Binding parameters and thermodynamics of the interaction of the human cytomegalovirus DNA polymerase accessory protein, UL44, with DNA: implications for the processivity mechanism.
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.
Loregian
Arianna
A
Department of Histology, Microbiology and Medical Biotechnologies, University of Padova, 35121 Padova, Italy. arianna.loregian@unipd.it
Sinigalia
Elisa
E
Mercorelli
Beatrice
B
Palù
Giorgio
G
Coen
Donald M
DM
D001665:Binding Sites; D002151:Calorimetry; D002412:Cations; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D024202:Electrophoretic Mobility Shift Assay; D008954:Models, Biological; D011485:Protein Binding; D012492:Salts / Q000737:chemistry; D055672:Static Electricity; D013816:Thermodynamics; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism
D002412:Cations; D004268:DNA-Binding Proteins; C072217:ICP36 protein, Cytomegalovirus; D012492:Salts; D014764:Viral Proteins; D004247:DNA