A hexameric helicase encircles one DNA strand and excludes the other during DNA unwinding.
The bacteriophage T7 DNA helicase/primase (gene 4 protein) is a ring-like hexamer that encircles ssDNA and requires forked DNA to catalyze DNA unwinding. We report that optimal rates of unwinding of forked DNA require ssDNA tails of 55 nucleotides on the 5'-to-3' strand and 15 nucleotides on the 3'-to-5' strand. Surprisingly, streptavidin bound to a biotinylated 3'-end fully substitutes for the 3'-to-5' ssDNA tail. This suggests that excluding the 3'-to-5' DNA strand from the center of the helicase is an essential aspect of the mechanism of hexameric helicase-catalyzed DNA unwinding. We also report that streptavidin bound to a biotinylated dT within the 5'-to-3' strand of the duplexed region abolishes DNA unwinding; whereas, streptavidin bound to a biotinylated dT within the duplexed region of the other strand has no effect. These results unambiguously demonstrate that the T7 gene 4 protein is a 5'-to-3' helicase and imply that during DNA unwinding the 5'-to-3' strand transverses the center of the ring while the 3'-to-5' strand is excluded from the center of the ring. Implications for collisions between a helicase and other protein-DNA complexes are discussed.
Hacker
K J
KJ
Department of Biochemistry and Molecular Biology, 106 Althouse Laboratory, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Johnson
K A
KA
D017123:Bacteriophage T7 / Q000201:enzymology*; D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D019915:DNA Primase / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011485:Protein Binding; D019809:Streptavidin / Q000378:metabolism; D013942:Thymine Nucleotides / Q000378:metabolism
D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides; D013942:Thymine Nucleotides; D004247:DNA; D019809:Streptavidin; D019915:DNA Primase; C024157:thymidine 5'-triphosphate