Replisome mediated DNA replication in bacteriophage T4 requires the coordinated effort of eight proteins to efficiently replicate DNA on both the leading and lagging-strands. This relatively simple DNA replication model system possesses functional analogs with prokaryotes and eukaryotes. For minimal leading-strand DNA synthesis, only four proteins are required to form the holoenzyme: the DNA polymerase (gp43) and two accessory factors, the sliding clamp (gp45) and the clamp-loader (a complex of gp44 and gp62). For lagging-strand DNA synthesis, the single-stranded DNA binding protein (gp32) and the primosome complex are also required. The primosome consists of the helicase (gp41) and the primase (gp61) proteins. The helicase loading protein (gp59) is required for loading helicase onto gp32-coated DNA.

Our specific research interests are the investigation of the kinetics of assembly and disassembly of the holoenzyme and primosome, the mechanism and dynamics of proteins within the holoenzyme and primosome, and the identification of the protein-protein interactions within and between the holoenzyme and primosome. These studies culminate in the in vitro assembly of a functional replisome capable of coordinated leading and lagging-strand synthesis. We use steady-state (fluorescence and UV-vis spectroscopy) as well as presteady-state (stopped flow and rapid quench) techniques to elucidate kinetic parameters and associations between the various components. Protein-protein interactions are investigated by a variety of techniques including isothermal titration calorimetry (ITC), analytical ultracentrifugation, surface plasmon resonance, cross-linking, tandem mass spectrometry, ensemble fluorescence resonance energy transfer (FRET), and single-molecule FRET. These experimental approaches are supplemented with computer modeling and molecular mechanics simulations to help elucidate a complete model of DNA replication.

SPECIFIC AREAS OF RESEARCH

The T4 DNA polymerase holoenzyme constitutes the core of the replisome and is responsible for the processive synthesis of complementary DNA in the 5’ to 3’ direction. The polymerase (gp43) alone can only synthesize short DNA strands. Another essential protein component, the sliding clamp (gp45), acts as the processivity factor by topologically tethering the polymerase to the DNA and greatly increasing its processivity. To load gp45 onto DNA the T4 clamp loader protein (gp44/62) is required. The gp44/62 protein functions as a ‘matchmaker’ and presumably opens and properly orients the clamp on DNA. The T4 gp44/62 belongs to the versatile AAA+ protein family (ATPases associated with a variety of cellular activities) that convert the energy from ATP hydrolysis to the mechanical force required to load gp45 onto DNA.

We have previously demonstrated the pathway resulting in the assembly of active holoenzyme (see Figure). Recently, we have found through both steady-state and time-resolved FRET experiments that gp45 exists as an open ring in solution. The observation of an open form of gp45 in solution raises an intriguing question regarding the role of gp44/62 in clamp loading. Since gp45 posseses a single opened interface large enough to allow the passage of dsDNA, why does the clamp loader (gp44/62) hydrolyze two equivalents of ATP to initiate loading of an already opened clamp? We are studying alternative pathways to load clamp using stopped-flow FRET and rapid-quench experiments to investigate the dynamics of clamp opening/closing and ATP utilization under various controlled mixing conditions.

Equally important to the assembly of holoenzyme is the disassembly of holoenzyme, which is counterintuitive given the stability of the holoenzyme, during the repetitive lagging strand Okazaki fragment synthesis. We are investigating the mechanism that triggers holoenzyme disassembly and the driving force for this process.

Two anti-parallel DNA strands are replicated in an asymmetrical manner during DNA replication. Leading strand synthesis is continuous whereas lagging strand synthesis is discontinuous and results in the formation of short Okazaki fragments. Both leading and lagging holoenzymes possess high processivity during replication, implying that the repeated dissociation of the lagging polymerase from DNA is a controlled process. We are currently investigating the mechanism responsible for signaling the recycling of the lagging strand polymerase during lagging strand synthesis.

Figure from: Xi J, Zhang Z, Zhuang Z, Yang J, Spiering MM, Hammes GG, Benkovic SJ. (2005) "Interaction between the T4 Helicase Loading Protein (gp59) and the                            DNA Polymerase (gp43): Unlocking of the gp59-gp43-DNA Complex to Initiate Assembly of A Fully Functional Replisome." Biochemistry. 44(21):7747-56.

The bacteriophage T4 replisome, which is responsible for highly processive and efficient DNA replication, is likely to be assembled in a stepwise fashion. The process is initiated by gp59 binding to the fork region of a D loop containing a gp32-coated lagging strand. Gp59 assists in the assembly of the leading-strand holoenzyme and the loading of gp41. Following the loading of gp41, gp59 dissociates and gp61 is loaded to form the primosome complex with gp41. The formation of the primosome complex subsequently triggers the assembly of the lagging strand holoenzyme resulting in a fully active replisome. We are currently investigating the formation of the primosome complex and how the loading of gp61 initiates the assembly of the lagging strand holoenzyme.