Human Kaposi's sarcoma herpesvirus processivity factor-8 functions as a dimer in DNA synthesis.
In the absence of other proteins, the DNA polymerase (Pol-8) of Kaposi's sarcoma herpesvirus incorporates only several nucleotides from a primer template. However, association with the Kaposi's sarcoma herpesvirus processivity factor (PF-8) enables Pol-8 to incorporate thousands of nucleotides. Unlike the well described sliding clamp processivity factors, eukaryotic proliferating cell nuclear antigen and Escherichia coli beta-subunit, PF-8 and other herpesvirus processivity factors do not require a clamp loader or ATP to bind to template DNA. To begin to understand the mechanism used by PF-8 to achieve processivity, we have now purified PF-8 and demonstrated that it is a dimer both in solution and on the DNA. Mutational analysis of the PF-8 protein (396R) indicates that residues between 277 and 304 as well as the N-terminal 21 amino acids are required for dimerization. The results further correlate PF-8 dimerization with binding to Pol-8 and stabilizing Pol-8 on primer template. Notably, although removal of only 26 residues from the C terminus of PF-8 does not affect its ability to form dimers on DNA or to bind Pol-8, only short DNA chains (<100 nucleotides) are synthesized. This indicates that full-length PF-8 is necessary to enable Pol-8 to incorporate thousands of nucleotides. Interestingly, cross-linking of the processivity factor UL44 of cytomegalovirus reveals that it is a dimer in solution also.
Chen
Xulin
X
Department of Microbiology, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Lin
Kai
K
Ricciardi
Robert P
RP
D000255:Adenosine Triphosphate / Q000737:chemistry; D019921:Biotinylation; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003587:Cytomegalovirus / Q000235:genetics; D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D004252:DNA Mutational Analysis; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D017353:Gene Deletion; D006564:Herpesviridae / Q000378:metabolism; D008954:Models, Biological; D009154:Mutation; D011233:Precipitin Tests; D011485:Protein Binding; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012514:Sarcoma, Kaposi / Q000821:virology*; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000737:chemistry / Q000502:physiology*
D003432:Cross-Linking Reagents; C110348:ORF59 protein, Human herpesvirus 8; D011994:Recombinant Proteins; D014764:Viral Proteins; D000255:Adenosine Triphosphate; D004247:DNA