胞嘧啶核苷酸單磷酸激酶和核苷酸二磷酸激酶3對於DNA修補之貢獻

Abstract

DNA在複製及修補時需有足量且平衡的去氧核醣核苷酸三磷酸（dNTPs）供給。其中，核糖核酸還原酶（RNR）為合成dNTP的關鍵酵素，可將核糖核苷酸二磷酸（NDPs）還原成去氧核醣核苷酸二磷酸（dNDPs），同時也是DNA修補時所必須。我的研究則進一步發現胞嘧啶單磷酸核苷酸激酶（CMPK）及核苷酸二磷酸激酶3（NME3）在DNA修補上扮演重要的角色。其中CMPK的功能為合成胞嘧啶核苷酸二磷酸（CDP或dCDP），NME3則為十種核苷酸二磷酸激酶（nucleoside diphosphate kinase）異構體的成員之一，負責將(d)NDP合成為(d)NTP。在無血清培養之下，降低CMPK表現量減緩細胞對UV造成DNA損傷的修補，但在增殖細胞中則否。降低NME3表現量則減緩細胞對阿黴素（doxorubicin）造成DNA損傷的修補。我的研究也進一步發現這兩個酵素皆會透過自身蛋白的Ｎ端區域與組蛋白乙醯化酶－Tip60及RNR形成蛋白複合體，而匯集到DNA損傷區域。去除蛋白質N端區域的CMPK及NME3則無法和Tip60形成蛋白複合體，同時也導致在DNA修補能力上的缺陷。因此，我認為多重酵素複合體在DNA損傷處的形成提供一個細胞內局部生成dNTP的方式，使細胞在無血清培養下有效促進DNA的修補。除此之外，NME3在增殖細胞中DNA修補的角色也牽涉到了核醣核苷酸三磷酸（rNTP）的合成。

Cellular supply of deoxynucleoside triphosphates (dNTPs) is crucial for DNA replication and repair. Ribonucleotide reducatse (RNR) is a rate-limiting enzyme for dNTP synthesis required for DNA repair, by reduction of four ribonucleoside diphosphates to generate corresponding dNDPs. In my thesis, the functional importance of UMP/CMP kinase (CMPK), an enzyme catalyzes (d)CDP formation and nucleoside diphosphate kinase 3 (NME3), one of ten isoforms of NME that converts (d)NDPs to (d)NTPs in DNA repair are demonstrated. Knockdown of CMPK delayed DNA repair after UV-irradiation in serum-deprived cells but had no effect on proliferating cells, while knockdown of NME3 declined DNA repair rate after doxorubicin exposure in proliferating cells. In addition, these two enzymes could form a complex with RNR and Tip60, a histone acetyltransferase by their N-terminal regions and recruited to DNA damages sites in a Tip60-depedent manner. The mutants of CMPK and NME3 with their N-terminal sequences deletion to eliminate their interaction with Tip60 also show the functional deficiency in DNA repair as compared to wild-type enzymes. I propose that the site-specific multienzyme complex formation provides a means for local synthesis of dNTPs to facilitate DNA repair in serum-deprived cells, which contain low cellular dNTP supply from cytosol. In addition to Tip60/RNR interaction for dNTPs supply at DNA damage sites, another function of NME3 in proliferating cells for DNA repair may involve the local production of ribonucleoside triphosphates (rNTPs).