| Summary: | 碩士 === 國立臺灣大學 === 微生物學研究所 === 88 === Epstein-Barr virus (EBV) DNase is encoded by the BGLF5 ORF and expressed in the early phase of viral lytic cycle. EBV DNase possesses both exonuclease and endonuclease activities . It digests processively on dsDNA but distributively on ssDNA. Divalent cations are absolutely required as cofactors. The herpesvirus DNases are evolutionary conserved. We compared the EBV DNase conserved sequences to those of the nucleases which crystal structures have been well-defined, such as EcoRI，EcoRV，PvuII，BamHI，l-exonuclease，and E. coli MutH. We found that the sequences of conserved regions B, C in the catalytic center were similar among these DNases, including EBV DNase. We also found the residues corresponding to the amino acids in the catalytic center of the type II restriction enzymes. Therefore, the site directed mutagenesis was used to define the roles of these region，D203(Xn)E225XK227. D203 residue was changed to Asn, Glu, Gln, Lys or His residues. E225 residue was changed to Asp or Gln residues. K227 was changed to Arg, Asp, Asn, Glu or Gly residues. EBV DNase activity was reduced when these residues were replaced. Other Asp and Glu residues in the conserved regions B, C were changed into Asn and Gln, and the activities retained except the mutant D195N. There was another acidic residue which is important to the catalytic mechanism in the upstream of the type II restriction enzyme catalytic center. Therefore, it is suggested that the D195(Xn)D203(Xn)E225XK227 may be the putative catalytic center of EBV DNase. In addition, the same method was used to mutate four His residues of the EBV DNase, H153, H306, H352 and H371. The H352 was found to be the conserved residue of the herpesvirus DNases that is also important to the EBV DNase nuclease activity.
EBV major DNA binding protein (mDBP) is also one of the gene products in the lytic cycle, which is encoded by BALF2. EBV mDBP has stronger binding activity to ssDNA than to dsDNA and ssRNA. The N-terminal truncated mutant (NDmDBP, from amino acids 423 to 1128) of EBV mDBP could inhibit the endonuclease and exonuclease activity of the EBV DNase. It also inhibited the DNA binding activity of the EBV DNase. Therefore, a series of the EBV mDBP deletion clones were generated to map the region responsible for interaction between EBV DNase and EBV mDBP. The results suggest that the C- terminus of the EBV mDBP might be important in inhibiting the activity of EBV DNase. The smallest region of EBV mDBP that could inhibit the activity of EBV DNase was defined to be between amino acids 989 to 1128.
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