UNWINDING OF CHROMATIN BY THE SV40 LARGE T-ANTIGEN DNA HELICASE

We have analysed the unwinding of nucleosomally organized DNA by simia n virus 40 large tumour (T) antigen. Isolated T antigen can bind to ex isting nucleosome cores containing the viral replication origin sequen ce, which results in displacement of the histone octamer and unwinding of the DNA. However, specific binding to nucleosome cores is salt sen sitive and nearly completely blocked under ionic conditions that other wise support DNA replication. Once started, the progressing T antigen helicase, like an elongating RNA polymerase, is not further repressed by histone octamers, irrespective of the presence or absence of linker histone H1. Disruption of the nucleosomal structure in the process of unwinding may be assisted by the demonstrated interaction of the hexa meric T antigen complex with histone proteins H1 and H3. Finally, our studies reveal the inability of topoisomerase I and/or II to continual ly relieve the superhelical tension of covalently closed circular mini chromosomes as generated during, their unwinding by T antigen. This ma y indicate that chromatin relaxation during the process of DNA replica tion can only be efficiently performed by a topoisomerase that is (tra ns)activated by other factors.