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首页> 外文期刊>Biochemistry >Evidence That DNA Polymerase #delta# Isolated by Immunoaffinity Chromatography Exhibits High-Molecular Weight Characteristics and Is Associated with the KIAA0039 Protein and RPA.
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Evidence That DNA Polymerase #delta# Isolated by Immunoaffinity Chromatography Exhibits High-Molecular Weight Characteristics and Is Associated with the KIAA0039 Protein and RPA.

机译：通过免疫亲和层析分离的DNA聚合酶#delta具有高分子量特征并与KIAA0039蛋白和RPA相关联的证据。

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DNA polymerase #delta#, the key enzyme for eukaryotic chromosomal replication, has been well characterized as consisitng of a core enzyme of a 125kDa catalytic subunit and a smaller 50 kDa subunit. However, less is known about the other proteins that may comprise additional subunits or participate in the macromolecular protein complex that is involved in chromosomal DNA replication. In this study, the properties of calf thymus pol #delta# preparations isolated by immunoaffinity chromatography were investigated. It is demonstrated for the first time using higly purified preparations that the pol #delta# heterodimer is associated with other polypeptides in high-molecular weight species that range from 260000 to > 500000 in size, as determined by FPLC gel filtration. These preparations are associated with polypeptides of ca. 68-70,34,32 and 25 kDa. Similar findings were revealed with glycerol gradient ultracentrifugation. The p68 polypeptide was shown to be a PCNA bindig protein by overlay methods with biotinylated PCNA. Protein sequencing of the p68, p34, and p25 polypeptide bands revealed sequences that correspond to the hypothetical protein KIAA0039. KIAA0039 displays a small but significant degree of homology to Schizosaccharomyces pombe Cdc27, which ,like Saccharomyces cerevisiae Pol32p, has been described as the third subunit of yeast pol #delta#. These studies provide evidence that p68 is a subunit of pol #delta#. In addition, the p68-70 and p32 polypeptides were found to be derived from the 70 and 32 kDa subunits of RPA, respectively.
机译：DNA聚合酶＃delta＃是真核染色体复制的关键酶，已被很好地表征为由125kDa催化亚基和一个较小的50kDa亚基的核心酶组成。但是，对于可能包含其他亚基或参与涉及染色体DNA复制的大分子蛋白质复合物的其他蛋白质知之甚少。在这项研究中，研究了通过免疫亲和色谱法分离的小牛胸腺pol＃delta＃制剂的特性。首次证明，通过FPLC凝胶过滤测定，pol＃delta＃异二聚体与高分子量物种中的其他多肽结合，其分子量范围为260000至> 500000。这些制品与约1，2，3，3，4，5，6，7，8，7，7，8，7，8，9，9，8，9，9，8，9，11，18，18，18，26，18，26，26，26，13，16，13，13，13，13，13，13，20，13，13，10，10，10，10，10，10，10，20，18，20，18，20，10 68-70、34、32和25 kDa。甘油梯度超速离心显示了相似的发现。通过用生物素化的PCNA的覆盖方法显示p68多肽是PCNA结合蛋白。 p68，p34和p25多肽条带的蛋白质测序揭示了对应于假设蛋白质KIAA0039的序列。 KIAA0039与粟酒裂殖酵母Cdc27表现出很小但显着的同源性，就像酿酒酵母Pol32p一样，它被描述为酵母pol＃delta＃的第三个亚基。这些研究证明p68是pol＃delta＃的一个亚基。另外，发现p68-70和p32多肽分别来自RPA的70和32kDa亚基。

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