DNA is most susceptible to damage during the process of replication. Therefore, complex mechanisms have evolved to ensure the accurate spatial and temporal initiation of DNA synthesis at replication origins, and the faithful copying of DNA during replication fork progression. Errors in the components of the multiprotein replication initiation complex, or the inability of tumor suppressor proteins to resolve blocks to fork movement lead to rearrangements, losses or duplications of DNA, and result in numerous genetic disorders. Our laboratory identified the replication origin 5'to the human c-myc oncogene, and we have shown that the structure, protein binding and function of the core c-myc replicator as a replication origin are the same at its endogenous chromosomal site and at ectopic sites in the human genome. In this application, the c-myc replicator will be used as a model mammalian replication origin to study the DNA sequences and protein factors that enable origin activity. In addition, the c-myc replicator will be used to initiate the replication of naturally occurring disease-related repeated DNA sequences that are impediments to replication fork progress. The proteins involved in sensing and stabilizing stalled replication forks are responsible for the suppression of genome instability, cancer, and neurodegenerative diseases. Because mammalian chromatin imposes constraints on replication that may not be recapitulated in other model systems, this is an innovative approach that allows the characterization of multiple sequences and DNA stress response proteins affecting replication fork stability in a chromatin environment. All three Aims will use site-directed FLP recombinase-mediated cassette exchange (FLP-RMCE) to integrate the c-myc replicator and its modified constructs into a unique site in the human genome.
Aim 1 will use deletion analysis to identify protein binding sites in the c-myc replicator by quantitative chromatin immunoprecipitation (ChIP), define the minimal c-myc replication origin by PCR quantitation (qPCR) of nascent DNA, and assess the effect of Gal4 recruitment of replication proteins to the origin by ChIP and qPCR of nascent DNA.
Aim 2 will test the effects of origin location, orientation, repeat sequence composition, and the effects of fork stabilizing proteins on the expansion or contraction of (CTG7CAG)n and (ATTCT7AGAAT)n microsatellites during replication.
In Aim 3, a naturally occurring asymmetric polypurine7polypyrimidine sequence derived from the human PKD1/TSC2 locus, which we have shown to form a natural stalled replication fork, will be replicated from the ectopic c-myc origin and the role of replisome, DNA stress response proteins, translocase and helicases in fork stabilization and restart will be assessed by siRNA knockdown, ChIP, and qPCR of nascent DNA. We anticipate that these studies will give novel insight into the processes of replication initiation, replication fork progression, and genome stabilization in a human chromosomal environment.
Inappropriate regulation of DNA replication initiation can lead to chromosome fragmentation, abnormal cell division and human disease. We have developed a novel system that mimics the genomic instability observed in cancers and neurodegenerative diseases. Thus, our studies are likely to provide fundamental new insight into the mechanisms of DNA replication, the origins of genetic diseases, and new models for disease treatment.
