Kim Do-Hyung, Lee Kyoung-Hwa, Kim Jeong-Hoon, Ryu Gi-Hyuck, Bae Sung-Ho, Lee Byung-Chul, Moon Kyeong-Yeop, Byun Si-Myung, Koo Hyeon-Sook, Seo Yeon-Soo
National Creative Research Initiative Center for Cell Cycle Control, Department of Biological Sciences, Korea Advanced Institute of Science and Technology Daejeon 305-701, Korea.
Nucleic Acids Res. 2005 Mar 3;33(4):1372-83. doi: 10.1093/nar/gki255. Print 2005.
In both budding and fission yeasts, a null mutation of the DNA2 gene is lethal. In contrast, a null mutation of Caenorhabditis elegans dna2+ causes a delayed lethality, allowing survival of some mutant C.elegans adults to F2 generation. In order to understand reasons for this difference in requirement of Dna2 between these organisms, we examined the enzymatic properties of the recombinant C.elegans Dna2 (CeDna2) and its interaction with replication-protein A (RPA) from various sources. Like budding yeast Dna2, CeDna2 possesses DNA-dependent ATPase, helicase and endonuclease activities. The specific activities of both ATPase and endonuclease activities of the CeDna2 were considerably higher than the yeast Dna2 (approximately 10- and 20-fold, respectively). CeDna2 endonuclease efficiently degraded a short 5' single-stranded DNA tail (<10 nt) that was hardly cleaved by ScDna2. Both endonuclease and helicase activities of CeDna2 were stimulated by CeRPA, but not by human or yeast RPA, demonstrating a species-specific interaction between Dna2 and RPA. These and other enzymatic properties of CeDna2 described in this paper may shed light on the observation that C.elegans is less stringently dependent on Dna2 for its viability than Saccharomyces cerevisiae. We propose that flaps generated by DNA polymerase delta-mediated displacement DNA synthesis are mostly short in C.elegans eukaryotes, and hence less dependent on Dna2 for viability.
在出芽酵母和裂殖酵母中，DNA2基因的无效突变都是致死的。相比之下，秀丽隐杆线虫dna2+的无效突变会导致延迟致死，使一些突变的秀丽隐杆线虫成虫存活至F2代。为了理解这些生物体对Dna2需求存在差异的原因，我们研究了重组秀丽隐杆线虫Dna2（CeDna2）的酶学特性及其与来自不同来源的复制蛋白A（RPA）的相互作用。与出芽酵母Dna2一样，CeDna2具有依赖于DNA的ATP酶、解旋酶和核酸内切酶活性。CeDna2的ATP酶和核酸内切酶活性的比活性均显著高于酵母Dna2（分别约为10倍和20倍）。CeDna2核酸内切酶能有效降解短的5'单链DNA尾巴（<10个核苷酸），而酿酒酵母Dna2几乎不能切割这种尾巴。CeDna2的核酸内切酶和解旋酶活性均受到CeRPA的刺激，但不受人或酵母RPA的刺激，这表明Dna2与RPA之间存在物种特异性相互作用。本文描述的CeDna2的这些及其他酶学特性，可能有助于解释秀丽隐杆线虫在生存能力上对Dna2的依赖程度不如酿酒酵母这一现象。我们推测，在秀丽隐杆线虫真核生物中，由DNA聚合酶δ介导的置换DNA合成产生的瓣状结构大多较短，因此在生存能力上对Dna2的依赖较小。
