The DNA-unwinding mechanism of the ring helicase of bacteriophage T7.
Helicases are motor proteins that use the chemical energy of NTP hydrolysis to drive mechanical processes such as translocation and nucleic acid strand separation. Bacteriophage T7 helicase functions as a hexameric ring to drive the replication complex by separating the DNA strands during genome replication. Our studies show that T7 helicase unwinds DNA with a low processivity, and the results indicate that the low processivity is due to ring opening and helicase dissociating from the DNA during unwinding. We have measured the single-turnover kinetics of DNA unwinding and globally fit the data to a modified stepping model to obtain the unwinding parameters. The comparison of the unwinding properties of T7 helicase with its translocation properties on single-stranded (ss)DNA has provided insights into the mechanism of strand separation that is likely to be general for ring helicases. T7 helicase unwinds DNA with a rate of 15 bp/s, which is 9-fold slower than the translocation speed along ssDNA. T7 helicase is therefore primarily an ssDNA translocase that does not directly destabilize duplex DNA. We propose that T7 helicase achieves DNA unwinding by its ability to bind ssDNA because it translocates unidirectionally, excluding the complementary strand from its central channel. The results also imply that T7 helicase by itself is not an efficient helicase and most likely becomes proficient at unwinding when it is engaged in a replication complex.
Jeong
Yong-Joo
YJ
Department of Biochemistry, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854, USA.
Levin
Mikhail K
MK
Patel
Smita S
SS
D017123:Bacteriophage T7 / Q000201:enzymology*; D001483:Base Sequence; D004247:DNA / Q000737:chemistry; D004265:DNA Helicases / Q000502:physiology*; D004264:DNA Topoisomerases, Type I / Q000502:physiology*; D007700:Kinetics; D008956:Models, Chemical
D004247:DNA; D004265:DNA Helicases; D004264:DNA Topoisomerases, Type I