The DNA double helix comprises two antiparallel strands, yet the polymerases responsible for its replication can synthesize new DNA only in the 5’-3’ direction. As a consequence, DNA replication is an intrinsically asymmetric process. Only one parental strand at the replication fork can be continuously replicated: the other ”“ the lagging strand ”“ is discontinuously synthesized via the iterative generation, processing and ligation of short DNA strands known as Okazaki fragments.
The fundamental mechanism by which DNA is replicated is almost universally conserved. However, the genetic, epigenetic and evolutionary consequences of asymmetric DNA replication ”“ as well as the molecular mechanisms by which lagging-strand synthesis occurs ”“ remain under-studied. My lab uses genomics to investigate several inter-related aspects of lagging-strand synthesis and replication asymmetry, generally using the budding yeast Saccharomyces cerevisiae ”“ a simple eukaryote ”“ as a model system.
The mechanism and regulation of lagging-strand synthesis
Around 40,000 Okazaki fragments are generated per S-phase in S. cerevisiae, and up to 20 million in a human cell. The synthesis of each Okazaki fragment requires the sequential action of several enzymatic activities to prime, extend, process and ligate the nascent fragment into the growing lagging strand. We are investigating what mediates the transitions between the three Okazaki polymerases (primase, Pol Î± and Pol Î´), and characterizing the specific contributions of several redundant helicases and nucleases to fragment processing.
Obstacle bypass by the eukaryotic replisome
A variety of impediments, including DNA damage and stable DNA-protein complexes, can stall DNA polymerases and lead to replication fork arrest. Okazaki fragment sequencing experiments allow us to infer the direction of replication fork movement across the genome and, thus, to investigate replication fork stalling at unprecedented resolution. In separate experiments, we are developing methods to analyze gapped DNA replication intermediates in order to define the mechanisms by which the eukaryotic replication machinery is able to tolerate unrepaired damage to the replication template.
Epigenetics and replication asymmetry
Histones and other DNA-bound proteins determine the expression state of genes, thereby controlling the behavior of the cell. Because replication requires a single-stranded template, all DNA-bound proteins must be removed prior to passage of the replication fork. Some, for example bulk histones, are tightly regulated such that they are present in one copy per daughter genome. Other proteins, for example transcription factors and enhancers, will be present in one copy per two daughter genomes immediately following replication. We aim to dissect the role played by the asymmetric mechanism of DNA replication in the generation and persistence of epigenetic asymmetry.