Billions of base pairs of DNA must be replicated trillions of times during a human lifetime. In addition to copying the DNA, all of the epigenetic information encoded in DNA methylation and histone modifications must also be copied. Adding to the difficulty, replication is challenged by stresses including DNA template lesions, difficult to replicate sequences, and conflicts with transcription. Cells employ multiple repair and signaling responses to replication stress depending on the type, persistence, and location of the problem. We have employed a new method that my lab developed called iPOND (isolation of proteins on nascent DNA) to study DNA replication, chromatin deposition and maturation, and the replication stress response. When combined with quantitative mass spectrometry, this method is particularly powerful at characterizing changes in replisome composition in response to perturbations. As an unbiased approach, it is also a discovery tool. In preliminary studies we identified several new proteins that function at active and damaged replication forks. This application focuses on two of these proteins, which we found to be involved in the replication stress response and in re- establishment of epigenetic information during the cell division cycle.
Aim 1 uses genetic and biochemical methods to test specific hypotheses and models about how these proteins maintain genome and epigenome stability.
Aim 2 examines how replication forks deal with DNA-protein crosslinks including those caused by common environmental toxins and cancer therapies. How a cell deals with these types of fork-stalling events is poorly characterized, and our quantitative iPOND-mass spectrometry approach provides a unique discovery opportunity to understand this significant threat to genome integrity. Since cancer cells have elevated levels of replication stress and many cancer therapeutics work by interfering with DNA replication, these studies will also generate discoveries that may be translated into the cancer clinic.

Public Health Relevance

Accurate and complete replication of the genome and its accompanying epigenetic information must happen trillions of times during a human lifetime. Completion of this research project will define new mechanisms by which cells ensure successful replication in the context of replication stress challenges, thereby maintaining genome stability and preventing disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM116616-03
Application #
9321262
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Reddy, Michael K
Project Start
2015-08-15
Project End
2019-07-31
Budget Start
2017-08-01
Budget End
2018-07-31
Support Year
3
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Biochemistry
Type
Schools of Medicine
DUNS #
965717143
City
Nashville
State
TN
Country
United States
Zip Code
37240
Dungrawala, Huzefa; Bhat, Kamakoti P; Le Meur, Rémy et al. (2017) RADX Promotes Genome Stability and Modulates Chemosensitivity by Regulating RAD51 at Replication Forks. Mol Cell 67:374-386.e5
Vujanovic, Marko; Krietsch, Jana; Raso, Maria Chiara et al. (2017) Replication Fork Slowing and Reversal upon DNA Damage Require PCNA Polyubiquitination and ZRANB3 DNA Translocase Activity. Mol Cell 67:882-890.e5
Reynolds, John J; Bicknell, Louise S; Carroll, Paula et al. (2017) Mutations in DONSON disrupt replication fork stability and cause microcephalic dwarfism. Nat Genet 49:537-549
Poole, Lisa A; Cortez, David (2016) SMARCAL1 and telomeres: Replicating the troublesome ends. Nucleus 7:270-4
Bass, Thomas E; Luzwick, Jessica W; Kavanaugh, Gina et al. (2016) ETAA1 acts at stalled replication forks to maintain genome integrity. Nat Cell Biol 18:1185-1195
Badu-Nkansah, Akosua; Mason, Aaron C; Eichman, Brandt F et al. (2016) Identification of a Substrate Recognition Domain in the Replication Stress Response Protein Zinc Finger Ran-binding Domain-containing Protein 3 (ZRANB3). J Biol Chem 291:8251-7
Kavanaugh, Gina; Ye, Fei; Mohni, Kareem N et al. (2015) A whole genome RNAi screen identifies replication stress response genes. DNA Repair (Amst) 35:55-62
Dungrawala, Huzefa; Rose, Kristie L; Bhat, Kamakoti P et al. (2015) The Replication Checkpoint Prevents Two Types of Fork Collapse without Regulating Replisome Stability. Mol Cell 59:998-1010