Studies on the regulation of cell growth and division are central to understanding human cell proliferative diseases such as cancer. One approach to identify these important regulatory factors is to first understand the mechanism of DNA replication in eukaryotic cells and identify the replication proteins that may be regulated. In turn, these proteins can be used as substrates to identify the key regulators. To this end, we have been studying DNA replication from the Simian Virus 40 (SV40) origin using a cell-free replication system. The DNA tumor virus, SV40 has long been a model for understanding macromolecular processes such as DNA replication and transcription and for studies on cell transformation by oncogenes. A cell free system for the replication of DNA containing the SV40 origin of DNA replication has been extensively characterized and a number of replication factors have been purified from human cells. These in turn have been used to characterize the mechanism of DNA replication in eukaryotes. One such protein, called replication factor A, is overproduced in human tumor and transformed cells. This protein functions in the initiation of DNA replication, but also stimulates the activity of both DNA polymerases alpha and delta, during the elongation stage of DNA replication. We have shown that RF-A is phosphorylated in a cell cycle dependent manner in both human and yeast cells. The goal of this proposed research is to understand the regulation of DNA replication in eukaryotic cells, using the SV40 DNA replication system as the foundation for the proposed research. In particular, the cell cycle dependent phosphorylation of replication factor A will be further characterized, particularly the identification of the cell cycle regulated protein kinase that phosphorylates it in S phase. How phosphorylation affects the function of the protein will also be investigated. Finally, the distribution of sites of DNA replication and RF-A in the nucleus will be studied to determine if temporal regulation of these structures occurs.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Molecular Cytology Study Section (CTY)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Cold Spring Harbor Laboratory
Cold Spring Harbor
United States
Zip Code
On, Kin Fan; Jaremko, Matt; Stillman, Bruce et al. (2018) A structural view of the initiators for chromosome replication. Curr Opin Struct Biol 53:131-139
Yuan, Zuanning; Riera, Alberto; Bai, Lin et al. (2017) Structural basis of Mcm2-7 replicative helicase loading by ORC-Cdc6 and Cdt1. Nat Struct Mol Biol 24:316-324
Tocilj, Ante; On, Kin Fan; Yuan, Zuanning et al. (2017) Structure of the active form of human origin recognition complex and its ATPase motor module. Elife 6:
Noguchi, Yasunori; Yuan, Zuanning; Bai, Lin et al. (2017) Cryo-EM structure of Mcm2-7 double hexamer on DNA suggests a lagging-strand DNA extrusion model. Proc Natl Acad Sci U S A 114:E9529-E9538
Sheu, Yi-Jun; Kinney, Justin B; Stillman, Bruce (2016) Concerted activities of Mcm4, Sld3, and Dbf4 in control of origin activation and DNA replication fork progression. Genome Res 26:315-30
Stillman, Bruce (2015) Reconsidering DNA Polymerases at the Replication Fork in Eukaryotes. Mol Cell 59:139-41
Sun, Jingchuan; Fernandez-Cid, Alejandra; Riera, Alberto et al. (2014) Structural and mechanistic insights into Mcm2-7 double-hexamer assembly and function. Genes Dev 28:2291-303
Sheu, Yi-Jun; Kinney, Justin B; Lengronne, Armelle et al. (2014) Domain within the helicase subunit Mcm4 integrates multiple kinase signals to control DNA replication initiation and fork progression. Proc Natl Acad Sci U S A 111:E1899-908
Sun, Jingchuan; Evrin, Cecile; Samel, Stefan A et al. (2013) Cryo-EM structure of a helicase loading intermediate containing ORC-Cdc6-Cdt1-MCM2-7 bound to DNA. Nat Struct Mol Biol 20:944-51
Rossmann, Marlies P; Stillman, Bruce (2013) Immunoblotting histones from yeast whole-cell protein extracts. Cold Spring Harb Protoc 2013:625-30

Showing the most recent 10 out of 34 publications