Oligomeric structure of bacteriophage T7 DNA primase/helicase proteins.
The oligomeric structure of bacteriophage T7 gene 4 helicase/primase proteins was investigated using protein cross-linking and high pressure gel-filtration chromatography. Studies were carried out with both 4A' and 4B proteins. 4A' is a M64L mutant of 4A which has similar helicase and primase activities as the wild-type mixture of 4A and 4B proteins (Patel, S. S., Rosenberg, A. H., Studier, F. W., and Johnson, K. A. (1992) J. Biol. Chem. 267, 15013-15021), and 4B is the smaller protein which has only helicase activity. Chemical cross-linking of 4A' and 4B proteins with dimethyl suberimidate resulted in cross-linked species ranging from dimers to hexamers and beyond. The cross-linking time course, however, indicated that hexamers were the predominant species to accumulate in both 4A' and 4B proteins. The effect of MgNTP and DNA binding on oligomerization of the gene 4 proteins was investigated using high pressure gel-filtration chromatography at increasing protein concentrations. In the absence of added ligands, close to 100 microM protein concentrations were required to form stable oligomers beyond dimers. However, in the presence of Mg-beta, gamma-methylene deoxythymidine triphosphate (nonhydrolyzable analog of dTTP), 4A' and 4B protein assembled into stable hexamers at protein concentrations less than 8 microM. Addition of single-stranded DNA further stabilized the hexamer structure. Therefore, in the presence of a 60-nucleotide-long single-stranded DNA, hexamers were observed at protein concentrations as low as 0.2 microM. Nuclease protection experiments indicated that the 4A' and 4B hexamers protect about 60-65 bases of single-stranded DNA.
Patel
S S
SS
Department of Biochemistry, Ohio State University, Columbus 43210.
Hingorani
M M
MM
D017123:Bacteriophage T7 / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002850:Chromatography, Gel; D019915:DNA Primase; D004120:Dimethyl Suberimidate; D004591:Electrophoresis, Polyacrylamide Gel; D005814:Genes, Viral; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D011071:Poly T / Q000378:metabolism; D012316:RNA Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011071:Poly T; D004120:Dimethyl Suberimidate; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases