Structural and functional characterizations of two enzymes involved in DNA breakdown and synthesis: Vibrio vulnificus nuclease (Vvn) and human cytosolic thymidine kinase (TK1)

• Main Authors: Chia-Lung Li, 李家隆
• Other Authors: Zee-Fen Chang
• Format: Others
• Language: en_US
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/58080889484224288746

博士 === 國立臺灣大學 === 生物化學暨分子生物學研究所 === 92 === Nucleic acids act as the genetic information material. Synthesis and breakdown of nucleic acids play many important roles during cell growth, proliferation and defense. Two enzymes involved in nucleic acids breakdown and synthesis are studied in this thesis: Vibrio vulnificus nuclease (Vvn) and human cytosolic thymidine kinase (TK1). Vvn is a non-specific periplasmic nuclease capable of digesting DNA and RNA. Vvn likely protects cells by preventing the uptake of foreign DNA during transformation. We found that Vvn cleaves DNA at the 3’ side of a phosphodiester bond, leaving a 5’-phosphonucleotide product. The crystal structure of the magnesium ion-bound Vvn and the crystal structure of Vvn mutant H80A in complex with a duplex DNA and a calcium ion were resolved both at 2.3 Å resolution. Vvn has a novel mixed  topology containing four disulfide bridges; consistent with the finding that Vvn is not active under reducing conditions in the cytoplasm. The overall structure of Vvn shows no similarity to other endonucleases, however, a known endonuclease motif containing a “-metal” fold is identified in the central cleft region. The magnesium ion is located in the active site, bound to Asn127, Glu77 and four water molecules in an octahedral geometry. The crystal structure of the mutant Vvn-H80A/DNA complex demonstrates that Vvn binds mainly at the minor groove of DNA, resulting in duplex bending towards the major groove by about 20o. Only the DNA phosphate backbones make hydrogen bonds with Vvn, suggesting at structural basis for its sequence-independent recognition of DNA and RNA. Based on the enzyme/substrate and enzyme/product structures observed in the mutant Vvn/DNA crystal structure, a catalytic mechanism is proposed in which the His80 functions as a general base that activates a water molecule to attack on the scissile phosphate, with a magnesium ion involved in stabilization of the phosphoanion transition state and in protonation of the 3’ oxygen. This structural study suggests that Vvn hydrolyzes DNA by a general single-metal ion mechanism, and indicates how sugar non-specific DNA-binding proteins may recognize DNA. In addition, we also studied the functional effect of phosphorylation on the human cytosolic thymidine kinase (TK1). Regulation of TK1 plays a major role in controlling a correct dTTP pool in the cell cycle to coordinate with the process of DNA replication. The human TK1 has been shown previously that it is phosphorylated on serine-13 at the G2/M phase and the phosphorylation may be important for the regulation of its enzyme activity. In this study, serine-13 was replaced by aspartic acid (S13D) to mimic phosphorylation to elucidate the role of phosphorylation on serine-13 in regulation of human TK1. Comparison of the kinetic properties of purified wild-type and S13D TK1, the S13D mutant showed a significant decrease in thymidine substrate-binding affinity. Through size exclusion chromatography in the presence of ATP, wild-type TK1 appeared to be mostly in tetramer form, while a portion of S13D mutant existed in dimer form. A clear ATP-activating effect on TK1 activity was seen for wild-type TK1, but not for S13D mutant. Analysis using cross-linking reagents showed that S13D mutant synthesized in LM-tk- cells was predominantly in dimer form, while S13A mutant was in higher order of oligomeric form. As it has been well established that TK1 in a dimer form is less active and in a tetramer is more active, our results further suggest that mitotic phosphorylation on serine-13 is of physiological importance by counteracting ATP-dependent activation of TK1 through an effect on quaternary structure, thus attenuating its enzymatic function at the G2/M phase.