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Academic Background

Nick Rhind received his Ph.D. from the Department of Molecular and Cell Biology at U.C. Berkeley in 1995, where he studied worm sex determination with Barbara Meyer. He was a Leukemia and Lymphoma Society post-doctoral fellow with Paul Russell at the Scripps Research Institute from 1996 to 2001.

Regulation of DNA Replication

Photo: Nick Rhind

Amajor current interest of my lab is how replication is regulated during S phase. We are interested both in how normal S phase is organized to insure efficient replication of the entire genome, and how cells respond to DNA damage during S phase to coordinate replication and repair.

Most of the work in my lab focuses on the fission yeast Schizosaccharomyces pombe (Figure 1). Fission yeast is a great organism for studying replication because it has a simple, well-understood cell cycle and is amenable to genetic, molecular and biochemical approaches. It has the added attraction that the mechanisms of cell cycle and checkpoint control in fission yeast are very similar to those used by human cells. In fact, much of what is known about human cell cycle and checkpoints was first discovered in yeast. My lab is currently pursuing two general areas of the regulation of replication.

Regulation of Origin Firing Kinetics

Eukaryotic genomes replicate in defined patterns, with some parts of the genome replicating early in S phase and other parts replicating later. Replication timing correlates with transcription, chromatin modification, sub-nuclear localization and genome evolution, suggesting an intimate association between replication timing and other important aspects of chromosome metabolism. However, the mechanism of replication timing is currently unknown.

We developed an computational approach to extract replication kinetics from genome-wide replication timecourses (Figure 2). Our results support a model where earlier-firing origins have more MCM complexes loaded and a more-accessible chromatin environment (Figure 3). The MCM complex is the replicative helicase; its loading is what establishes sites as potential replication origins. We propose that the timing of origin firing is regulated in by the number of MCM complexes loaded at an origin. Thus, for the first time, our model suggests a detailed, testable, biochemically plausible mechanism for the regulation of replication timing in eukaryotes. ChIP-seq validation of our model confirms that early firing origins have more MCM loaded. We are currently exploring how MCM loading is regulated at different origins.

Checkpoint Regulation of Replication

Checkpoints are mechanisms that cells use to deal with problems during the cell cycle, such as DNA damage, replication errors and misattachment of chromosomes to the mitotic spindle. By actively responding to these problems, cells can fix most of them. In contrast, cells that lack proper checkpoints are very sensitive to DNA damage and show increased rates of mutation and other chromosomal abnormalities. Loss of checkpoints is an important step in the development of cancer.

The S-phase DNA damage checkpoint slows the rate of replication in response to DNA damage. The simple model is that this checkpoint prevents damaged DNA from being replicated before it is repaired. However, the checkpoint is clearly more subtle than that. Recent results from yeast and human cells suggest that this checkpoint coordinates recombinational repair and replication during S-phase. In particular, we have established an epistatic pathway of recombinational-repair proteins that regulate replication-fork progress in response to DNA damage. We think this pathway regulates a choice the fork makes when it encounters damage: it can replicate quickly in an error-prone manner or, via a checkpoint-dependant mechanism, it can use recombinational strand switching to replicate the damaged template accurately, but slowly (Figure 4). Furthermore, via phospho-proteomics, we have identified candidate checkpoint targets, which maybe responsible for making this decision.

 

 

 

 

 

Figures

 
 
Fission Yeast
 
Figure 1.Fission Yeast
The fission yeast Schizosaccharomyces pombeis a simple, single-cell eukaryote that has proven to be an excellent model for cell cycle and checkpoint regulation. It divides by medial fission, distinguishing it from the budding yeast Saccharomyces cerevisiae, another popular lab yeast.
 
 

Measuring Replication Kinetics

Figure 2. Measuring Replication Kinetics
A) Using deep sequencing to assay the change from one copy to two copies during replication, we can create replication profiles in which each origin is a peaks. Shown is the replication profile for Chromosome 3. The blue dots represent the computer-identified location and height of the origin peaks.

