A Comparative Study of Uracil-DNA Glycosylases from Human and Herpes Simplex Virus Type 1

Kuakarun Krusong,Elisabeth P Carpenter,Stuart R.W Bellamy,Renos Savva,Geoff S Baldwin

doi:10.1074/jbc.m509137200

Abstract

Uracil-DNA glycosylase (UNG) is the key enzyme responsible for initiation of base excision repair. We have used both kinetic and binding assays for comparative analysis of UNG enzymes from humans and herpes simplex virus type 1 (HSV-1). Steady-state fluorescence assays showed that hUNG has a much higher specificity constant (k(cat)/K(m)) compared with the viral enzyme due to a lower K(m). The binding of UNG to DNA was also studied using a catalytically inactive mutant of UNG and non-cleavable substrate analogs (2'-deoxypseudouridine and 2'-alpha-fluoro-2'-deoxyuridine). Equilibrium DNA binding revealed that both human and HSV-1 UNG enzymes bind to abasic DNA and both substrate analogs more weakly than to uracil-containing DNA. Structure determination of HSV-1 D88N/H210N UNG in complex with uracil revealed detailed information on substrate binding. Together, these results suggest that a significant proportion of the binding energy is provided by specific interactions with the target uracil. The kinetic parameters for human UNG indicate that it is likely to have activity against both U.A and U.G mismatches in vivo. Weak binding to abasic DNA also suggests that UNG activity is unlikely to be coupled to the subsequent common steps of base excision repair.

Highlights

Uracil-DNA glycosylase (UNG) is one of the initiating DNA glycosylases of the base excision repair pathway and is responsible for the specific recognition and removal of uracil
Steady-state Analysis—Steady-state reactions were performed with hUNG to determine its kinetic parameters under the same conditions that had been previously used for herpes simplex virus type 1 (HSV-1) UNG [10]
We determined that removal of the N-terminal His tag from hUNG had no impact on the catalytic constants

Summary

Introduction

UNG is one of the initiating DNA glycosylases of the base excision repair pathway and is responsible for the specific recognition and removal of uracil. UNG enzymes have been identified in a wide variety of organisms, including human, Escherichia coli, and herpes simplex virus type 1 (HSV-1). HUNG has been cited as having a higher Km value [8, 12, 13] than we observed for HSV-1 UNG [10], and this has informed the description of hUNG as having a single function in post-replicative repair of U1⁄7A mismatches [14] In light of these reported differences between the viral and human UNG enzymes, we decided to investigate both enzymes in kinetic and DNA binding studies. We wished to investigate the DNA binding of both human and HSV-1 UNG enzymes to non-cleavable substrate analogs both as a probe to investigate protein-DNA interactions and to further explore the possibility of finding specific inhibitors of viral UNG

Methods

Results

Discussion

Conclusion