ISOLATION OF DNA HELICASES REQUIRED FOR HUMAN CHROMOSOMAL DNA REPLICATION

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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.