DNA-Induced Switch from Independent to Sequential dTTP Hydrolysis in the Bacteriophage T7 DNA Helicase

Abstract

We show that the mechanisms of DNA-dependent and -independent dTTP hydrolysis by the gene 4 protein of bacteriophage T7 differ in the pathways by which these reactions are catalyzed. In the presence of dTTP, gene 4 protein monomers assemble as a ring that binds single-stranded DNA and couples the hydrolysis of dTTP to unidirectional translocation and the unwinding of duplex DNA. When mixing wild-type monomers with monomers lacking the catalytic base for the dTTPase reaction, we observe that each wild-type subunit hydrolyzes dTTP independently in the absence of single-stranded DNA. Conversely, when either these catalytically inactive monomers or altered monomers incapable of binding single-stranded DNA are mixed with wild-type monomers, a small fraction of altered to wild-type monomers causes a sharp decline in DNA-dependent dTTP hydrolysis. We propose that sequential hydrolysis of dTTP is coupled to the transfer of single-stranded DNA from subunit to adjacent subunit.

On helicases and other motor proteins

“…A sequential hydrolysis mechanism with coordination among the subunits has also been proposed for the f12 dsRNA packaging motor P4 [80,81,82 ]. In the case of T7gp4, DNAdependent ATPase activity has been shown to require active ATPase sites at all six subunit interfaces, inconsistent with a probabilistic hydrolysis model [66 ]. For T7gp4, it has been suggested that the reaction cycle may not proceed by one unique pathway and that multiple (perhaps similar) pathways could operate simultaneously [83 ].…”

“…For example, with one defective cylinder, the machine may continue to operate with the remaining five. In the case of T7gp4, DNA-dependent ATPase activity has been shown to require an active catalytic base (E343) at all six subunit interfaces [66 ], but some inactive arginine fingers are permitted [87]. The most direct studies of a multisubunit machine's tolerance to individual ATP site disruption involve the bacterial unfoldase ClpX [79 ].…”

“…It is not yet clear whether these two helicases operate by an identical mechanism or whether a single universal mechanism operates for hexameric helicases because the mode of DNA coordination by T7gp4 and the other hexameric helicases, such as MCM, are unknown. Bacteriophage T7gp4 requires intact lysine residues on the DNA-binding loop II region for all six subunits in order to achieve DNA-dependent ATP hydrolysis [66 ], demonstrating that all six subunits require the capacity to participate in DNA binding, but it is not known how many subunits bind DNA simultaneously. In the case of the homologous DnaB, single-stranded DNA coordination is dramatically tighter for 7-mer oligonucleotides than for 5-mer oligonucleotides [88].…”

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

“…The sequence of these states around the hexamer led Singleton et al (6) to propose a sequential pathway in which all subunits participated in dTTP hydrolysis. Subsequent biochemical studies have largely validated this model (14,16). A structure of the bovine papillomavirus (BPV) E1 helicase, a member of the AAAϩ family, bound to an oligonucleotide and ADP, has provided insight into the actual coupling of nucleotide hydrolysis to translocation on ssDNA (17).…”

“…Upon binding of the nucleotide, motif 4 assumes a helical conformation, suggesting a role for this motif in coupling NTP binding to DNA binding (6). In addition, loop II that protrudes inside the central hole of the hexamer also plays a role in DNA binding (14). Motif 4 from one subunit contacts loop II from an adjacent subunit at the interface of hexameric helicase.…”