Interaction of Bacteriophage T7 Gene 4 Primase with Its Template Recognition Site

Qimron

Proc. Natl. Acad. Sci. U.S.A.

2008

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The DNA helicase encoded by bacteriophage T7 consists of six identical subunits that form a ring through which the DNA passes. Binding of ssDNA is a prior step to translocation and unwinding of DNA by the helicase. Arg-493 is located at a conserved structural motif within the interior cavity of the helicase and plays an important role in DNA binding. Replacement of Arg-493 with lysine or histidine reduces the ability of the helicase to bind DNA, hydrolyze dTTP, and unwind dsDNA. In contrast, replacement of Arg-493 with glutamine abolishes these activities, suggesting that positive charge at the position is essential. Based on the crystallographic structure of the helicase, Asp-468 is in the range to form a hydrogen bonding with Arg-493 on the adjacent subunit. In vivo complementation results indicate that an interaction between Asp-468 and Arg-493 is critical for a functional helicase and those residues can be swapped without losing the helicase activity. This study suggests that hydrogen bonding between Arg-493 and Asp-468 from adjacent subunits is critical for DNA binding ability of the T7 hexameric helicase.DNA replication ͉ hydrogen bonding ͉ subunit interaction ͉ residue swappping T he replication, recombination, and repair of DNA all require the unwinding of duplex DNA. Such an important transformation of dsDNA into ssDNA is carried out by a group of proteins designated as DNA helicases. Underlying the overall process of unwinding dsDNA is the ability of the protein to bind to ssDNA and use the hydrolysis of a nucleoside triphosphate to translocate unidirectionally on the DNA (1, 2). Not surprisingly, DNA helicases differ in the mechanism by which they bind to DNA, translocate on ssDNA, and unwind the duplex. On the basis of their amino acid sequences and 3D structures, DNA helicases can be divided into several families or superfamilies (2, 3).The helicase encoded by gene 4 of bacteriophage T7 is one of the most extensively characterized helicases. As a member of the DnaB-like family (family 4), the gene 4 protein forms a hexameric toroid. The oligomeric structure not only gives rise to the nucleotide bindings at the subunit interfaces but also provides a central cavity through which the ssDNA can pass. Binding sites for DNA and nucleotide span between subunits, thus allowing cooperative binding of DNA and efficient coupling of nucleotide hydrolysis to movement of the DNA strand.Electron microscopy and x-ray crystallography illustrated that the protein forms a closed ring-shaped oligomeric structure composed of six or seven subunits (4-7). Biochemical studies have identified regions critical to oligomerization of the protein, including a linker region that connects the C-terminal helicase domain of the protein to the N-terminal primase domain (8, 9). Four motifs were defined based on conserved amino acid sequences among family 4 helicases. Strictly conserved residues in motifs 1 and 2 contact the nucleotide at the nucleotide binding site located at the interface of subunits. These residues participate in h...

Section: Resultsmentioning

confidence: 99%

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

Qimron

Proc. Natl. Acad. Sci. U.S.A.

2008

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“…These two advantages are intimately related in the case of the T7 gene 4 helicase-primase since the covalent association assures both translocation of the primase and the immediate access to the ssDNA template generated behind the translocating helicase at the replication fork. Aside from the replication fork, primase activity on ssDNA is stimulated greatly by the helicase activity in that the hexameric helicase in the presence of NTP binds tightly to ssDNA whereas the primase has a low affinity for ssDNA including its recognition site (33). Not surprisingly, alterations in the helicase domain of gene 4 protein that affect translocation or helicase activity have drastic effects on primase activity (17,34).…”

Section: Fig 5 Oligoribonucleotide Synthesismentioning

confidence: 99%

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

Journal of Biological Chemistry

2004

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The gene 4 protein of bacteriophage T7 provides both helicase and primase activities. The C-terminal helicase domain is responsible for DNA-dependent dTTP hydrolysis, translocation, and DNA unwinding whereas the N-terminal primase domain is responsible for templatedirected oligoribonucleotide synthesis. A 26 amino acid linker region (residues 246 -271) connects the two domains and is essential for the formation of functional hexamers. In order to further dissect the role of the linker region, three residues (Ala 257 , Pro 259 , and Asp 263 ) that was disordered in the crystal structure of the hexameric helicase fragment were substituted with all amino acids, and the altered proteins were analyzed for their ability to support growth of T7 phage lacking gene 4. The in vivo screening revealed Ala 257 and Asp 263 to be essential whereas Pro 259 could be replaced with any amino acid without loss of function. Selected gene 4 proteins with substitution for Ala 257 or Asp 263 were purified and examined for their ability to unwind DNA, hydrolyze dTTP, translocate on ssDNA, and oligomerize. In the presence of Mg 2؉ , all of the altered proteins oligomerize. However, in the absence of divalent ion, alterations at position 257 increase the extent of oligomerization whereas those at position 263 reduce oligomer formation. Although dTTP hydrolysis activity is reduced only 2-3-fold, none of the altered gene 4 proteins can translocate effectively on single-strand DNA, and they cannot mediate the unwinding of duplex DNA. Primer synthesis catalyzed by the altered proteins is relatively normal on a short DNA template but it is severely impaired on longer templates where translocation is required. The results suggest that the linker region not only connects the two domains of the gene 4 protein and participates in oligomerization, but also contributes to helicase activity by mediating conformations within the functional hexamer.

“…The primase activity of the 63-kDa gene 4 protein is increased by the presence of dTTP on a long DNA template. The stimulatory effect of dTTP on the 63-kDa protein derives from its ability to promote the formation of hexamers, which bind to the template and increase the affinity of the primase for primase recognition sites (23). The primase fragment is unable to form hexamers and thus no stimulation by dTTP is detected (3).…”

Section: Requirement Of Hexamer Formation For Reconstitution Of Primasementioning

confidence: 99%

Interaction of adjacent primase domains within the hexameric gene 4 helicase-primase of bacteriophage T7

Proc. Natl. Acad. Sci. U.S.A.

2002

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The interaction of primase monomers within the hexameric gene 4 helicase-primase of bacteriophage T7 has been examined by using two genetically distinct gene 4 proteins. The T7 56-kDa gene 4 protein differs from the full-length 63-kDa protein in that it lacks the N-terminal zinc motif essential for the recognition of primase recognition sites. A second gene 4 protein, gp4-K122A, is unable to catalyze the synthesis of phosphodiester bonds as the result of an amino acid change in the catalytic site. Although each protein alone is inactive, the two together catalyze the synthesis of RNA primers. Reconstitution of activity depends on hexamer formation. We propose that the zinc motif of one subunit in the hexamer interacts with the catalytic sites of adjacent subunits.