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. 2007;35(14):4779-91.
doi: 10.1093/nar/gkm506. Epub 2007 Jul 7.

Binding parameters and thermodynamics of the interaction of the human cytomegalovirus DNA polymerase accessory protein, UL44, with DNA: implications for the processivity mechanism

Arianna Loregian  1 , Elisa Sinigalia, Beatrice Mercorelli, Giorgio Palù, Donald M Coen
Affiliations

Binding parameters and thermodynamics of the interaction of the human cytomegalovirus DNA polymerase accessory protein, UL44, with DNA: implications for the processivity mechanism

Arianna Loregian et al. Nucleic Acids Res. 2007.

Abstract

The mechanisms of processivity factors of herpesvirus DNA polymerases remain poorly understood. The proposed processivity factor for human cytomegalovirus DNA polymerase is a DNA-binding protein, UL44. Previous findings, including the crystal structure of UL44, have led to the hypothesis that UL44 binds DNA as a dimer via lysine residues. To understand how UL44 interacts with DNA, we used filter-binding and electrophoretic mobility shift assays and isothermal titration calorimetry (ITC) analysis of binding to oligonucleotides. UL44 bound directly to double-stranded DNA as short as 12 bp, with apparent dissociation constants in the nanomolar range for DNAs >18 bp, suggesting a minimum DNA length for UL44 interaction. UL44 also bound single-stranded DNA, albeit with lower affinity, and for either single- or double-stranded DNA, there was no apparent sequence specificity. ITC analysis revealed that UL44 binds to duplex DNA as a dimer. Binding was endothermic, indicating an entropically driven process, likely due to release of bound ions. Consistent with this hypothesis, analysis of the relationship between binding and ionic strength indicated that, on average, 4 +/- 1 monovalent ions are released in the interaction of each monomer of UL44 with DNA. The results taken together reveal interesting implications for how UL44 may mediate processivity.

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Figures

Figure 1.
Figure 1.
UL44 binding to double-stranded and single-stranded oligonucleotides of different lengths. Increasing concentrations of UL44ΔC290 were incubated with 1 fmol of radiolabeled double-stranded (A) or single-stranded (B) oligonucleotides (0.1 nM final concentration). Free and protein-bound DNA were quantified by filter-binding assays, and the fraction of protein-bound DNA was plotted against the protein concentration (calculated as a monomer). Apparent dissociation constants correspond to protein concentrations that led to half-maximal occupation of binding sites, as described in legend to Table 1.
Figure 2.
Figure 2.
DNA-binding activity of baculovirus-expressed full-length UL44. Reactions were performed as in Figure 1, but contained increasing concentrations of baculovirus-expressed, full-length UL44 protein. Apparent dissociation constants, which correspond to protein concentrations that led to half-maximal occupation of binding sites, are reported in Table 2.
Figure 3.
Figure 3.
Electrophoretic mobility shift analysis of UL44 binding to double-stranded oligonucleotides. Binding reactions, which contained 1 nM ds 18-bp oligonucleotide and the indicated concentrations of truncated, wild-type (wt) UL44ΔC290 (A) or 1 nM ds 30-bp oligonucleotide and truncated, wt UL44ΔC290 (B) or full-length, wt UL44 (C) or truncated, mutant UL44ΔC290 L86A/L87A protein (D), were incubated for 10 min at room temperature in binding buffer prior to separation on a native polyacrylamide gel. The ds templates were formed by annealing synthetic oligonucleotides with their complement. The annealed oligonucleotides were radiolabeled and gel-purified as described in the Materials and Methods section. The arrows in panel D point at faster migrating species, likely corresponding to UL44 monomer–DNA complexes, observed in EMSA analysis of DNA binding of the UL44ΔC290 L86A/L87A mutant, which is defective in dimerization, but not in EMSA with wild-type UL44ΔC290.
Figure 4.
Figure 4.
ITC analysis of the binding of UL44 to ds 18-bp DNA. Titrations were performed with 10-μl injections of ds 18mer into a sample cell containing UL44ΔC290 (left panel), or, as a control, buffer only (right panel). (A) Raw data for the titrations, in which the power output in microcalories per second is measured as a function of time in minutes. (B) The heats of dilution of both protein and DNA were subtracted, and the area under each injection curve was integrated to generate the points, which represent heat exchange in kilocalories per mole and are plotted against the cumulative DNA-to-protein molar ratio for each injection. The solid line represents the best-fit curve for the data. The thermodynamic parameters describing the fit are presented in Table 3.
Figure 5.
Figure 5.
ITC analysis of the binding of UL44 to ds 30-bp DNA. Titrations were performed with 10-μl injections of ds 30mer into a sample cell containing UL44ΔC290 (left panel), or, as a control, buffer only (right panel). (A) Raw data for the titrations, in which the power output in microcalories per second is measured as a function of time in minutes. (B) The heats of dilution of both protein and DNA were subtracted, and the area under each injection curve was integrated to generate the points, which represent heat exchange in kilocalories per mole and are plotted against the cumulative DNA-to-protein molar ratio for each injection. The solid line represents the best-fit curve for the data. The thermodynamic parameters describing the fit are presented in Table 4.
Figure 6.
Figure 6.
The dependence of the observed binding constant of the UL44–DNA interaction on the concentration of sodium chloride or sodium acetate. Experiments were performed by incubating increasing concentrations of UL44ΔC290 with 1 fmol of radiolabeled ds 18-bp oligonucleotide and in the presence of varying concentrations of sodium chloride (A) or sodium acetate (B). Free and protein-bound DNA were quantified by filter-binding assays, and, for each salt concentration, a plot such as those shown in Figure 1 was created, wherein the Kd value corresponds to protein concentrations that led to half-maximal occupation of binding sites. The Kobs at each salt concentration was calculated as the reciprocal of Kd value. The logarithm of Kobs was then plotted as a function of the logarithm of the Na+ concentration (lower X-axis). Salt concentrations used in these experiments are indicated on the upper X-axis.
Figure 7.
Figure 7.
The dependence of the observed binding constant of the UL44–DNA interaction on the concentration of MgC12. Reactions were performed as in Figure 6, but contained various concentrations of MgC12. The log Kobs was plotted against the log of Mg2+ concentration (lower X-axis). MgC12 concentrations used in these experiments are indicated on the upper X-axis.
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