Residues in the central beta-hairpin of the DNA helicase of bacteriophage T7 are important in DNA unwinding.
The ring-shaped helicase of bacteriophage T7 (gp4), the product of gene 4, has basic beta-hairpin loops lining its central core where they are postulated to be the major sites of DNA interaction. We have altered multiple residues within the beta-hairpin loop to determine their role during dTTPase-driven DNA unwinding. Residues His-465, Leu-466, and Asn-468 are essential for both DNA unwinding and DNA synthesis mediated by T7 DNA polymerase during leading-strand DNA synthesis. Gp4-K467A, gp4-K471A, and gp4-K473A form fewer hexamers than heptamers compared to wild-type helicase and alone are deficient in DNA unwinding. However, they complement for the growth of T7 bacteriophage lacking gene 4. Single-molecule studies show that these three altered helicases support rates of leading-strand DNA synthesis comparable to that observed with wild-type gp4. Gp4-K467A, devoid of unwinding activity alone, supports leading-strand synthesis in the presence of T7 DNA polymerase. We propose that DNA polymerase limits the backward movement of the helicase during unwinding as well as assisting the forward movement necessary for strand separation.
Satapathy
Ajit K
AK
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
Kochaniak
Anna B
AB
Mukherjee
Sourav
S
Crampton
Donald J
DJ
van Oijen
Antoine
A
Richardson
Charles C
CC
D000595:Amino Acid Sequence; D017123:Bacteriophage T7 / Q000201:enzymology* / Q000378:metabolism; D018360:Crystallography, X-Ray / Q000379:methods; D004247:DNA / Q000737:chemistry; D004265:DNA Helicases / Q000737:chemistry* / Q000378:metabolism; D005816:Genetic Complementation Test; D006868:Hydrolysis; D007700:Kinetics; D008957:Models, Genetic; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D004247:DNA; D004265:DNA Helicases