Polymerase and Exonuclease Activities in Herpes Simplex Virus Type 1 DNA Polymerase Are Not Highly Coordinated
| Autor: | Robert D. Kuchta, Ashwani Kumar Vashishtha |
|---|---|
| Rok vydání: | 2015 |
| Předmět: |
Models
Molecular Exonuclease DNA polymerase DNA polymerase II DNA-Directed DNA Polymerase Herpesvirus 1 Human Biochemistry Article Viral Proteins 03 medical and health sciences Catalytic Domain Humans Polymerase 030304 developmental biology Klenow fragment 0303 health sciences DNA clamp biology 030302 biochemistry & molecular biology Herpes Simplex DNA Processivity Molecular biology Exodeoxyribonucleases biology.protein Primase |
| Zdroj: | Biochemistry |
| ISSN: | 1520-4995 0006-2960 |
| Popis: | The herpes polymerase–processivity factor complex consists of the catalytic UL30 subunit containing both polymerase and proofreading exonuclease activities and the UL42 subunit that acts as a processivity factor. Curiously, the highly active exonuclease has minimal impact on the accumulation of mismatches generated by the polymerase activity. We utilized a series of oligonucleotides of defined sequence to define the interactions between the polymerase and exonuclease active sites. Exonuclease activity requires unwinding of two nucleotides of the duplex primer–template. Surprisingly, even though the exonuclease rate is much higher than the rate of DNA dissociation, the exonuclease degrades both single- and double-stranded DNA in a nonprocessive manner. Efficient proofreading of incorrect nucleotides incorporated by the polymerase would seem to require efficient translocation of DNA between the exonuclease and polymerase active sites. However, we found that translocation of DNA from the exonuclease to polymerase active site is remarkably inefficient. Consistent with inefficient translocation, the DNA binding sites for the exonuclease and polymerase active sites appear to be largely independent, such that the two activities appear noncoordinated. Finally, the presence or absence of UL42 did not impact the coordination of the polymerase and exonuclease activities. In addition to providing fundamental insights into how the polymerase and exonuclease function together, these activities provide a rationale for understanding why the exonuclease minimally impacts accumulation of mismatches by the purified polymerase and raise the question of how these two activities function together in vivo. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|