B) A expanded view of the well-studied left arm of Chromosome 3. The green bars represent confirmed origins in the region. The numbered origins were previously described. We have confirmed the activity of the two unnumbered origins by single-molecule DNA fiber origin mapping.

C) Using replication profile timecourses, we can study replication kinetics The extent of replication is determined at timepoints throughout S phase and plotted along the chromosome; for clarity, only 4 time points are show here. Peaks in the profiles (a, b and c) correspond to origins; the slope of the curve leading away from each peak is affected by fork velocity. Fork pause site are recognized as very steep curves that are only replicated by forks coming from the other direction. The individual profiles are assembled into a kinetic profile that reveals the dynamics of replication over time. In particular, the kinetics of origin firing distinguish early efficient origins (a) from early inefficient origins (b) and late efficient origins (c).

 

A Model for the Regulation of Replication Timing

Figure 3. A Model for the Regulation of Replication Timing
We propose that the timing of origin firing is regulated by the number of MCMs loaded. Origins at which many MCMs are loaded are more likely to fire in early S and therefore have an early average firing time; origins at which fewer MCMs are loaded are less likely to fire in early S and therefore have a later average firing time. We further propose that the number of MCMs loaded is regulated by the affinity of ORC for the origin. High-affinity origins are bound by ORC for more of G1 and thus have more MCMs loaded. Heterochromatin could provide a second layer of regulation, on top of our proposed MCM-based mechanism. Heterochromatin could delay origin firing by inhibiting any step in MCM loading or activation. However, based on our preliminary results that many MCMs are loaded at late-firing heterochromatic subtelomeric origins, we propose that heterochromatin acts mainly to inhibit MCM activation.

 

A Model for Checkpoint Regulation of Replication

 

Figure 4. A Model for Checkpoint Regulation of Replication
We have identified three classes of proteins required for the S-phase DNA damage checkpoint, which we think are involved in a checkpoint-dependent choice between slow and fast replication through DNA damage. Mus81 and other fork-metabolism proteins are required for Cds1-dependent checkpoint slowing of replication forks, presumably by recombinational template switching. Rad51 and other recombination protein antagonize the Mus81-dependent pathway and allow fast replication, possibly by leading-strand repriming. Sfr1, Rqh1 and other recombination regulator inhibit the Rad51-dependent pathway and allow the Mus81-dependent pathway to function.

 


 

 

One or more keywords matched the following items that are connected to Rhind, Nicholas

Item TypeName
Academic Article The DNA replication checkpoint directly regulates MBF-dependent G1/S transcription.
Academic Article Incorporation of thymidine analogs for studying replication kinetics in fission yeast.
Academic Article The fission yeast Rad32(Mre11)-Rad50-Nbs1 complex acts both upstream and downstream of checkpoint signaling in the S-phase DNA damage checkpoint.
Academic Article Modeling genome-wide replication kinetics reveals a mechanism for regulation of replication timing.
Academic Article Genome-wide identification and characterization of replication origins by deep sequencing.
Academic Article Signaling pathways that regulate cell division.
Academic Article DNA replication timing.
Academic Article DNA replication origins fire stochastically in fission yeast.
Academic Article DNA replication timing: random thoughts about origin firing.
Academic Article An intrinsic checkpoint model for regulation of replication origins.
Academic Article The Hsk1(Cdc7) replication kinase regulates origin efficiency.
Academic Article Regulation of DNA replication by the S-phase DNA damage checkpoint.
Academic Article The role of specific checkpoint-induced S-phase transcripts in resistance to replicative stress.
Academic Article Replication timing and its emergence from stochastic processes.
Concept DNA Replication Timing
Concept DNA Replication
Academic Article Checkpoint regulation of replication forks: global or local?
Academic Article The three most important things about origins: location, location, location.
Academic Article Incorporation of thymidine analogs for studying replication kinetics in fission yeast.
Academic Article Replication timing is regulated by the number of MCMs loaded at origins.
Academic Article How and why multiple MCMs are loaded at origins of DNA replication.
Academic Article Replication fork slowing and stalling are distinct, checkpoint-independent consequences of replicating damaged DNA.

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  • DNA Replication