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The role of the associated 3' to 5' exonuclease activity and processivity factor (UL42) or herpes simplex virus type 1 DNA polymerase on the fidelity of DNA replication

Song, Liping
http://rave.ohiolink.edu/etdc/view?acc_num=osu1083958702

Abstract Details

2004, Doctor of Philosophy, Ohio State University, Medical Microbiology and Immunology.
Herpes simplex virus type 1 (HSV-1) DNA polymerase (pol) has an associated 3’ to 5’ exonuclease (exo) activity that plays an important role in maintaining the fidelity of DNA replication and viability of the virus. To assess the functional significance of the exo activity, the aspects of fidelity and translesion DNA synthesis affected by HSV-1 pol deficient in this exo activity (D368A) were examined and compared with those of wild-type pol. Because HSV-1 pol forms a stable heterodimer with UL42, a protein that stimulates the polymerizing activity and increases the processivity of pol, the impact of UL42 on fidelity was also evaluated. Pre-steady-state kinetic analysis revealed that the D368A mutation destroyed exo activity without significantly altering the inherent polymerization function. It was demonstrated that both wild-type and mutant pols could selectively incorporate correct vs. incorrect nucleotides, and exo activity enhanced nucleotide selectivity by roughly an order of magnitude under steady-state conditions. However, the primary effect of exo activity on fidelity was manifested during processive DNA synthesis, because it functioned to remove incorporated mismatches, the latter which impeded the extension ability of pol, particularly in the presence of UL42 processivity factor. UL42 did not alter the pre-steady-state rate constants for correct nucleotide incorporation, misincorporation, translocation, or nucleotide excision. However, UL42 decreased dissociation of pol from DNA, which not only made the enzyme more processive but also allowed enough time to excise replication errors. Lesions, such as abasic (AP) sites, also impeded extension beyond the sites, although both HSV-1 wild-type and exo-deficient pols could incorporate dATP opposite the AP site with reduced efficiencies compared to that for normal DNA synthesis. The exo activity facilitated sensing of the lesion through increased excision rate, thereby preventing extension through repeated incorporation and excision events. Taken together, the results of this study suggest that the associated exo activity of HSV-1 pol coordinates with the polymerizing activity by partitioning the primer terminus between the exo and polymerizing sites to proofread errors and prevent translesion synthesis, ultimately increasing the fidelity of HSV-1 DNA synthesis.
Deborah Parris (Advisor)
220 p.
Chemistry, Biochemistry
HSV-1 DNA polymerase, exo activity, processivity factor UL42, DNA replication fidelity, misincorporation, mismatch extension,; translesion DNA synthesis, abasic site

Recommended Citations

Citations

  • Song, L. (2004). The role of the associated 3' to 5' exonuclease activity and processivity factor (UL42) or herpes simplex virus type 1 DNA polymerase on the fidelity of DNA replication [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1083958702

    APA Style (7th edition)

  • Song, Liping. The role of the associated 3' to 5' exonuclease activity and processivity factor (UL42) or herpes simplex virus type 1 DNA polymerase on the fidelity of DNA replication. 2004. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1083958702.

    MLA Style (8th edition)

  • Song, Liping. "The role of the associated 3' to 5' exonuclease activity and processivity factor (UL42) or herpes simplex virus type 1 DNA polymerase on the fidelity of DNA replication." Doctoral dissertation, Ohio State University, 2004. http://rave.ohiolink.edu/etdc/view?acc_num=osu1083958702

    Chicago Manual of Style (17th edition)

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This open access ETD is published by The Ohio State University and OhioLINK.