T7 DNA helicase: a molecular motor that processively and unidirectionally translocates along single-stranded DNA.
DNA helicases are molecular motors that use the energy from NTP hydrolysis to drive the process of duplex DNA strand separation. Here, we measure the translocation and energy coupling efficiency of a replicative DNA helicase from bacteriophage T7 that is a member of a class of helicases that assembles into ring-shaped hexamers. Presteady state kinetics of DNA-stimulated dTTP hydrolysis activity of T7 helicase were measured using a real time assay as a function of ssDNA length, which provided evidence for unidirectional translocation of T7 helicase along ssDNA. Global fitting of the kinetic data provided an average translocation rate of 132 bases per second per hexamer at 18 degrees C. While translocating along ssDNA, T7 helicase hydrolyzes dTTP at a rate of 49 dTTP per second per hexamer, which indicates that the energy from hydrolysis of one dTTP drives unidirectional movement of T7 helicase along two to three bases of ssDNA. One of the features that distinguishes this ring helicase is its processivity, which was determined to be 0.99996, which indicated that T7 helicase travels on an average about 75kb of ssDNA before dissociating. We propose that the ability of T7 helicase to translocate unidirectionally along ssDNA in an efficient manner plays a crucial role in DNA unwinding.
Department of Biochemistry, UMDNJ-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854, USA.
Dong-Eun
DE
Kim
Murli
M
Narayan
Smita S
SS
Patel
D017104:Bacteriophage M13 / Q000235:genetics; D017123:Bacteriophage T7 / Q000201:enzymology*; D001692:Biological Transport; D004265:DNA Helicases / Q000378:metabolism*; D019915:DNA Primase / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008957:Models, Genetic; D020409:Molecular Motor Proteins / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010710:Phosphates / Q000378:metabolism; D021381:Protein Transport; D011755:Pyrophosphatases / Q000378:metabolism; D013942:Thymine Nucleotides / Q000378:metabolism
D004277:DNA, Single-Stranded; D020409:Molecular Motor Proteins; D009838:Oligodeoxyribonucleotides; D010710:Phosphates; D013942:Thymine Nucleotides; C027903:oligo (dT); D019915:DNA Primase; D011755:Pyrophosphatases; C023117:thymidine-triphosphatase; D004265:DNA Helicases; C024157:thymidine 5'-triphosphate