In eukaryotic cells, checkpoint control mechanisms prevent cell division if the genome contains stalled DNA replication forks or damaged DNA. Checkpoint pathways contain various proteins that detect problems in the genome and thereupon activate kinase-signaling pathways that control cell cycle progression. In vertebrates, the master regulatory kinase ATR functions at the apex of key checkpoint responses. ATR phosphorylates the checkpoint effector kinase Chk1 with the assistance of the mediator protein Claspin. The phosphorylated, activated form of Chk1 modulates the activity of pivotal cell cycle control enzymes in order to prohibit mitotic entry. ATR possesses a binding partner called ATRIP that interacts directly with RPA. This property enables the ATR-ATRIP complex to accumulate at various DNA lesions that share RPA-coated, single- stranded DNA as an important structural feature. However, the association of ATR-ATRIP with RPA-coated DNA is not sufficient for its activation. This observation suggested that ATR-ATRIP must interact with one or more additional components at DNA lesions in order to undergo checkpoint-dependent stimulation of its kinase activity. Recently, it has been shown that a protein known as TopBP1 functions as the direct upstream activator of the ATR-ATRIP complex. TopBP1 is a multi-functional protein that is necessary for both DNA replication and checkpoint control. Moreover, the association of TopBP1 with the checkpoint clamp comprised of Rad9-Hus1-Rad1 (the 9-1-1 complex) regulates the interaction of TopBP1 with ATR- ATRIP. These studies have revealed critical early steps in the initiation of checkpoint responses. In the upcoming grant period, a variety of studies will be carried out to examine the structure, function, and regulation of TopBP1. These investigations will be performed mostly with Xenopus egg extracts, a system that allows detailed biochemical analysis of checkpoint control mechanisms. This system also provides an excellent model for checkpoint regulation in human cells. Structure-function analyses will be carried out to elucidate the various functional domains of TopBP1 and their contribution to its regulation. In addition, mechanistic studies will be conducted to reveal how the 9-1-1 complex regulates the ability of TopBP1 to carry out the activation of ATR-ATRIP. A newly identified regulatory interaction between TopBP1 and the Mre11-Rad50-Nbs1 (MRN) complex will be also investigated. Finally, novel interactions and functions of TopBP1 at stalled replication forks will be explored. Through the study of TopBP1 in a vertebrate system that is amenable to intensive functional analysis, important insights may be gleaned into the mechanisms by which animal cells prevent the occurrence of chromosomal aberrations.

Public Health Relevance

Cells utilize intricate surveillance or checkpoint mechanisms to ensure that their genetic material remains intact throughout life. If these regulatory mechanisms do not function properly, cells accumulate defects in their chromosomes that may ultimately result in cancer. Therefore, a thorough knowledge of checkpoint mechanisms is essential for understanding the root causes of cancer.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM070891-08
Application #
8325690
Study Section
Cellular Signaling and Regulatory Systems Study Section (CSRS)
Program Officer
Reddy, Michael K
Project Start
2004-09-01
Project End
2014-08-31
Budget Start
2012-09-01
Budget End
2014-08-31
Support Year
8
Fiscal Year
2012
Total Cost
$472,877
Indirect Cost
$180,978
Name
California Institute of Technology
Department
None
Type
Schools of Arts and Sciences
DUNS #
009584210
City
Pasadena
State
CA
Country
United States
Zip Code
91125
Lee, Joon; Dunphy, William G (2013) The Mre11-Rad50-Nbs1 (MRN) complex has a specific role in the activation of Chk1 in response to stalled replication forks. Mol Biol Cell 24:1343-53
Kumar, Sanjay; Yoo, Hae Yong; Kumagai, Akiko et al. (2012) Role for Rif1 in the checkpoint response to damaged DNA in Xenopus egg extracts. Cell Cycle 11:1183-94
Kumagai, Akiko; Shevchenko, Anna; Shevchenko, Andrej et al. (2011) Direct regulation of Treslin by cyclin-dependent kinase is essential for the onset of DNA replication. J Cell Biol 193:995-1007
Meng, Zheng; Capalbo, Luisa; Glover, David M et al. (2011) Role for casein kinase 1 in the phosphorylation of Claspin on critical residues necessary for the activation of Chk1. Mol Biol Cell 22:2834-47
Ramirez-Lugo, Juan S; Yoo, Hae Yong; Yoon, Su Jin et al. (2011) CtIP interacts with TopBP1 and Nbs1 in the response to double-stranded DNA breaks (DSBs) in Xenopus egg extracts. Cell Cycle 10:469-80
Wawrousek, Karen E; Fortini, Barbara K; Polaczek, Piotr et al. (2010) Xenopus DNA2 is a helicase/nuclease that is found in complexes with replication proteins And-1/Ctf4 and Mcm10 and DSB response proteins Nbs1 and ATM. Cell Cycle 9:1156-66
Gold, Daniel A; Dunphy, William G (2010) Drf1-dependent kinase interacts with Claspin through a conserved protein motif. J Biol Chem 285:12638-46
Kumagai, Akiko; Shevchenko, Anna; Shevchenko, Andrej et al. (2010) Treslin collaborates with TopBP1 in triggering the initiation of DNA replication. Cell 140:349-59
Lee, Joon; Dunphy, William G (2010) Rad17 plays a central role in establishment of the interaction between TopBP1 and the Rad9-Hus1-Rad1 complex at stalled replication forks. Mol Biol Cell 21:926-35
Yoo, Hae Yong; Kumagai, Akiko; Shevchenko, Anna et al. (2009) The Mre11-Rad50-Nbs1 complex mediates activation of TopBP1 by ATM. Mol Biol Cell 20:2351-60

Showing the most recent 10 out of 21 publications