Processive and unidirectional translocation of monomeric UvsW helicase on single-stranded DNA.
UvsW protein from bacteriophage T4 controls the transition from origin-dependent to origin-independent initiation of replication through the unwinding of R-loops bound to the T4 origins of replication. UvsW has also been implicated through genetic and biochemical experiments to play a role in DNA repair processes such as replication fork regression and Holliday junction branch migration. UvsW is capable of unwinding a wide variety of substrates, many of which contain only duplex DNA without single-stranded regions. Based on this observation, it has been suggested that UvsW is a dsDNA translocase. In this work we examine the ability of UvsW to translocate on ssDNA. Kinetic analysis indicates that the rate of ATP hydrolysis is strongly dependent on the length of the ssDNA lattice, whereas the K(M)-DNA remains relatively constant, demonstrating that UvsW translocates on ssDNA in an ATP-dependent fashion. Experiments using streptavidin blocks or poly dT sequences located at either end of the ssDNA substrate indicate that UvsW translocates in a 3' to 5' direction. Mutant competition and heparin trapping experiments reveal that UvsW is extremely processive during ATP-driven translocation with a half-life on the order of several minutes. Finally, functional assays provide evidence that UvsW is monomeric while translocating on ssDNA. The ability of UvsW to unwind DNA duplexes is likely to be mechanistically linked to its ability to processively translocate on ssDNA in a 3' to 5' unidirectional fashion.
Nelson
Scott W
SW
Department of Chemistry, 414 Wartik Laboratory, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Perumal
Senthil K
SK
Benkovic
Stephen J
SJ
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000235:genetics; D017122:Bacteriophage T4 / Q000201:enzymology*; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000235:genetics*; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry / Q000235:genetics; D011499:Protein Processing, Post-Translational / Q000235:genetics; D014178:Translocation, Genetic; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics*
D004277:DNA, Single-Stranded; D009692:Nucleic Acid Heteroduplexes; D014764:Viral Proteins; D000255:Adenosine Triphosphate; D004265:DNA Helicases; C107414:UvsW protein, Bacteriophage T4