Divergent structures of Mammalian and gammaherpesvirus uracil DNA glycosylases confer distinct DNA binding and substrate activity

Abstract

Uracil DNA glycosylase (UNG) removes mutagenic uracil base from DNA to initiate base excision repair (BER). The result is an abasic site (AP site) that is further processed by the high-fidelity BER pathway to complete repair and maintain genome integrity. The gammaherpesviruses (GHVs), human Kaposi sarcoma herpesvirus (KSHV), Epstein-Barr virus (EBV), and murine gammaherpesvirus 68 (MHV68) encode functional UNGs that have a role in viral genome replication. Mammalian and GHVs UNG share overall structure and sequence similarity except for a divergent amino-terminal domain and a leucine loop motif in the DNA binding domain that varies in sequence and length. To determine if divergent domains contribute to functional differences between GHV and mammalian UNGs, we analyzed their roles in DNA interaction and catalysis. By utilizing chimeric UNGs with swapped domains we found that the leucine loop in GHV, but not mammalian UNGs facilitates interaction with AP sites and that the amino-terminal domain modulates this interaction. We also found that the leucine loop structure contributes to differential UDGase activity on uracil in single- versus double-stranded DNA. Taken together we demonstrate that the GHV UNGs evolved divergent domains from their mammalian counterparts that contribute to differential biochemical properties from their mammalian counterparts.

Keywords: Abasic site; Gammaherpesvirus; Uracil DNA glycosylase.

Figure 1.

GHV UNG AP site binding. (A) EMSA assay with dsDNA probe containing an AP site (AP:G) and indicated amounts of purified recombinant MHV68UNG or mUNG. Shifted (*) probe is indicated. (B) EMSA assay with equivalent non-AP containing probe (C:G). (C) EMSA assay with AP:G probe, 500nM MHV68 UNG and indicated fold addition of unlabeled AP:G probe DNA or (D) unlabeled C:G probe DNA. Uncropped gel images included in supplemental data.

Figure 2.

Alignment of GHV UNGs and mammalian UNGs and diagram for domain swap mutants of UNGs. (A) Sequence alignment of uracil-DNA glycosylases encoded by MHV68, KSHV, EBV, human, and mouse. Denotation of secondary structural elements, alpha helix (horizontal bar) and beta sheet (arrow) are based on PDB structures of KSHV, EBV and human UNG. Sequence of these elements are shaded. Catalytic site aspartic acid and histidine residues are labeled with asterisk. Leucine loop extension is boxed. (B) Diagram for UNG variants used in this study. MHV68UNG sequence is depicted as white bar and mUNG with gray bar. Numbers on the bar denote the start and stop amino acids for chimeric UNGs as indicated.

Figure 3.

Contribution of leucine loop extension and N-terminus of GHV UNG to AP sites binding. EMSA assays with dsDNA probe containing an AP site (AP:G) and indicated amounts of recombinant UNGs corresponding to (A) MHV68UNG, mUNG, KSHVUNG and EBVUNG. (B) Amino-terminal deletion mutants ΔN, (C) Chimeras with the N-terminus swapped (m/MHV68UNG, MHV68/mUNG) (D) Chimeras with the leucine loop extension swapped (MHV68UNG-Ls, mUNG-Ls) or 7 amino acids of the leucine loop extension motif deleted (MHV68UNGΔL-Loop) (E) ΔN mutants with leucine loop extension swapped (MHV68UNG ΔN-Ls, mUNG ΔN-Ls) or (F) EBVUNG ΔN with indicated leucine loop extension residues to mutated to the equivalent residues in KSHVUNG, N220L and 219QNST222 to SLGG. All panels representative of n = 3 independent experiments. Shifted (*) probe is indicated. Uncropped gel images included in supplemental data.

Figure 4.

