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.
Highlights
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]
Cross-linking and fluorescence resonance energy transfer experiments (FRET) studies indicate that a complex between these two proteins is formed at the replication fork prior to the initiation of DNA replication [9, 16, 27]
Following the first few rounds of origin-initiated replication, the majority of DNA replication in T4 phage is initiated through strand invasion of ssDNA into homologous dsDNA templates catalyzed through the combined action of UvsX, UvsY, and gp32 [43]
Summary
[␣-32P]dCTP was purchased from PerkinElmer Life Sciences. Unlabeled ribonucleotides were purchased from Roche Applied Science. The reaction was carried out at 37 °C, and various aliquots were removed at the time points indicated and quenched with an equal volume of 250 mM EDTA, 0.2% SDS, and loading buffer (50% glycerol, 1 g/ml bromphenol blue, 1 g/ml xylene cyanol FF). D-loop Initiated Replication—For the polymerase inhibition reactions, the 5Ј-32P-labeled ss80mer (10 nM) was preincubated with 1.5 M UvsX and 0.25 M UvsY for 10 min at 37 °C prior to mixing with a 3-fold molar excess of dsM131500 (30 nM) in the presence of 2 mM ATP, 50 M dNTPs, 10 mM creatine phosphate, and 5 units/ml of creatine kinase followed by the immediate addition of the replisomal proteins at the following concentrations: 200 nM gp exoϪ, gp44/62, and gp45; 400 nM gp (where indicated); 2 M gp; and 100 nM wild-type or Y122A gp mutant. Aliquots were removed at the indicated time points, quenched with 500 mM EDTA, and separated on 6% denaturing PAGE for 2.5 h
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