The cell cycle in eukaryotic organisms is a highly regulated network of processes that contains numerous checks and balances to ensure its proper and timely execution. Cell cycle checkpoints ensure that the events during a specific period of the cell cycle are carried out appropriately before proceeding to the next phase of the cell cycle. The checkpoint mechanisms of interest in this proposal are those that respond to DNA damage and to problems arising during DNA replication. Defects in checkpoints result in increased chromosome instability and such defects are known cancer predisposition conditions in human. DNA damage and DNA replication checkpoint pathways have been conserved from yeast to human which makes the former an ideal organisms for fundamental checkpoint studies. The primary focus of this proposal will be on the sensor protein kinase Mec1, the ortholog of human ATR, that initiates the checkpoint. Mec1/ATR kinase activity is activated as the initiating step of the checkpoint pathway, through interaction with specific activators that show cell-cycle phase specificity. A second protein kinase, Rad53, which is related to human Chk1 and Chk2, acts downstream in halting the cell cycle and activating other response pathways. While numerous genetic and molecular biological studies have identified many of the factors involved in the various checkpoints, the biochemical mechanisms of activation of Mec1 and of Rad53 have remained relatively obscure. The biochemical studies in this proposal are aimed at understanding Mec1 activation by the replication protein Dna2 (Aim 1);to study the structure of Mec1 by cryo-electron microscopy and crystallogaphy and the nature of activation of Mec1 by structure-function analysis using small peptides as probes (Aim 2);and to study the role of DNA binding in activation of Rad53 (Aim 3). These biochemical experiments will be complemented by genetic studies to obtain an integrated view of checkpoint activation.

Public Health Relevance

In human cells, so-called checkpoints monitor the intactness of the DNA, our genetic material, and the correctness of DNA duplication prior to cell division. Patients with known defects in checkpoint pathways, such as Ataxia telangiectasia and Seckel syndrome, are at a greatly increased risk for developing cancer. Checkpoint pathways are highly conserved from human to yeast, and we are proposing to study these pathways in yeast, because this model organism is more approachable to genetic and biochemical analysis.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Molecular Genetics B Study Section (MGB)
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Reddy, Michael K
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Washington University
Schools of Medicine
Saint Louis
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Burgers, Peter M J; Kunkel, Thomas A (2017) Eukaryotic DNA Replication Fork. Annu Rev Biochem 86:417-438
Wanrooij, Paulina H; Tannous, Elias; Kumar, Sandeep et al. (2016) Probing the Mec1ATR Checkpoint Activation Mechanism with Small Peptides. J Biol Chem 291:393-401
Sawicka, Marta; Wanrooij, Paulina H; Darbari, Vidya C et al. (2016) The Dimeric Architecture of Checkpoint Kinases Mec1ATR and Tel1ATM Reveal a Common Structural Organization. J Biol Chem 291:13436-47
Wanrooij, Paulina H; Burgers, Peter M (2015) Yet another job for Dna2: Checkpoint activation. DNA Repair (Amst) 32:17-23
Kumar, Sandeep; Burgers, Peter M (2013) Lagging strand maturation factor Dna2 is a component of the replication checkpoint initiation machinery. Genes Dev 27:313-21
Sparks, Justin L; Kumar, Rakesh; Singh, Mayank et al. (2012) Human exonuclease 5 is a novel sliding exonuclease required for genome stability. J Biol Chem 287:42773-83
Navadgi-Patil, Vasundhara M; Burgers, Peter M (2011) Cell-cycle-specific activators of the Mec1/ATR checkpoint kinase. Biochem Soc Trans 39:600-5
Burgers, Peter M (2011) It's all about flaps: Dna2 and checkpoint activation. Cell Cycle 10:2417-8
Navadgi-Patil, Vasundhara M; Kumar, Sandeep; Burgers, Peter M (2011) The unstructured C-terminal tail of yeast Dpb11 (human TopBP1) protein is dispensable for DNA replication and the S phase checkpoint but required for the G2/M checkpoint. J Biol Chem 286:40999-1007
Navadgi-Patil, Vasundhara M; Burgers, Peter M (2009) The unstructured C-terminal tail of the 9-1-1 clamp subunit Ddc1 activates Mec1/ATR via two distinct mechanisms. Mol Cell 36:743-53

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