If one were to rank cellular processes of critical importance, duplication of the genome must surely be near the top. Its significance as a fundamental process has prompted studies on specific aspects that have revealed that distinct molecular machines operate during different stages of genome duplication. Other studies show that DNA replication is highly regulated so that the genome is duplicated only once per cell cycle. This cell cycle dependence is the result of separate biochemical mechanisms that control DNA replication by modulating the frequency of replication initiation. However, our understanding of these regulatory processes is incomplete. We also know that DNA replication is strictly coordinated with cell growth, but we do not have an understanding of the genetics or biochemistry of this coordination. Hence, major questions remain unanswered.
In metabolically active cells that are not engaged in a cycle of DNA replication, the genome acts as a repository for those genes that are expressed, whether that cell is terminally differentiated, pluripotent, or a microbe in a holding pattern (stationary phase). Bad things happen when pathways that control DNA replication are disrupted, leading to the duplication of an organism’s genome in an unregulated manner. For example, uncontrolled DNA replication gives rise to double strand breaks (DSBs) in DNA that apparently result when newly formed replication forks collide from behind with stalled or more slowly moving forks. The DSBs are lethal if not repaired. If their repair is imprecise in mammalian cells, the errors in DNA can alter the function of genes, pseudogenes, or small noncoding RNAs that control cell proliferation, thus aberrantly inducing malignant cell growth. These observations support the importance of studies on how DNA replication is regulated.
Recently, our lab has worked on pathways that, when disrupted by mutation or overcome by experimentally altering conditions, lead to hyperinitiation of DNA replication. We use E. coli as a model organism because the molecular events of E. coli DNA replication are remarkably similar to those occurring in eukaryotic cells. For example, the replication initiators named DnaA and DnaC of E. coli are comparable in structure by X-ray crystallography to Cdc6 and Orc1, eukaryotic proteins required for replication initiation. Like DnaC, eukaryotic Cdc6 is required to load the replicative helicase at a replication origin. In both E. coli and eukaryotic cells, the replicative helicase is recruited in an inactive form at a replication origin, and must be activated after loading. Hence, our studies in E. coli very likely will provide important insight into the mechanism of DNA replication and its regulation in higher organisms.
Our studies have revealed apparently novel strategies whereby the cell minimizes DSBs by lowering the frequency of initiation, and have also identified new factors that appear to decrease the likelihood of fork collision. Via an independent approach to identify proteins that modulate the frequency of replication initiation, we have discovered several that inhibit the activity of the replication initiator by physically interacting with DnaA protein. A third major approach centers on DnaC. Recent critical studies show that DnaC acts as a checkpoint in the transition from the stage of initiation to the elongation phase of DNA replication. Current experiments focus on the molecular mechanisms of these regulatory proteins on the initiation process, and we anticipate new and exciting findings.