DNA-induced switch from independent to sequential dTTP hydrolysis in the bacteriophage T7 DNA helicase.
We show that the mechanisms of DNA-dependent and -independent dTTP hydrolysis by the gene 4 protein of bacteriophage T7 differ in the pathways by which these reactions are catalyzed. In the presence of dTTP, gene 4 protein monomers assemble as a ring that binds single-stranded DNA and couples the hydrolysis of dTTP to unidirectional translocation and the unwinding of duplex DNA. When mixing wild-type monomers with monomers lacking the catalytic base for the dTTPase reaction, we observe that each wild-type subunit hydrolyzes dTTP independently in the absence of single-stranded DNA. Conversely, when either these catalytically inactive monomers or altered monomers incapable of binding single-stranded DNA are mixed with wild-type monomers, a small fraction of altered to wild-type monomers causes a sharp decline in DNA-dependent dTTP hydrolysis. We propose that sequential hydrolysis of dTTP is coupled to the transfer of single-stranded DNA from subunit to adjacent subunit.
Crampton
Donald J
DJ
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Mukherjee
Sourav
S
Richardson
Charles C
CC
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017123:Bacteriophage T7 / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism*; D005814:Genes, Viral; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D021122:Protein Subunits; D017386:Sequence Homology, Amino Acid; D013942:Thymine Nucleotides / Q000378:metabolism*
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D021122:Protein Subunits; D013942:Thymine Nucleotides; D004265:DNA Helicases; C024157:thymidine 5'-triphosphate