UNGs binding to AP:G in dsDNA monitored by fluorescence anisotropy. Anisotropy data for equilibrium binding of recombinant UNGs to 36-nt fluorescein-dT labeled dsDNA containing AP:G. Kd determined by nonlinear hyperbolic binding mode. Anisotropy profiles for (A) full length MHV68UNG, EBVUNG, KSHVUNG AND mUNG. (B) Amino-terminal deletion mutants ΔN. (C) Chimeras with the leucine loop extension swapped (MHV68UNG-Ls), ΔN mutants with leucine loop extension swapped (MHV68UNG ΔN-Ls, mUNG ΔN-Ls) or 7 amino acids of the leucine loop extension motif deleted (MHV68UNGΔL-Loop). (D) EBVUNG ΔN with indicated leucine loop extension residues to mutated to the equivalent residues in KSHVUNG, N220L and 219QNST222 to SLGG. Data represents average of n=3 independent experiments.

Figure 5.

UDGase activity of mUNG vs. GHVUNG. (A) Summary of specific activity of mUNG, MHV68UNG, and EBVUNG on uracil in ssDNA (black bar) or G:U mismatch in dsDNA (grey bar) in UDGase assay from n=3 experiments. (B) Representative denaturing PAGE gels from the UDGase assay resolving indicated dosage of mUNG, MHV68UNG, or EBVUNG incubated with 333 nM G:U mismatch in dsDNA or (C) the ssDNA substrate with uracil incubated for the indicated time. Upper band (intact substrate) and lower band (cleaved product of UDGase activity) are resolved on the gel images. Uncropped gel images included in supplemental data.

Figure 6.

Effects of N-terminus motif on UDGase activity of mUNG and MHV68UNG. Summary of specific activity on uracil in ssDNA (black bar) or G:U mismatch in dsDNA (grey bar) in UDGase assay of mUNG and MHV68UNG compared to (A) mutants with deleted amino-terminal domains (mUNGΔ, MHV68UNGΔ) or (B) chimeras with swapped amino-terminal domains (m/MHV68UNG, MHV68UNG/mUNG). Average from n=3 independent experiments. (C) Representative denaturing PAGE gels from the UDGase assay resolving indicated dosage of mUNG, MHV68UNG or mutant incubated with 333 nM G:U mismatch in dsDNA or (D) ssDNA substrate containing an uracil for the indicated time. Upper band (intact substrate) and lower band (cleaved product of UDGase activity) are resolved on the gel images. Uncropped gel images included in supplemental data.

Figure 7.

Effects of leucine loop extension motif on UDGase activity of mUNG and MHV68UNG. Summary of specific activity on uracil in ssDNA (black bar) or G:U mismatch in dsDNA (grey bar) in UDGase assay of mUNG and MHV68UNG compared to chimeras with leucine loop extension motif swapped from the other UNG (mUNG-Ls, MHV68UNG-Ls) from n=3 independent experiments. (B) Representative denaturing PAGE gels from the UDGase assay resolving indicated dosage of mUNG, or mUNG-Ls with 333 nM G:U mismatch in dsDNA (upper panels) or the ssDNA substrate with uracil (lower panels) incubated for the indicated time. Upper band (intact substrate) and lower band (cleaved product of UDGase activity) are resolved on the gel images. (C) Plot of percentage uracil removal as a function of time. The percentage of processed substrates at each time point for the indicated concentrations are displayed. (D) Specific activity of MHV68UNG compared to MHV68ΔL-Loop (deletion of the first 7 amino acids of the 12 amino acid leucine loop extension) on uracil in ssDNA (ssU) or G:U mismatch in dsDNA from n=2 independent experiments. (E) Representative denaturing PAGE gels from the UDGase assay and (F) Plot of percentage uracil removal as a function of time. Uncropped gel images included in supplemental data.

Figure 8.

Stimulation of MHV68UNG turnover by recombinant murine APE1. (A) denaturing PAGE gels from the UDGase assay resolving 333 nM G:U mismatch incubated with 5 nM MHV68UNG for the indicated time in the presence of 0, 150nM, 300 nM or 600 nM murine APE1. (B) Plot of percentage uracil removal as a function of time. Uncropped gel images included in supplemental data.