Biochemical Analysis of Mutant T7 Primase/Helicase Proteins Defective in DNA Binding, Nucleotide Hydrolysis, and the Coupling of Hydrolysis with DNA Unwinding

Abstract

We characterized nine helicase-deficient mutants of bacteriophage T7 helicase-primase protein (4A) prepared by random mutagenesis as reported in the accompanying paper (Rosenberg, DNA helicases catalyze unwinding of duplex DNA to singlestranded DNA, a process energetically coupled to NTP hydrolysis. Helicases are an important class of proteins required in almost all the processes of DNA and RNA metabolism. Recently, a large number of putative helicases have been identified mainly from amino acid sequence homologies. Known helicases have homologous amino acid sequences, confined to small regions in the protein, that are used as signature motifs for identifying helicases (1, 2). Since a high resolution structure of a helicase is not known at the present time, the roles of these conserved motifs remain largely unclear.AWe are studying the mechanism of bacteriophage T7 DNA helicase, which is involved in DNA replication. Bacteriophage T7 is a model system used to study the detailed mechanisms of DNA replication because of its simplicity. A minimum of two proteins, T7 DNA polymerase and T7 DNA primase/helicase, have been shown to reconstitute duplex DNA replication in vitro. T7 gene 4 encodes the two primase/helicase proteins, 4A and 4B (3). The full-length 63-kDa 4A protein has both helicase and primase activities, whereas the shorter 56-kDa 4B protein that begins at a second initiation codon has only helicase activity (4, 5). The helicase activity unwinds double-stranded DNA during leading strand DNA replication, and the primase catalyzes synthesis of tetraribonucleotides that serve as primers for lagging strand DNA replication (4).T7 DNA helicase belongs to the general class of hexameric helicases. Its low resolution structure, studied in detail using electron microscopy and image averaging, shows that both 4AЈ and 4B proteins form ring-shaped hexamers, and the ssDNA 1 binds through the central hole of the ring (6). This mode of DNA binding results in protection of about 25 bases of ssDNA from nuclease digestion (7) and likely confers high processivity to DNA unwinding. The helicase forms hexamers only in the presence of nucleotide ligands such as dTDP, dTTP, ATP, and dTMP-PCP (8, 9). DNA binds tightly only to the hexameric species and requires the presence of dTTP or dTMP-PCP (7). The various activities of the helicase protein such as NTP binding/hydrolysis, protein oligomerization, and DNA binding are linked (9). Therefore, it is likely that amino acids responsible for these activities also may be close in space or perhaps lie in the same motif.Regions of T7 gene 4 protein show sequence homology to several bacterial and bacteriophage primase/helicase and primase-related helicases that belong to the DnaB family of helicases. Comparison of amino acid sequences in this family of helicases has led to the identification of five conserved motifs denoted 1A, 1a, 2B, 3, and 4 (2). Conserved motif 1A is the well known GXXGXGKT/S sequence found in numerous nucleotidebinding proteins and shown in many ATPases to be involved...

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

“…However, all of the altered proteins were more abundant in extracts and the overall yield after purification was 2-5-fold greater than for wildtype gene 4 protein. Similar increases in overexpression have been observed with altered gene 4 proteins defective in dTTPase and helicase activity (8,17).…”

“…As shown in Fig. 5B, the wild-type gene 4 protein requires the presence of dTTP to synthesize primers effectively on a 65-mer oligonucleotide, 5Ј-(N) 43 TGGTC(N) 17 -3Ј. In this assay where translocation is important, the primase activities of gp4-A257V and gp4-D263N are not stimulated by dTTP.…”

“…Residues Pro 259 and Asp 263 were chosen since they are conserved in gene 4 protein of phage T3 where the primase is also fused to the helicase. Residue Ala 257 was included in this study since alteration at this position had been shown previously to affect DNA-dependent dTTP hydrolysis and DNA unwinding (17).…”

“…On relatively long templates primase activity is dependent on the translocation of the protein to the recognition site, a reaction mediated by the helicase domain of gene 4 protein. A 65-mer (5Ј-(N) 43 TGGTC(N) 17 -3Ј) containing a primase recognition site 5Ј-TGGTC-3Ј was incubated with CTP, ATP, and the indicated concentrations of dTTP or its non-hydrolyzable analog ␤,␥-methylene dTTP. The reactions contained 20 nM wild-type gene 4 protein, gp4-A257V, or gp4-D263N.…”

Regulation of mitochondrial DNA content and cancer

“…Therefore, this protein regulates concentration of adenine nucleotides in the cytoplasm and mitochondria. Twinkle is a mitochondrial protein with homology to phage T7 primase/helicase, and the mutation to Twinkle enhances dNTPase activity (Washington et al, 1996). The identification of mutations of genes encoding ANT1 and Twinkle in patients with adPEO and thymidine phosphorylase in patients with MINGIE indicate that imbalance of mitochondrial nucleotide pools may cause multiple deletion of mtDNA and depletion of mtDNA.…”