研究领域

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Isolation and characterization of mammalian origins of DNA replication Our primary research interest is in the molecular basis of the mechanisms regulating mammalian DNA replication. Eukaryotic chromosomes are organized into multiple replication units that initiate replication only one per cell cycle. The mechanism that prevents the reinitiation of replication of DNA that has been previously replicated is unknown. Among the major questions about mammalian DNA replication are: 1) whether initiation occurs at specific DNA sequences (replication origins), and 2) what are the molecular features of these sequences. Using the instability of replication loops as a method for the isolation of active replication origins, we have purified and cloned DNA sequences that contain origins of replication. In this manner we have generated libraries of monkey and human cell DNA replication origin-enriched sequences (ors) that are activated at the onset of S phase. We have characterized several of these ors in terms of their genomic complexity, molecular structure and ability to initiate replication, using a variety of biochemical and molecular biological techniques. We have also established a cell-free (in vitro) DNA replication system using the orsclones as DNA templates. Our current objectives are to: a) further characterize, in vivo and in vitro, mammalian cis-acting sequences directing the initiation of DNA replication, including studying the functional requirements of the ors at their chromosomal locus; b) clone the human homologues of the functional monkey ors that we have identified; c) use our in vitro replication system to analyze in detail the mechanism of initiation of mamalian DNA replication and its control; d) carry out deletion/mutation analyses of monkey and human ors with demonstrated biological activity in order to identify the specific sequence requirements for origin function and define common features shared by mammalian core origins; and e) identify DNA elements and factors which can modulate origin function in vivo and in vitro. These projects are directly pertinent to underlying fundamental mechanisms of cell neoplasia, differentiation, development and senescence. Purification and characterization of human DNA replication proteins The general objective of this project is to: a) identify and purify proteins involved in mammalian DNA replication and determine their function, and b) characterize these proteins and clone the genes that encode them. To achieve this, we are using the cloned origins of DNA replication (ors) that we isolated from mammalian (monkey and human) cells, in conjunction with our mammalian in vitro DNA replication system. We recently identified and purified two human proteins: 1) OBA, a sequence-specific binding protein, and 2) CBP, a cruciform-specific binding protein. We have found that both of these proteins associate in vivo with mammalian origins of DNA replication. We are currently analyzing their interaction with the ors by in vitro and in vivo footprinting and studying their involvement in initiation events of DNA replication using our in vitro replication system. We are also analyzing their interactions with other replication proteins. These studies will enable us to dissect the regulatory steps that lead to the initiation of DNA replication and help us define those steps involved in the abnormal replication of cell neoplasia. Role of cruciform DNA in the regulation of DNA replication The general objective of this project is to analyze the role of DNA cruciforms in the regulation of DNA replication. To achieve this, among other methods, we are using our in vitro DNA replication system and the cruciform binding protein (CBP/14-3-3) that we recently purified. These projects will enable us to dissect the regulatory steps that lead to the initiation of DNA replication and help us define those steps involved in the abnormal replication of cell neoplasia.