Genetic analyses of Schizosaccharomyces pombe dna2(+) reveal that dna2 plays an essential role in Okazaki fragment metabolism.
Genetic analyses of Schizosaccharomyces pombe dna2(+) reveal that dna2 plays an essential role in Okazaki fragment metabolism.
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发表时间：
2000-07
期刊：
影响因子：
3.3
通讯作者：
Hoyoung Kang;Hoyoung Kang;Eunjoo Choi;Sung-Ho Bae;Kyoung-Hwa Lee;B. Gim;Hee-Dai Kim;Chankyu Park;S. MacNeill;Y. Seo
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文献类型：
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作者：
Hoyoung Kang;Hoyoung Kang;Eunjoo Choi;Sung-Ho Bae;Kyoung-Hwa Lee;B. Gim;Hee-Dai Kim;Chankyu Park;S. MacNeill;Y. Seo
在本报告中，我们研究了粟酒裂殖酵母 dna2(+) 的温度敏感 (ts) 突变等位基因（芽殖酵母 DNA2 的同源物）引起的表型，试图进一步确定其在细胞周期 S 期滞后链合成方面的体内功能。在限制温度下，dna2 (ts) 细胞停滞在 S 期晚期，但大量 DNA 合成不受影响。此外，当与检查点突变结合时，它们表现出异常的有丝分裂，这与 Dna2 在冈崎片段成熟中的作用一致。类似地，dna2(+) 被破坏的孢子在萌发期间复制了其 DNA 含量，并且也在 S 期后期停滞。 dna2(+) 失活导致的染色体断裂与 DNA 连接酶 I 基因 cdc17(+) 失活时所见的情况极为相似。编码聚合酶δ（cdc1（+）和cdc27（+））、DNA连接酶I（cdc17（+））和Fen-1（rad2（+））亚基的基因过表达抑制了dna2（ts）突变体的温度依赖性致死率。这些基因产物中的每一种都在冈崎片段的延伸或成熟中发挥作用。此外，它们在酵母双杂交检测中都与粟酒裂殖酵母 Dna2 相互作用，尽管程度不同。根据这些结果，我们得出结论，dna2(+) 在冈崎片段的延伸和成熟中起着直接作用。我们建议 dna2(+) 作为中心蛋白质与其他蛋白质形成复合物，协调冈崎片段延伸和成熟的多酶过程。
In this report, we investigated the phenotypes caused by temperature-sensitive (ts) mutant alleles of dna2(+) of Schizosaccharomyces pombe, a homologue of DNA2 of budding yeast, in an attempt to further define its function in vivo with respect to lagging-strand synthesis during the S-phase of the cell cycle. At the restrictive temperature, dna2 (ts) cells arrested at late S-phase but were unaffected in bulk DNA synthesis. Moreover, they exhibited aberrant mitosis when combined with checkpoint mutations, in keeping with a role for Dna2 in Okazaki fragment maturation. Similarly, spores in which dna2(+) was disrupted duplicated their DNA content during germination and also arrested at late S-phase. Inactivation of dna2(+) led to chromosome fragmentation strikingly similar to that seen when cdc17(+), the DNA ligase I gene, is inactivated. The temperature-dependent lethality of dna2 (ts) mutants was suppressed by overexpression of genes encoding subunits of polymerase delta (cdc1(+) and cdc27(+)), DNA ligase I (cdc17(+)), and Fen-1 (rad2(+)). Each of these gene products plays a role in the elongation or maturation of Okazaki fragments. Moreover, they all interacted with S. pombe Dna2 in a yeast two-hybrid assay, albeit to different extents. On the basis of these results, we conclude that dna2(+) plays a direct role in the Okazaki fragment elongation and maturation. We propose that dna2(+) acts as a central protein to form a complex with other proteins required to coordinate the multienzyme process for Okazaki fragment elongation and maturation.