Identification of a novel protein, PDIP38, that interacts with the p50 subunit of DNA polymerase δ and proliferating cell nuclear antigen
Identification of a novel protein, PDIP38, that interacts with the p50 subunit of DNA polymerase δ and proliferating cell nuclear antigen
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DOI：
10.1074/jbc.m208694200
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发表时间：
2003-03-21
影响因子：
4.8
通讯作者：
Lee, MYWT
中科院分区：
文献类型：
--
作者：
Liu, L;Rodriguez-Bèlmonte, EM;Lee, MYWT
酵母双杂交筛选方法用于鉴定与人DNA聚合酶5（pol δ）相关的新蛋白。在这项研究中使用了两种诱饵。这些是核心pol δ异二聚体的大亚基（p125）和小亚基（p50）。p50是以p125为诱饵分离的唯一阳性。从p50筛选中鉴定出两种新的蛋白质伴侣，命名为PDIP 38和PDIP 46。在本研究中，进一步表征了PDIP 38与pol δ的相互作用。PDIP 38编码368个氨基酸的蛋白质，其C末端与细菌APAG蛋白和F盒A蛋白保守。发现PDIP 38还与增殖细胞核抗原（PCNA）相互作用。PDIP 38与pol δ的p50亚基和PCNA两者相互作用的能力通过使用谷胱甘肽S-转移酶（GST）-PDIP 38融合蛋白的下拉测定来证实。PCNA-PDIP 38相互作用也通过PCNA覆盖实验证明。通过GST-PDIP 38下拉和免疫共沉淀实验，证明PDIP 38与pol δ的结合发生在小牛胸腺组织和哺乳动物细胞提取物中。PDIP 38与通过免疫亲和层析分离的pol δ结合。PDIP 38与pol δ的结合也可以通过天然凝胶电泳证明。
The yeast two-hybrid screening method was used to identify novel proteins that associate with human DNA polymerase 5 (pol delta). Two baits were used in this study. These were the large (p125) and small (p50) subunits of the core pol delta heterodimer. p50 was the only positive isolated with p125 as the bait. Two novel protein partners, named PDIP38 and PDIP46, were identified from the p50 screen. In this study, the interaction of PDIP38 with pol delta was further characterized. PDIP38 encodes a protein of 368 amino acids whose C terminus is conserved with the bacterial APAG protein and with the F box A protein. It was found that PDIP38 also interacts with proliferating cell nuclear antigen (PCNA). The ability of PDIP38 to interact with both the p50 subunit of pol delta and with PCNA was confirmed by pull-down assays using glutathione S-transferase (GST)-PDIP38 fusion proteins. The PCNA-PDIP38 interaction was also demonstrated by PCNA overlay experiments. The association of PDIP38 with pol delta was shown to occur in calf thymus tissue and mammalian cell extracts by GST-PDIP38 pull-down and coimmunoprecipitation experiments. PDIP38 was associated with pol delta isolated by immunoaffinity chromatography. The association of PDIP38 with pol delta could also be demonstrated by native gel electrophoresis.