Startstellen der DNA-Replikation und Bindestellen des 'Origin Recognition Complex' im Humangenom

Abstract

The first part investigated the temporal and spatial control of the initiation of DNA replication in a human cell free system. I showed that the lamin B2 origin is activated upon entry of the cells into S-phase whereas the rDNA origin is active through progression of mid S-phase. The date present evidence for a spatial and temporal control of replication initiation in human cells that is maintained from one cell cycle to the next. The second part investigated human origin recogntion complex (ORC) proteins which are key player in the initiation of DNA replication. I showed that all six subunits reside at same chromatin sites in vivo. A new version of the chromatin immunoprecipitation assay in combination with quantitative real-time PCR was used for the identification of ORC binding sites in the human genome. A ORC binding site is located close to transcription factor SP1 binding sites in the human TOP1 gene promoter. The region contains two matrix attachment region and the promoter is located within a CpG island. By nascent DNA strand analyses using quantitative real-time PCR it was demonstrated that the ORC binding site coincide with an active of origin of bidirectional DNA replication. ORC was also found to be bound to heterochromatic regions of alphoid satellite DNA which, however, did not serve as a replication origin. This suggests that ORC may have additional functions in heterochromatin despite its role in DNA replication.