In eukaryotic cells, chromosomal replication is carefully coordinated
with the program of the cell division cycle. During S phase,
the cell must replicate the entire
genome as well as the associated chromatin and transcriptional
machinery in an accurate and regulated manner. Despite the importance
of these events in maintaining the genetic integrity of the cell,
the proteins and mechanisms that direct and regulate the duplication
of eukaryotic chromosomes are not yet well understood.
DNA replication in S. cerevisiae: The short (150-300 bp),
well characterized origins of replication derived from the yeast
S.
cerevisiae and the ability to combine biochemical and genetic approaches
have led us to study chromosome duplication in this eukaryote.
In particular, we are studying a six-protein complex, referred
to as the origin recognition complex (ORC), that recognizes the
only essential element of yeast origins. This complex is responsible
for the initial selection of origins of replication through its
DNA binding activity and the subsequent recruitment of other essential
replication factors to the origin. Our strategy is to use a combination
of approaches to determine the role of ORC in yeast DNA replication
and to identify proteins that cooperate with or regulate these
activities.
Recognition of origin DNA by ORC: Because ORC is required for the assembly of higher order complexes at yeast origins of replication, we have investigated the architecture of ORC bound to yeast origins of replication. These studies have determined that the interface between ORC and the DNA is a complex one involving four of the six ORC subunits. In addition to binding DNA, ORC also binds and hydrolyzes ATP and we observe a reciprocal relationship between these events. ORC requires ATP to specifically bind to origin DNA. Association of ORC with origin DNA stabilizes ATP binding and inhibits ATP hydrolysis by the complex. Binding of ORC to DNA does not require ATP hydrolysis, arguing that hydrolysis is important for a downstream event in the initiation of replication. Interestingly, ORC also binds ssDNA in a sequence non-specific manner, and this interaction stimulates ATP hydrolysis. We are investigating how these biochemical findings are related to the formation of ssDNA during the origin-DNA unwinding step of replication initiation.
Assembly and Movement of DNA Replication Complexes: We have investigated the in vivo association of yeast DNA replication proteins with origins of replication and non-origin DNA sequences. During G1 we have identified ORC and other replication proteins as components of a pre-replication complex (pre-RC) at the origin. During S phase, two of these factors (the MCM complex and Cdc45p) dissociate from origin DNA and associate with non-origin DNA with similar kinetics as DNA polymerases. Based on these and other results, we hypothesize that MCM proteins and Cdc45p are components of the protein complexes assembled at both the origin and the replication fork. Using a purified preparation of the six-protein MCM complex, we have assessed its enzymatic activities. These studies identified two distinct enzymatic classes of MCM subunits and make predictions of the architecture and function of this critical replication factor. We are exploiting these and other assays to identify the biochemical function of the MCM proteins and Cdc45p.
ORC Function in Drosophila melanogaster: The DNA sequences that direct the initiation of replication in higher eukaryotes remain poorly understood. In addition, origin selection in higher eukaryotes is under developmental control. We have extended our studies of ORC to the fruit fly Drosophila melanogaster to identify these sequences and to determine how the origin selection process is altered during development. Using protein-DNA crosslinking procedures, we have demonstrated that Drosophila ORC (DmORC) is capable of recognizing an origin involved in chromosomal amplification in vivo. We have purified DmORC and found that this complex can directly recognize the same origin in vitro. We are now using genomic approaches (e.g. Wyrick et al. below) and other tools to identify Drosophila origins across the genome and to dissect the developmental control of origin selection.
Chromatin Structure and Origin Function: Numerous studies of origin function and chromatin assembly suggest an intimate connection between these two processes. To gain a molecular understanding of their interaction, we have undertaken a molecular analysis in yeast. We find that origin sequences are nucleosome free whereas the adjacent sequences are packaged in precisely positioned nucleosomes. We have investigated the proteins responsible for this structure at the ARS1 origin of replication and have found that ORC and a second ARS1 binding factor (Abf1p) are sufficient in vitro and necessary in vivo to establish the appropriate nucleosome positioning. By manipulating the positioning of nucleosomes, we have found that nucleosome location has important consequences for pre-RC formation and origin function. We are currently defining the steps of replication initiation most influenced by local nucleosomes.