The C-terminal Residues of Bacteriophage T7 Gene 4 Helicase-Primase Coordinate Helicase and DNA Polymerase Activities

Seung-Joo Lee,Boriana Marintcheva,Samir M Hamdan,Charles C Richardson

doi:10.1074/jbc.m604602200

Abstract

The gene 4 protein of bacteriophage T7 plays a central role in DNA replication by providing both helicase and primase activities. The C-terminal helicase domain is not only responsible for DNA-dependent dTTP hydrolysis, translocation, and DNA unwinding, but it also interacts with T7 DNA polymerase to coordinate helicase and polymerase activities. The C-terminal 17 residues of gene 4 protein are critical for its interaction with the T7 DNA polymerase/thioredoxin complex. This C terminus is highly acidic; replacement of these residues with uncharged residues leads to a loss of interaction with T7 DNA polymerase/thioredoxin and an increase in oligomerization of the gene 4 protein. Such an alteration on the C terminus results in a reduced efficiency in strand displacement DNA synthesis catalyzed by gene 4 protein and T7 DNA polymerase/thioredoxin. Replacement of the C-terminal amino acid, phenylalanine, with non-aromatic residues also leads to a loss of interaction of gene 4 protein with T7 DNA polymerase/thioredoxin. However, neither of these modifications of the C terminus affects helicase and primase activities. A chimeric gene 4 protein containing the acidic C terminus of the T7 gene 2.5 single-stranded DNA-binding protein is more active in strand displacement synthesis. Gene 4 hexamers containing even one subunit of a defective C terminus are defective in their interaction with T7 DNA polymerase.

Highlights

Vidual protein functions in several capacities via multiple interactions with the other components of the replisome
C-terminal region abolished a proper interaction with T7 DNA pol/trx, a loss that resulted in a failure of the proteins to carry out strand displacement DNA synthesis
The early study clearly demonstrated the importance of the C-terminal of gene 4 protein in strand displacement DNA synthesis, it is not known what features of the C-terminal region are responsible for its critical interaction with T7 DNA polymerase

Summary

C Terminus of T7 Gene 4 Protein

We showed that deletion of the 17 C-terminal residues of gene 4 protein results in an inability to physically interact with T7 DNA polymerase. The absence of this interaction prevents the helicase and T7 DNA pol/trx from mediating strand displacement DNA synthesis [8]. Whereas this earlier study clearly showed that the helicase domain of gene 4 protein interacts with polymerase via its C-terminal during leading strand DNA synthesis, it did not address the properties of the C terminus important for this interaction. To investigate mechanistic details of the interaction with T7 DNA pol/trx at the C terminus of gene 4 protein, we constructed a variety of alterations on the C-terminal region and their effects on the protein function were examined

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION