We are studying the proteins responsible for the initiation of eukaryotic DNA replication using a combination of biochemistry, genetics, and molecular biology. In particular, we wish to understand how the origin recognition protein, ORC, promotes the initiation of DNA synthesis from yeast origins of replication. The relationship between DNA replication and transcriptional control is investigated.
In eukaryotic cells, replication of the genome is carefully coordinated with the program of the cell division cycle. The initiation of DNA replication only ensues after the cell has committed itself to division and entered the S phase of the cell 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, little is known about the proteins and mechanisms that direct and regulate the initiation of replication at eukaryotic chromosomes.
DNA Replication in S. cerevisiae. The short (150-300 bp), well characterized origins of replication derived from S. cerevisiae chromosomes and the ability to combine biochemical and genetic approaches have led us to study DNA replication in this eukaryote. In particular, we are studying a six protein assembly, referred to as the origin recognition complex (ORC), that recognizes the only essential element of yeast origins. We have identified the genes encoding each of the subunits in the complex and found that each is essential for yeast cell viability. When expressed simultaneously in insect cells, these six genes support the reconstitution of ORC that is indistinguishable from ORC purified from yeast cells. Using this expression system we are able to produce and study mutant forms of ORC that cannot be produced in yeast cells because of their lethal phenotype. Both biochemical and genetic evidence supports the hypothesis that ORC selects yeast origins of replication and plays a critical role in the subsequent initiation of DNA synthesis from these sites. 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.
Although a relatively small region of the origin (15-20 bp) is required to direct ORC DNA binding, ORC contacts over five turns of the DNA helix. These interactions include a series of contacts that suggest that ORC wraps or distorts the origin DNA. DNA distortion is frequently observed at origins of replication as a precursor to unwinding of the DNA duplex. The alterations of the origin DNA by ORC and the ORC subunits involved in mediating origin binding remain poorly understood. We are using a combination of protein-DNA crosslinking and analysis of ORC complexes missing one or more subunits to identify the proteins required for origin binding. Detailed DNA binding studies are being performed to determine the nature of the ORC-origin interactions.
In addition to binding and distorting origin DNA, origin recognition proteins frequently direct the assembly of the DNA synthesis machinery at the origin. In vivo footprinting studies argue that ORC is bound to the origin throughout most of the cell cycle, implying that binding of ORC to an origin is unlikely to be sufficient to initiate DNA replication. This suggests that like bacterial and viral origin recognition proteins, interactions between ORC and other replication or regulatory proteins are critical to initiate DNA replication at yeast chromosomes. We are using both protein affinity chromatography and genetic screens to identify ORC interacting proteins that regulate DNA replication.
Regulation of ORC by ATP. ORC requires ATP to specifically bind to origin DNA. DNA binding studies using non-hydrolyzable analogs of ATP suggest ATP hydrolysis is required to direct specific DNA binding by ORC. The presence of nucleotide binding motifs in two of the ORC subunits is consistent with a role for nucleotide binding in ORC function and mutations in these motifs result in defective ORC function in vivo. The function of ATP in regulating ORC in vivo remains unknown. ATP binding and hydrolysis may act as a clock, regulating the replicative potential of ORC during the cell cycle. Alternatively, ATP may provide the energy necessary for subsequent events of replication such as unwinding the origin DNA. To study ATP regulation in vivo we are using genetic approaches to generate new alleles of the genes encoding subunits most likely to interact with ATP. In addition, to determine how ATP regulates ORC DNA binding we are studying the biochemical properties of ORC complexes that are altered in the putative ATP binding sites.
Transcriptional Silencing and DNA Replication: In addition, to its DNA replication functions ORC has also been implicated in transcriptional silencing at the yeast silent mating type loci. ORC binds to each of the cis-acting elements required to maintain mating type silencing and mutations in ORC genes result in derepression of the silent mating type loci. Intriguingly, the largest subunit of ORC is closely related to SIR3, a protein required for transcriptional silencing at both the silent mating type loci and yeast telomeres. By comparing the functions of ORC at origins of replication and the mating type silencers we hope to gain insights into the relationship between transcriptional regulation and DNA replication in the nucleus.