Heterohexamer of 56- and 63-kDa Gene 4 Helicase-Primase of Bacteriophage T7 in DNA Replication.
Bacteriophage T7 expresses two forms of gene 4 protein (gp4). The 63-kDa full-length gp4 contains both the helicase and primase domains. T7 phage also express a 56-kDa truncated gp4 lacking the zinc binding domain of the primase; the protein has helicase activity but no DNA-dependent primase activity. Although T7 phage grow better when both forms are present, the role of the 56-kDa gp4 is unknown. The two molecular weight forms oligomerize by virtue of the helicase domain to form heterohexamers. The 56-kDa gp4 and any mixture of 56- and 63-kDa gp4 show higher helicase activity in DNA unwinding and strand-displacement DNA synthesis than that observed for the 63-kDa gp4. However, single-molecule measurements show that heterohexamers have helicase activity similar to the 63-kDa gp4 hexamers. In oligomerization assays the 56-kDa gp4 and any mixture of the 56- and 63-kDa gp4 oligomerize to form more hexamers than does the 63-kDa gp4. The zinc binding domain of the 63-kDa gp4 interferes with hexamer formation, an inhibition that is relieved by the insertion of the 56-kDa species. Compared with the 63-kDa gp4, heterohexamers synthesize a reduced amount of oligoribonucleotides, mediated predominately by the 63-kDa subunits via a cis mode. During coordinated DNA synthesis 7% of the tetraribonucleotides synthesized are used as primers by both heterohexamers and hexamers of the 63-kDa gp4. Overall, an equimolar mixture of the two forms of gp4 shows the highest rate of DNA synthesis during coordinated DNA synthesis.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Huidong
H
Zhang
Seung-Joo
SJ
Lee
Arkadiusz W
AW
Kulczyk
Bin
B
Zhu
Charles C
CC
Richardson
D017123:Bacteriophage T7 / Q000201:enzymology* / Q000235:genetics; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D019915:DNA Primase / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication / Q000502:physiology*; D004279:DNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D055503:Protein Multimerization / Q000502:physiology*; D017434:Protein Structure, Tertiary
D004279:DNA, Viral; D019915:DNA Primase; D004265:DNA Helicases