To preserve the integrity of their genomic DNA, eukaryotic cells utilize a variety of surveillance mechanisms known as checkpoint controls. For example, during cell duplication, cells must make sure that they have replicated their DNA accurately. To cope with the challenges of copying the DNA precisely and rectifying any problems that arise in the process, cells utilize a myriad of regulatory proteins. In vertebrates, the kinase ATR acts as a pivotal regulator in the mechanisms that detect problems with DNA replication and enable cells to address such defects. A key feature of this kinase is that it undergoes precisely regulated activation upon genomic perturbation. Accordingly, this activation involves elaborate control mechanisms. For example, a binding partner known as ATRIP helps to recruit the ATR-ATRIP complex to RPA-coated regions of single-stranded DNA, which are characteristic of numerous detrimental DNA lesions. However, the ATR- ATRIP complex still remains weakly active upon associating with RPA on the DNA. At some point thereafter, ATR-ATRIP interacts with another protein called TopBP1. This binding results in a massive increase in the kinase activity of ATR and represents the culmination of numerous steps that trigger activation of a checkpoint response. Past studies have indicated that the Rad9-Hus1-Rad1 (9-1-1) complex plays a role in controlling the interaction of TopBP1 with ATR-ATRIP. However, many aspects of this overall process have remained nebulous. It has also been unclear whether activation of ATR occurs solely through this route in animal cells. We have recently observed that the Mre11-Rad50-Nbs1 (MRN) complex plays a novel role in the activation of ATR in response to aberrant DNA replication. The MRN complex had been best known for its role in another type of checkpoint mechanism, namely, the response to double-stranded DNA breaks (DSBs). In the upcoming grant period, a variety of studies will be carried out to elucidate the mechanism by which MRN collaborates with TopBP1 and other checkpoint control proteins to promote the activation of ATR. A systematic series of experiments will be conducted to explore: (1) how the nuclease activity of MRN affects checkpoint induction at replication forks; (2) how MRN and TopBP1 interact with one another and with replication forks during the checkpoint response; and (3) how these steps ultimately lead to the activation of ATR. Moreover, searches will be undertaken for novel regulators in these pathways. These studies will be performed with both Xenopus egg extracts and human tissue culture cells. This strategy will capitalize upon the complementary advantages of each experimental system. Overall, these studies will exploit new perspectives on checkpoint signaling and hence promise to uncover original insights into the mechanisms that safegaurd genomic integrity. This information would be invaluable for understanding how cells forestall cancer-promoting mutations and other disease-inducing genetic abnormalities.

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 progressively accumulate defects in their chromosomes that may ultimately result in cancer. Therefore, a thorough knowledge of checkpoint mechanisms is essential both for understanding the root causes of cancer and approaching its treatment.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM070891-12
Application #
9413920
Study Section
Cellular Signaling and Regulatory Systems Study Section (CSRS)
Program Officer
Reddy, Michael K
Project Start
2004-09-01
Project End
2018-12-31
Budget Start
2018-01-01
Budget End
2018-12-31
Support Year
12
Fiscal Year
2018
Total Cost
Indirect Cost
Name
California Institute of Technology
Department
Type
Schools of Arts and Sciences
DUNS #
009584210
City
Pasadena
State
CA
Country
United States
Zip Code
91125
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Mu, Ruiling; Tat, John; Zamudio, Robert et al. (2017) CKS Proteins Promote Checkpoint Recovery by Stimulating Phosphorylation of Treslin. Mol Cell Biol 37:
Guo, Cai; Kumagai, Akiko; Schlacher, Katharina et al. (2015) Interaction of Chk1 with Treslin negatively regulates the initiation of chromosomal DNA replication. Mol Cell 57:492-505
Ryu, Hyunju; Yoshida, Makoto M; Sridharan, Vinidhra et al. (2015) SUMOylation of the C-terminal domain of DNA topoisomerase II? regulates the centromeric localization of Claspin. Cell Cycle 14:2777-84
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
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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

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