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

The Contribution of CMP Kinase and Nucleoside Diphosphate Kinase 3 in DNA Repair

曹甯(Ning Tsao)

指導教授 ： 張智芬

共同指導教授 ： 詹迺立(Nei-Li Chan)

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/博士(2015年)

https://doi.org/10.6342/NTU.2015.00522

摘要

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。

關鍵字

去氧核醣核苷酸三磷酸（dNTPs） ； 核糖核酸還原酶（RNR） ； 胞嘧啶單磷酸核苷酸激酶（CMPK） ； 核苷酸二磷酸激酶3（NME3） ； Tip60 ； DNA修補

並列摘要

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).

並列關鍵字

deoxynucleotide triphosphate (dNTP) ； ribonucleotide reductase (RNR) ； CMPK ； NME3 ； Tip60 ； DNA repair

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