This application whose overall goal is to gain insights into DNA replication domains, fragile sites and how cells maintain chromosome stability. This goal will be achieved through the study of the major breakpoint region (mbr) of the BCL2 oncogene on chromosome 18. This proposal involves several specific aims: 1) characterize the behavior of mbr and its associated protein complexes with a particular emphasis on the effects of DNA replication and BCL2 expression; 2) demonstrate and characterize the occurrence of ds DNA breaks within the mbr and investigate the relationship, if any, between mbr ds breaks, radiomimetic DNA damage and the onset of differentiation and programmed cell death; 3) determine, through FISH analysis, if the FRA18B fragile site occurs within the mbr region of BCL2; 4); establish and study YAC- based BCL2 model systems that will allow further analysis of the functional roles of the mbr sequence and mbr-binding factors, as well as permit, if specific aim 3 is successful, sequence and functional analyses of the FRA18B fragile site. The mbr region of the BCL2 gene is frequently involved in the t(14,18) translocations found in follicular lymphomas, with most translocations containing junction sequences that map within a 100 bp region of exon 3 of BCL2. This clustering of breakpoints suggests that this mbr region has unique structural or functional properties that renders it a target for chromosomal rearrangements. At the same time, cytogenetic studies have documented a constitutive fragile site at 18q21.3, raising the possibility that the BCL2 mbr region also may be a fragile site. In this grant, Dr. Krontiris proposes that that the mbr region of BCL2 is a """"""""boundary"""""""" between two replication domains. Dr. Krontiris further contends that the same properties of the mbr region that give it a special role in replication also render it a hotspot for ds DNA breaks, resulting in t(14,18) chromosome translocations and, perhaps, fragile site formation. In this revised grant he also proposes that the mbr region may be involved in triggering DNA damage-sensitive cell cycle checkpoints.
