Differential requirement for the N-terminal catalytic domain of the DNA polymerase ε p255 subunit in the mitotic cell cycle and the endocycle.
Differential requirement for the N-terminal catalytic domain of the DNA polymerase ε p255 subunit in the mitotic cell cycle and the endocycle.
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DOI：
10.1016/j.gene.2011.12.056
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发表时间：
2012-03
期刊：
影响因子：
3.5
通讯作者：
Osamu Suyari;M. Kawai;Hiroyuki Ida;H. Yoshida;K. Sakaguchi;M. Yamaguchi
中科院分区：
文献类型：
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作者：
Osamu Suyari;M. Kawai;Hiroyuki Ida;H. Yoshida;K. Sakaguchi;M. Yamaguchi
在果蝇中，已鉴定出255 kDa的催化亚基（dpolεp255）和58 kDa的DNA聚合酶ε亚基（dpolεp58）。dpolεp255的N-末端携带6个高度保守的DNA聚合酶亚结构域和5个3′→5′外切核酸酶基序。我们利用表达双链RNA（dsRNA）的转基因果蝇品系研究了dpolεp255在果蝇发育过程中的作用。dpolεp255 dsRNA在眼盘中的表达诱导小眼表型并抑制DNA合成，表明在有丝分裂周期的G1-S转换和/或S期进展中的作用。类似地，dpolεp255 dsRNA在唾液腺中的表达导致小尺寸和内复制缺陷，证明在内循环进展中起关键作用。在眼盘中，dpolεp255敲低诱导的缺陷通过dpolεp255的C-末端区域的过表达而被挽救，表明该非催化结构域的功能在酵母和果蝇之间是保守的。然而，唾液腺的情况并非如此，这表明催化N-末端区域对于内复制至关重要，并且其缺陷不能被其他DNA聚合酶补充。此外，还发现了与dpolεp255相互作用的基因，包括RFC、DNA引发酶、DNA polη、Mcm 10和Psf 2等DNA复制相关基因以及Iswi等染色质重塑相关基因。
In Drosophila, the 255kDa catalytic subunit (dpolεp255) and the 58kDa subunit of DNA polymerase ε (dpolεp58) have been identified. The N-terminus of dpolεp255 carries well-conserved six DNA polymerase subdomains and five 3′→5′ exonuclease motifs as observed with Polε in other species. We here examined roles of dpolεp255 during Drosophila development using transgenic fly lines expressing double stranded RNA (dsRNA). Expression of dpolεp255 dsRNA in eye discs induced a small eye phenotype and inhibited DNA synthesis, indicating a role in the G1–S transition and/or S-phase progression of the mitotic cycle. Similarly, expression of dpolεp255 dsRNA in the salivary glands resulted in small size and endoreplication defects, demonstrating a critical role in endocycle progression. In the eye disc, defects induced by knockdown of dpolεp255 were rescued by overexpression of the C-terminal region of dpolεp255, indicating that the function of this non-catalytic domain is conserved between yeast and Drosophila. However, this was not the case for the salivary gland, suggesting that the catalytic N-terminal region is crucial for endoreplication and its defect cannot be complemented by other DNA polymerases. In addition, several genetic interactants with dpolεp255 including genes related to DNA replication such as RFC, DNA primase, DNA polη, Mcm10 and Psf2 and chromatin remodeling such as Iswi were also identified.