| Summary: | 博士 === 國立臺灣大學 === 生物化學暨分子生物學研究所 === 93 === Proper control of intracellular dTTP pool size is critical for maintenance of high fidelity of DNA replication and DNA repair. Concurrent supply of dTTP relies on functions of both the de novo and salvage pathways. In the de novo pathway, thymidyltae synthase (TS) catalyzes the rate-limiting step of converting dUMP to dTMP. Alternatively, thymidine kinase (TK), the key enzyme in the salvage pathway, catalyzes the reaction transferring the terminal phosphate of ATP to the 5’-hydroxyl group of thymidine to form dTMP. Subsequent phosphorylation of dTMP by thymidylate kinase (TMPK) gives rise to dTDP, which is then converted to dTTP by dNDP kinase (NDK) for DNA synthesis. Intracellular production of dTTP is a highly regulated process, which is coordinated with DNA replication in the cell cycle. As cell approaches S phase, dTTP pool size is increased to 20-fold than that in G0-phase cells. This up-regulation of dTTP can be attributed to cell cycle-dependent expression of TS, TK, and TMPK through transcriptional control. After the completion of DNA replication, dTTP is no longer demanded, the forward synthesis must be inhibited. However, little is known about how the dTTP-synthesis enzymes are down-regulated. Anaphase promoting complex/cyclosome (APC/C)-mediated proteolysis is essential for chromosome segregation, mitotic exit and G1 entry. Two activators, Cdc20 and Cdh1 target substrates for APC/C-mediated proteolysis in mitosis and G1 phase, respectively. APC/C-Cdc20 prefers to recognize the D-box (RXXL), while APC/C-Cdh1 binds to the D-box and KEN-box (KEN). In this thesis, I demonstrate that the APC/C plays an important role in controlling dTTP pool size by targeting degradation of human cytosolic TK (hTK1) and TMPK (hTMPK). In chapter 2, I show that hTK1 is degraded via a ubiquitin-proteasome pathway in mitotic exit phase and that APC/C-Cdh1 is not only a necessary but also a rate-limiting factor for mitotic degradation of hTK1. A KEN-box sequence located in the C-terminal region of hTK1 is required for its recognition by Cdh1. By in vitro polyubiquitinylation assays, I provide direct evidence that APC/C-Cdh1 is a direct ubiquitin E3 ligase of hTK1 by recognizing its KEN box. In chapter 3, I further identify that hTMPK is another target of APC/C. By in vivo and in vitro experiments, I demonstrate that both APC/C-Cdc20 and APC/C-Cdh1 mediate hTMPK for degradation. APC/C-Cdc20 recognizes hTMPK through D box in M phase whereas APC/C-Cdh1 binds to D- and KEN boxes in early G1 phase. Simultaneous expression of wild type hTK1 and hTMPK leads to a 4-5-fold increase of the dTTP pool without promoting the spontaneous mutation in the hprt (hypoxanthine-guanine phosphoribosyltransferase) gene. In contrast, co-expression of nondegradable hTK1 and hTMPK expands the dTTP pool size ten-fold and induces a drastic dNTP pool imbalance, accompanied by a decrease in growth rate and a delay in SàG2/M transition. Most interestingly, cells co-expressing nondegradable TK1 and TMPK display a striking increase in gene mutation rate. I conclude that down-regulation of dTTP pool size by APC/C pathway during mitosis plays a critical role in keeping a balanced dNTP pool, which is essential for the S phase progression and the maintenance of genome integrity.
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