Regulation of the bacteriophage T4 Dda helicase by Gp32 single-stranded DNA–binding protein

Abstract

Dda, one of three helicases encoded by bacteriophage T4, has been well-characterized biochemically but its biological role remains unclear. It is thought to be involved in origin dependent DNA replication, recombination-dependent replication, anti-recombination, and recombination repair. The Gp32 protein of bacteriophage T4 plays critical roles in DNA replication, recombination, and repair by coordinating protein components of the replication fork and by stabilizing ssDNA. Previous work demonstrated that stimulation of DNA synthesis by Dda helicase appears to require direct Gp32-Dda protein-protein interactions and that Gp32 and Dda form a tight complex in the absence of ssDNA. Here we characterize the effects of Gp32-Dda physical and functional interactions through changes in the duplex DNA unwinding and ATPase activities of Dda helicase in the presence of different variants of Gp32 and different DNA repair and replication intermediate structures. Results show that Gp32-Dda interactions can be enhancing or inhibitory, depending on the Gp32 domain seen by Dda. Protein-protein interactions with Gp32 stimulate the unwinding activity of Dda, an effect associated with increased turnover of ATP, suggesting a higher rate of ATPase-driven translocation. Dda-Gp32 interactions also promote the unwinding of DNA substrates at higher salt concentrations and in the presence of substrate-bound DNA polymerase. Conversely, the formation of Gp32 clusters on ssDNA can inhibit unwinding, suggesting that Gp32-ssDNA formation sterically regulates which portions of replication and recombination intermediates are accessible for processing by Dda helicase. The data suggest a mechanism of replication fork restart in which Gp32 promotes Dda activity in template switching while preventing premature fork progression.

Using in silico modelling and FRET-based assays in the discovery of novel FDA-approved drugs as inhibitors of MERS-CoV helicase

“…Helicases are also essential for RNA transcription, mRNA splicing, mRNA export, translation, and RNA stability [13][14][15]. Helicases are involved in molecular processes, including protein-nucleic acid complexes dissociation [16][17][18], Holliday junction displacement [19], chromatin remodelling [20], and conformational modifications of nucleic acid [21][22][23]. MERS-CoV is a positive single-stranded RNA (ssRNA) virus belonging to the Coronaviridae family, and has one of the largest known RNA genomes (~31.7 kb) [24,25].…”

Efficient Genome Editing with Chimeric Oligonucleotide-Directed Editing

“…2a,b). T4 gp32 is a single-stranded binding protein (SSB) that is crucial for T4 replication and repair 19,20 . However, we observed reduced activity compared to CODE-T4v3, possibly due to gp32 SSB being inactive in a fusion format or being sterically hindered by nCas9 and T4 DNAP.…”

Single molecule technique unveils the role of electrostatic interactions in ssDNA–gp32 molecular complex stability

“… 50 Biochemical insights obtained from studies of gp32 continue to serve as an important basis for understanding the function of these proteins in bacteria and other higher organisms. 51 The gp32 protein has an N-terminal domain, a C-terminal domain, and a core domain. The N-terminal domain is necessary for the cooperative binding of the gp32 protein through its interactions with the core domain of an adjacent gp32 protein.…”