helicases self-assemble into hexamers with six potential nucleotide binding sites that are nonequivalent both in the absence and in the presence of single-stranded DNA. All nucleotides tested bind with high affinity to three sites (K(d) = 5 x 10(-6) M, dTTP; 6 x 10(-7) M, dTMP-PCP; 4 x 10(-6) M, dTDP; 3 x 10(-5) M, ATP; 2 x 10(-6) M, ATP gamma S), while binding to the remaining sites is undetectable. Interestingly, nucleotide binding to the high-affinity sites exhibits positive cooperativity which is sensitive to protein concentration. This effect is a result of ligand binding-linked oligomerization wherein helicase oligomer equilibrium changes as a function of both nucleotide and protein concentration. A study of DNA binding shows that 1-2 NTPs bound per hexamer are sufficient for stoichiometric interaction between the helicase and DNA. Thus, the ring-shaped helicase hexamers assemble around DNA with one, two, or three NTPs bound to each hexamer. This study also examines the preferred use of dTTP for T7 helicase-catalyzed DNA unwinding by comparison with ATP, the more commonly used nucleotide ligand. ATP binds to the helicase with 6-fold weaker affinity than dTTP and promotes hexamerization as well as DNA binding. Nevertheless, DNA unwinding with ATP is at least 100-fold slower than with dTTP. Thus, the difference in ATP and dTTP utilization probably lies in a highly specific step in the coupling of NTP hydrolysis to DNA unwinding.
Hingorani
M M
MM
Department of Biochemistry, Ohio State University, Columbus 43210, USA.
Patel
S S
SS
D017123:Bacteriophage T7 / Q000201:enzymology*; D001483:Base Sequence; D001665:Binding Sites; D002850:Chromatography, Gel; D004247:DNA / Q000378:metabolism*; D019915:DNA Primase; D004277:DNA, Single-Stranded / Q000378:metabolism; D006868:Hydrolysis; D008024:Ligands; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism*; D012316:RNA Nucleotidyltransferases / Q000378:metabolism*; D013379:Substrate Specificity
D004277:DNA, Single-Stranded; D008024:Ligands; D009711:Nucleotides; D004247:DNA; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases