The timing of DNA replication is a critical parameter of cellular growth. It correlates with patters of transcriptional regulation, chromatin modification, chromosome structure and genome evolution. Furthermore, replication timing changes as cells differentiate, and disruption of replication timing correlates with genome instability, suggesting an intimate relation between replication timing and other important aspects of chromosome metabolism. However, the mechanisms that regulate replication timing are still largely mysterious. We have developed, and proposes to test, a detailed, generally-applicable model for the mechanism of replication timing. Our model posits stochastic regulation of origin firing, in which each origin has a characteristic probability of firing, and the average time of origin firing is regulated by that orgin firing probability. We propose that the probability of origin firing is regulated by the number of MCM complexes - the replicative helicase which establishes an origin as a site of replication initiation - loaded during G1. Origins with more MCMs loaded are more likely to fire and thus, on average, fire earlier. Further, we propose that the number of MCMs loaded is regulated by the affinity with which ORC - the MCM loader - binds the origin. Higher affinity origins bind ORC for a greater fraction of G1, thus allowing more MCM complexes to be loaded. Finally, we propose that heterochromatin provides a second level of regulation on top of the MCM-based mechanism of origin timing regulation, such that in heterochromatic regions origin firing is delayed at one or more of the basic steps: ORC binding, MCM loading or MCM activation. We will test our model by mapping ORC binding, MCM binding and origin timing across both the budding and fission yeast genomes using deep-sequencing-based approaches. The complimentary strengths of these evolutionarily distant yeasts allow for a more rigorous test of our model. Furthermore, any mechanisms that are conserved between the two are good candidates for general principles of eukaryotic biology. If our model is confirmed, it will change the way people think about replication timing. Moreover, it will change the direction of the field from a focus on trying to discover the mechanisms of replication timing, to being able to directly test how MCM loading is regulated to control replication timing in metazoan genomes. Furthermore, accurate information about the mechanism of replication timing is essential to understand how replication timing influences the genome reprogramming required for stem-cell maintenance and cellular differentiation, as well as its role in maintaining genome stability and preventing cancer.

Public Health Relevance

Many of the genetic changes that lead to cancer are caused by errors during DNA replication. Proper organization of DNA replication is essential to prevent such errors. The proposed research will elucidate the mechanisms that organize replication, allowing for the identification o new therapeutic targets and diagnostic tools for the treatment and prevention of human cancer.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM098815-04
Application #
8811443
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Reddy, Michael K
Project Start
2012-05-15
Project End
2017-02-28
Budget Start
2015-03-01
Budget End
2017-02-28
Support Year
4
Fiscal Year
2015
Total Cost
Indirect Cost
Name
University of Massachusetts Medical School Worcester
Department
Biochemistry
Type
Schools of Medicine
DUNS #
603847393
City
Worcester
State
MA
Country
United States
Zip Code
Hwang, Yung; Futran, Melinda; Hidalgo, Daniel et al. (2017) Global increase in replication fork speed during a p57KIP2-regulated erythroid cell fate switch. Sci Adv 3:e1700298
Keifenheim, Daniel; Sun, Xi-Ming; D'Souza, Edridge et al. (2017) Size-Dependent Expression of the Mitotic Activator Cdc25 Suggests a Mechanism of Size Control in Fission Yeast. Curr Biol 27:1491-1497.e4
Iyer, Divya Ramalingam; Rhind, Nicholas (2017) The Intra-S Checkpoint Responses to DNA Damage. Genes (Basel) 8:
Santaguida, Stefano; Richardson, Amelia; Iyer, Divya Ramalingam et al. (2017) Chromosome Mis-segregation Generates Cell-Cycle-Arrested Cells with Complex Karyotypes that Are Eliminated by the Immune System. Dev Cell 41:638-651.e5
Ohira, Makoto J; Hendrickson, David G; Scott McIsaac, R et al. (2017) An estradiol-inducible promoter enables fast, graduated control of gene expression in fission yeast. Yeast 34:323-334
Willis, Nicholas A; Zhou, Chunshui; Elia, Andrew E H et al. (2016) Identification of S-phase DNA damage-response targets in fission yeast reveals conservation of damage-response networks. Proc Natl Acad Sci U S A 113:E3676-85
Das, Shankar P; Rhind, Nicholas (2016) How and why multiple MCMs are loaded at origins of DNA replication. Bioessays 38:613-7
Das, Shankar P; Borrman, Tyler; Liu, Victor W T et al. (2015) Replication timing is regulated by the number of MCMs loaded at origins. Genome Res 25:1886-92
Rhind, Nicholas (2015) Incorporation of thymidine analogs for studying replication kinetics in fission yeast. Methods Mol Biol 1300:99-104
Rhind, Nicholas (2014) The three most important things about origins: location, location, location. Mol Syst Biol 10:723

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