Site-directed Mutations of T4 Helicase Loading Protein (gp59) Reveal Multiple Modes of DNA Polymerase Inhibition and the Mechanism of Unlocking by gp41 Helicase

Abstract

The T4 helicase loading protein (gp59) interacts with a multitude of DNA replication proteins. In an effort to determine the functional consequences of these protein-protein interactions, point mutations were introduced into the gp59 protein. Mutations were chosen based on the available crystal structure and focused on hydrophobic residues with a high degree of solvent accessibility. Characterization of the mutant proteins revealed a single mutation, Y122A, which is defective in polymerase binding and has weakened affinity for the helicase. The interaction between single-stranded DNA-binding protein and Y122A is unaffected, as is the affinity of Y122A for DNA substrates. When standard concentrations of helicase are employed, Y122A is unable to productively load the helicase onto forked DNA substrates. As a result of the loss of polymerase binding, Y122A cannot inhibit the polymerase during nucleotide idling or prevent it from removing the primer strand of a D-loop. However, Y122A is capable of inhibiting strand displacement synthesis by polymerase. The retention of strand displacement inhibition by Y122A, even in the absence of a gp59-polymerase interaction, indicates that there are two modes of polymerase inhibition by gp59. Inhibition of the polymerase activity only requires gp59 to bind to the replication fork, whereas inhibition of the exonuclease activity requires an interaction between the polymerase and gp59. The inability of Y122A to interact with both the polymerase and the helicase suggests a mechanism for polymerase unlocking by the helicase based on a direct competition between the helicase and polymerase for an overlapping binding site on gp59.The T4 replisome is considered to be a model for the more complex replication systems (1, 2). The eight protein replisome can be divided into three modules, the leading strand holoenzyme, the lagging strand holoenzyme, and the primosome (3). The leading and lagging strand holoenzymes are formed by the polymerase (gp43) 4 and the polymerase clamp (gp45) with the aid of the clamp loader protein (gp44/62). Contained within gp43 polymerase are two distinct active sites. One is the polymerization site that catalyzes the 5Ј to 3Ј incorporation of deoxynucleotides (dNTPs) into the growing primer strand, and the other is the exonuclease site that is responsible for the removal of dNMPs in the 3Ј to 5Ј direction (4). gp45 is bound to the polymerase and encircles the DNA duplex, thereby increasing its processivity during DNA synthesis (5, 6). gp44/62 aids in the assembly of the polymeraseclamp complex by positioning the clamp at the 3Ј-OH of the primertemplate junction and acting as a chaperone for the interaction between gp45 and gp43 (7,8). The primosome is made up of the helicase (gp41), primase (gp61), single-stranded DNA-binding protein (gp32), and the helicase loading protein (gp59) (9). gp41 is a hexameric protein that travels along the ssDNA in a 5Ј to 3Ј manner using the energy of ATP hydrolysis to unwind duplex DNA (10, 11). gp61 is also hexameric and is respo...

RNA Primer Handoff in Bacteriophage T4 DNA Replication

“…The helicase is loaded onto the lagging strand template by the helicase loader protein (gp59) (9,10). gp59 plays an additional role as a "gatekeeper" of the replisome by coordinating the assembly of the primosome with the initiation of leading strand DNA synthesis through a direct interaction with the leading strand polymerase (11)(12)(13). Finally, gp32 plays a central role in most aspects of DNA metabolism, including DNA replication (14).…”

Investigation of Stoichiometry of T4 Bacteriophage Helicase Loader Protein (gp59)

“…3 Isothermal titration calorimetry experiments were consistent with a stoichiometry of 0.75 gp59 subunit to 1 gp41 subunit with a dissociation constant of ϳ150 nM. 3 The electron microscopic studies on the structure of the gp41-gp61 complex revealed two hexamers in a 1:1 stoichiometry (19). Based on the collective data, it could be postulated that gp59 should form a functional higher order complex ranging from tetramer to hexamer.…”

“…Cross-linking experiments observed complexes of gp59 up to a pentamer in the presence of gp32, gp41, and DNA. 3 Isothermal titration calorimetry experiments were consistent with a stoichiometry of 0.75 gp59 subunit to 1 gp41 subunit with a dissociation constant of ϳ150 nM. 3 The electron microscopic studies on the structure of the gp41-gp61 complex revealed two hexamers in a 1:1 stoichiometry (19).…”

“…In addition, gp59 inhibits the initiation of polymerization by gp43 (2,3). Furthermore, it acts in recombination and recombination-dependent DNA replication (4,5) and functions as a gatekeeper in origin-dependent replication in vivo (6).…”

“…3 The electron microscopic studies on the structure of the gp41-gp61 complex revealed two hexamers in a 1:1 stoichiometry (19). Based on the collective data, it could be postulated that gp59 should form a functional higher order complex ranging from tetramer to hexamer.…”