Chromosomes are the permanent repositories of the information that directs cell function. Each chromosome consists of a single DNA molecule with an array of associated proteins. Each time a cell divides it must accurately and completely replicate the DNA at the foundation of each chromosome and reassemble the proteins that interpret this essential cellular blueprint. Remarkably, during an average human lifetime this process accurately synthesizes trillions of miles of DNA. Inaccurate, incomplete, or excessive DNA replication can lead to cell death, developmental abnormalities, or cancer. Consistent with its critical role, chromosome duplication is carefully coordinated with cell division.
Our studies focus on the events that occur at the starting points of chromosome duplication, called origins of DNA replication. These DNA sequences are found at multiple sites on each eukaryotic chromosome and direct the assembly of a bidirectional pair of replication machines, or replisomes, that replicate the DNA on both sides of the origin. Each replisome must coordinate the activity of multiple enzymes, including a DNA helicase, three DNA polymerases, and an RNA polymerase. The events that occur at origins can be broken down into four steps: origin selection, helicase loading, helicase activation, and replisome assembly. To maintain proper chromosome numbers, dividing cells separate origin selection and helicase loading from helicase activation and replisome assembly. This separation ensures that each origin of replication can initiate replication only once per cell division, preventing rereplication of the genome.
The six-protein origin recognition complex (ORC) mediates the initial selection of origin DNA sequences. ORC was initially discovered in the budding yeast Saccharomyces cerevisiae, through its ability to bind to a conserved sequence present in this organism's origins of DNA replication. Although these sites are present in many positions across the genome, only a subset are bound by ORC. In collaboration with David MacAlpine's lab (Duke University), we used genome-wide mapping of nucleosomes to demonstrate that nucleosome-depleted regions are a critical determinant of the sites bound by ORC. Once bound to origin DNA, ORC establishes the precise position of nucleosomes on both sides of the origin. Our previous experiments indicate that this positioning is critical for helicase loading. We are currently focused on understanding how these nucleosome-depleted regions are encoded at origins of replication and how the positioned nucleosomes influence helicase loading and replication initiation.
As cells enter the G1 phase of the cell cycle, ORC recruits two other helicase-loading factors (Cdc6 and Cdt1) and the replicative DNA helicase (the Mcm2-7 complex) to the origin DNA. In a series of ATP-dependent events, ORC, Cdc6, and Cdt1 load the DNA helicase onto the adjacent DNA. After loading, two ring-shaped, hexameric Mcm2-7 complexes encircle double-stranded DNA (dsDNA) as a head-to-head double hexamer. Although inactive, these loaded double hexamers mark all potential origins of replication and are poised for bidirectional initiation.
We have investigated ATP control of helicase loading extensively. Analysis of Cdc6, ORC, and Mcm2-7 ATPase mutants reveals different roles for each protein. Mcm2-7 ATP binding and hydrolysis are primarily responsible for the ATP requirement for helicase loading. Although Cdc6 ATP hydrolysis mutants also contribute to helicase loading, this function is primarily involved in the release of Mcm2-7 complexes that fail to load. ORC ATP hydrolysis is not required for helicase loading but is important for multiple rounds of Mcm2-7 loading and is essential in vivo.
Our latest studies use single-molecule approaches to address protein dynamics during helicase loading and provide insights into the mechanisms that establish bidirectional replication initiation (Figure). In collaboration with Jeff Gelles's laboratory at Brandeis University, we have developed methods to monitor the association of multiple helicase-loading proteins with DNA by using colocalization single-molecule spectroscopy (CoSMoS). Our studies reveal that the sequential loading of the two Mcm2-7 helicases in a double hexamer involves the ordered binding and release of distinct Cdc6 and Cdt1 molecules. In contrast, only one ORC molecule directs loading of both helicases in a double hexamer. Single-molecule fluorescence resonance energy transfer (FRET) detection of Mcm-Mcm interactions shows that double hexamer formation occurs rapidly after arrival of the second helicase. Together, our studies suggest that distinct mechanisms load the first and second helicase in each double hexamer and ensure that they are in the head-to-head conformation required for subsequent bidirectional initiation. We are expanding these studies to monitor additional events during helicase loading (e.g. opening and closing of the Mcm2-7 ring) and to study mutant helicase-loading proteins to gain insights into the many new steps in helicase loading we have uncovered.
Helicase Activation and Replisome Assembly
Mcm2-7 complexes loaded onto the origin DNA during G1 are only activated upon entry into the S phase of the cell cycle. Helicase activation requires the action of the Dbf4-dependent Cdc7 kinase (DDK) and S-phase cyclin-dependent kinase (S-CDK). These kinases trigger recruitment of the helicase-activating factors Cdc45 and GINS, forming the Cdc45-Mcm2-7-GINS (CMG) activated helicase. In addition to activating the helicase, the cell must assemble the rest of the DNA replisome, including the three replicative DNA polymerases that are dedicated to leading-strand (Pol e) or lagging-strand (Pol a/primase, Pol d) synthesis.
We used assays that recapitulate the origin-dependent initiation of replication to determine that DDK and S-CDK trigger distinct and sequential events during helicase activation and that DDK is active prior to S phase at early origins of replication. By analyzing replisome assembly, we found that the leading-strand DNA polymerase is recruited before its lagging-strand counterparts. This order of events ensures that the leading-strand DNA polymerase is present before any RNA primer synthesis so that it can elongate the first primers.
We have used this replication assay to understand the functions of Mcm2-7 during replication initiation. Although many of the Mcm2-7 ATPase motif mutations we studied were defective in helicase loading, a subset of these mutant complexes showed only minor defects. Analysis of these mutants in the replication initiation assay revealed defects in CMG complex formation, suggesting roles for specific Mcm2-7 ATPase motifs in helicase activation. We have also collaborated with Eric Enemark's lab (St. Jude Children's Research Hospital) to demonstrate that a newly identified single-stranded DNA–binding domain in the N-terminal region of Mcm2-7 is also involved in helicase activation. Intriguingly, the polarity of this interaction predicts preferential binding to the single-stranded DNA that the Mcm2-7 complex encircles during replisome movement. This observation suggests a mechanism for Mcm2-7 to retain the translocating strand in the central channel during Mcm2-7's transition from encircling double-strand to encircling single-stranded DNA. Currently, we are using a fully reconstituted assay that recapitulates all the events of replication initiation and elongation to gain insights into the mechanics of replication initiation and we are pursuing single-molecule assays for the events downstream of helicase loading.
Impact of Chromatin on Replication Initiation
We are also studying the impact of nucleosomal DNA templates on DNA replication initiation. Using nucleosomal DNA templates assembled with purified histones, a histone chaperone and a chromatin remodeling enzyme, we are exploring the events of replication initiation that are altered in response to different nucleosome positioning or density. Interestingly, we have identified conditions that alter helicase loading without effecting subsequent helicase activation and vice versa. Are aim is to identify the mechanisms by which nucleosomes are effecting these events. In the future, we will extend these studies to investigate the effect of histone modifications and chromatin remodeling complexes on these events.
A grant from the National Institutes of Health provided partial support for these projects.
As of March 14, 2016