ISOLATION OF DNA HELICASES REQUIRED FOR HUMAN CHROMOSOMAL DNA REPLICATION

我从培养的人类细胞中寻找染色体DNA复制所需的DNA解旋酶。通过对这些活性的筛选，我获得了以下三个主要结果：1.我克隆了在病毒DNA解旋酶存在下允许DNA合成的DNA片段。所获得的DNA片段含有二核苷酸重复序列，将用于分析DNA复制所需的DNA解旋酶。2.我从与抗DNA聚合酶α抗体相关的蛋白质组分中分离出DNA解旋酶。该解旋酶的表观分子量为210kDa，方向为3 '至5'，依赖于单链结合蛋白RPA，但在存在二核苷酸重复序列的情况下不具有DNA合成活性。利用此反应，我分离到一种与细胞核紧密结合的DNA解旋酶。这种核解旋酶（解旋酶n）的表观分子量为190kDa，方向为3 '至5'。其110kDa肽具有ATP结合活性。这种解旋酶也表现出对RPA的强烈依赖性，这可能表明它与这种复制蛋白的特异性相互作用。这些特征表明，这是一种新的解旋酶，将参与DNA复制。

I have looked for DNA helicases from cultured human cells which will be required for chromosomal DNA replication. Through the screening of such activities, I have obtained following three major results.1.I have cloned DNA fragments which allow DNA synthesis in the presence of a viral DNA helicase. The obtained DNA fragments contain dinucleotide repeat sequences and will be useful to assay DNA helicases required for DNA replication.2.I have isolated a DNA helicase from a protein fraction assocated with anti-DNA polymerase alpha antibody. This helicase has an apparent molecular weight of 210kDa, the direction of 3'to 5'and dependency on a single stranded binding protein, RPA.However, this does not have a DNA synthesis activity in the presence of a dinucleotide repeat sequence.3.I have developed an assay system to screen a processive DNA helicase. Using this reaction, I have isolated a DNA helicase tightly associating with cell nuclei. This nuclear helicase (helicase n) has an apparent molecular weight of 190kDa and the direction of 3'to 5'. Its 110kDa peptide has an ATP binding activity. This helicase also exhibits strong dependency on RPA,and this may indicate its specific interaction with this replication protein. These characteristics suggest that this is a novel helicase and will be involved in DNA replication.