Essential Lysine Residues in the RNA Polymerase Domain of the Gene 4 Primase-Helicase of Bacteriophage T7

Abstract

At a replication fork DNA primase synthesizes oligoribonucleotides that serve as primers for the lagging strand DNA polymerase. In the bacteriophage T7 replication system, DNA primase is encoded by gene 4 of the phage. The 63-kDa gene 4 protein is composed of two major domains, a helicase domain and a primase domain located in the C-and N-terminal halves of the protein, respectively. T7 DNA primase recognizes the sequence 5-NNGTC-3 via a zinc motif and catalyzes the templatedirected synthesis of tetraribonucleotides pppACNN. T7 DNA primase, like other primases, shares limited homology with DNA-dependent RNA polymerases. To identify the catalytic core of the T7 DNA primase, single-point mutations were introduced into a basic region that shares sequence homology with RNA polymerases. The genetically altered gene 4 proteins were examined for their ability to support phage growth, to synthesize functional primers, and to recognize primase recognition sites. Two lysine residues, Lys-122 and Lys-128, are essential for phage growth. The two residues play a key role in the synthesis of phosphodiester bonds but are not involved in other activities mediated by the protein. The altered primases are unable to either synthesize or extend an oligoribonucleotide. However, the altered primases do recognize the primase recognition sequence, anneal an exogenous primer 5-ACCC-3 at the site, and transfer the primer to T7 DNA polymerase. Other lysines in the vicinity are not essential for the synthesis of primers.

The Linker Region between the Helicase and Primase Domains of the Gene 4 Protein of Bacteriophage T7

“…Biochemical Assays of Gene 4 Protein-Most of the assays used in this study have been described previously in detail (16). All assays (DNA unwinding, DNA binding, dTTP hydrolysis, oligomerization of gene 4 protein, primer synthesis, and DNA synthesis assays) used a reaction buffer containing 40 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 , 10 mM dithiothreitol, and 50 mM potassium glutamate plus additional components described in each assay.…”

“…The plasmids were transformed into E. coli HMS 174(DE3), and gene 4 proteins were overproduced by isopropyl-1-thio-␤-D-galactopyranoside induction. Genetically altered gene 4 proteins were purified following procedures described previously (16). The purified proteins were all greater than 95% pure as determined by SDS-PAGE analysis and staining with Coomassie Blue.…”

“…For example, a set of primers for Ala 257 was 5Ј-CAAGTGT-GGAATNNNGGTCCTTGGATT-3Ј and 5Ј-AATCCAAGGACCNNNAT-TCCACACTTG-3Ј (N, any base). Using these mutagenic primers and outside primers in a two-step overlap PCR procedure (16), a pool of DNA containing mutated codons was generated. After digestion with BstBI and Bsu36I, the DNA (0.62 pmol) was ligated into plasmid pET24gp4-63 (0.049 pmol) previously cut with the same restriction enzymes.…”

“…Each of the six altered gene 4 proteins as well as wild-type gene 4 protein were purified by a standard protocol used for purification of wild-type gene 4 protein described previously (16). All of the altered proteins had similar chromatographic properties to the wild-type gene 4 protein.…”

Communication between subunits critical to DNA binding by hexameric helicase of bacteriophage T7

“…Site-directed mutations were introduced into a plasmid, pET24-gp4, containing T7 gene 4 following a standard procedure. Altered gene 4 proteins were overproduced and purified as described (26).…”