Abstract
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 template-directed 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 (Ala257, Pro259, and Asp263) 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 Ala257 and Asp263 to be essential whereas Pro259 could be replaced with any amino acid without loss of function. Selected gene 4 proteins with substitution for Ala257 or Asp263 were purified and examined for their ability to unwind DNA, hydrolyze dTTP, translocate on ssDNA, and oligomerize. In the presence of Mg2+, 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.
Highlights
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
In order to further dissect the role of the linker region, three residues (Ala257, Pro259, and Asp263) 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
Summary
In addition to these interactions at the interface, a series of loops near the nucleotide binding site make contact between adjacent subunits with potential contacts of adjacent subunits with the bound nucleotide While it is clear from the biochemical and structural data that the linker region is essential for oligomerization of the gene 4 protein, the identity of the key residues critical for protein-protein interaction are unknown since previous studies have made use of truncated helicase domains. It is not known if the linker region via its contact with adjacent subunits assists in coordinating helicase and primase activities at the replication fork. We find that two of these residues, Ala257 and Asp263, play a critical role in oligomerization and translocation of the gene 4 protein
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