Drosophila RecQ4 Has a 3′-5′ DNA Helicase Activity That Is Essential for Viability
Drosophila RecQ4 Has a 3′-5′ DNA Helicase Activity That Is Essential for Viability
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DOI：
10.1074/jbc.m109.008052
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发表时间：
2009-11-06
影响因子：
4.8
通讯作者：
Hsieh, Tao-shih
中科院分区：
文献类型：
--
作者：
Capp, Christopher;Wu, Jianhong;Hsieh, Tao-shih
RecQ蛋白家族的成员是高度保守的DNA解旋酶，在维持基因组稳定性方面具有重要功能。RecQ4的缺陷与包括Rothmund-Thomson、RAPADILINO和Baller-Gerold综合征在内的人类疾病有关，所有这些疾病的特征都是发育缺陷、肿瘤倾向和遗传不稳定。然而，关于RecQ4的DNA解旋酶活性的文献中有相互矛盾的结果。我们报道了利用杆状病毒载体表达黑腹果蝇RecQ4的方法，并将其纯化到接近均一的水平。纯化的蛋白具有DNA依赖的ATPase活性，是依赖于ATP水解酶的3‘-5’DNA解旋酶。5‘-腺苷二磷酸(AMPPNP)是一种不可水解的ATP类似物，它的存在促进了RecQ4与单链DNA之间稳定的复合体的形成。果蝇RecQ4也可以退火互补的单链；这种活性在AMPNP存在下降低，可能是因为在这种条件下形成了稳定的蛋白质-DNA复合体。解旋酶结构域中高度保守的赖氨酸残基的点突变，尽管保留了野生型水平的退火酶活性，但灭活了ATPase和解旋酶活性，并消除了稳定的复合体形成。这些结果表明，解旋酶结构域本身就对果蝇酶的DNA解旋作用负责。我们产生了一个纯合子致死的空recq4突变体，我们用它来测试解旋酶死亡突变体在果蝇中的遗传功能。互补试验表明，解旋酶死亡突变体recq4不能挽救零突变，说明解旋酶活性具有重要的生物学功能。
Members of the RecQ family of proteins are highly conserved DNA helicases that have important functions in the maintenance of genomic stability. Deficiencies in RecQ4 have been linked to human diseases including Rothmund-Thomson, RAPADILINO, and Baller-Gerold syndromes, all of which are characterized by developmental defects, tumor propensity, and genetic instability. However, there are conflicting results shown in the literature regarding the DNA helicase activity of RecQ4. We report here the expression of Drosophila melanogaster RecQ4 with a baculoviral vector and its purification to near homogeneity. The purified protein has a DNA-dependent ATPase activity and is a 3'-5' DNA helicase dependent on hydrolysis of ATP. The presence of 5'-adenylyl-beta, gamma-imidodiphosphate (AMPPNP), a nonhydrolyzable ATP analog, promotes stable complex formation between RecQ4 and single-stranded DNA. Drosophila RecQ4 can also anneal complementary single strands; this activity was reduced in the presence of AMPPNP, possibly because of the stable protein-DNA complex formed under such conditions. A point mutation of the highly conserved lysine residue in the helicase domain, although retaining the wild type level of annealing activity, inactivated ATPase and helicase activities and eliminated stable complex formation. These results suggest that the helicase domain alone is responsible for the DNA unwinding action of the Drosophila enzyme. We generated a null recq4 mutant that is homozygous lethal, which we used to test the genetic function of the helicase-dead mutant in flies. Complementation tests showed that the helicase-dead mutant recq4 transgenes are incapable of rescuing the null mutation, demonstrating that the helicase activity has an essential biological function.