Kaposi's sarcoma associated herpesvirus (KSHV) is an emerging pathogen whose mechanism of replication is poorly understood. PF-8, the presumed processivity factor of the KSHV DNA polymerase, acts in combination with the catalytic subunit, Pol-8, to synthesize viral DNA. We have solved the crystal structure of residues 1-304 of PF-8 at a resolution of 2.8 Å. We found that like the human cytomegalovirus protein UL44, PF-8 forms a head-to-head dimer in the form of a C-clamp with its concave face containing a number of basic residues predicted to be important for DNA binding. However, PF-8 shows several specific features when compared with related proteins. Using the crystal structures of PF-8, of the herpes simplex virus catalytic subunit UL30, and of the RB69 bacteriophage DNA polymerase in complex with DNA, we created a model of how PF-8 might form a ternary complex with Poi-8 and DNA. The structure and the model suggest interesting similarities and differences in how PF-8 functions relative to structurally similar proteins.
To explore the interactions between PF-8 and DNA further, we used filter-binding and single-molecule assays to compare PF-8 directly to UL44. Despite the presence of long loops that are thought to wrap around DNA, the diffusion coefficient of UL44 was found to be more sensitive to salt than that of PF-8. UL44 appeared to hop, similarly to the previously characterized HSV-1 processivity factor UL42. In contrast, PF-8 appeared to predominantly slide. In addition, the diffusion coefficient of UL44 was more affected by an increase in its radius, reflecting a helical path of diffusion while the diffusion coefficient of PF-8 was more consistent with a non-helical path of diffusion, although in the presence of viscogens there was evidence of both helical and non-helical diffusion for PF-8.
Together, these data support the importance of the characteristic features found in the crystal structure of PF-8 and serve as a starting point for a better understanding of how PF-8 might bind to Pol-8 and diffuse on DNA. These results also highlight how seemingly minor differences in similar structures can result in significant distinctions at the functional level.
|Advisor:||Coen, Donald, Hogle, James|
|School Location:||United States -- Massachusetts|
|Source:||DAI-B 72/01, Dissertation Abstracts International|
|Subjects:||Biology, Molecular biology, Biophysics|
|Keywords:||Accessory subunit, DNA Polymerases, Herpesvirus, KSHV, PF-8, Replication|
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