RESEARCH

MRN:

Current studies in the lab are primarily focused on the biochemical activities of a complex of proteins, Mrell/Rad50/Nbs1 (MRN), which are critical components in the repair of DNA double-strand breaks. We study the activities of recombinant M/R/N complexes in vitro to characterize its functions on different types of DNA substrates and recombination intermediates. We are interested in how the MRN complex accomplishes the many reactions it performs at double-strand break sites: DNA end recognition, end tethering, initiation of DNA resection, and activation of DNA damage signaling. The Rad50 protein is a member of the Structural Maintenance of Chromosomes (SMC) family of proteins that includes cohesin and condensin complexes. All of these proteins have ATP-binding domains composed of Walker A and Walker B motifs separated by long coiled-coil domains. Some of the most interesting questions about MRN function involve the structure of Rad50: what is accomplished by ATP binding and ATP hydrolysis? What is the purpose of the coiled-coil domain? How do ATP-dependent changes in Rad50 translate into functional activities of the Mre11 protein?

diagram

One of the recent areas we have focused on in MRN function is in double-strand break resection. In preparation of homologous recombination, the 5' strand at a double-strand break site is removed (from a few hundred nucleotides to several thousand nucleotides). Studies from many laboratories have shown that MRN complexes promote resection by downstream helicase and nucleases in vivo, and we have studied this process of resection in vitro with purified archaeal, budding yeast, and human MRN proteins 1-3. Our work has shown that MRN promotes end resection through at least three different mechanisms: through 5' strand endonucleolytic degradation by Mre11, through recruitment of other enzymes, and through displacement of the Ku heterodimer that promotes non-homologous end joining.

diagram
Three mechanisms by which MRN(X) complexes stimulate resection by downstream nucleases4.

The Sae2 protein in S. cerevisae (and the ortholog of Sae2 in mammalian cells, CtIP) are also intimately involved in the initiation of DNA 5' strand resection. We characterized the Sae2 protein in vitro and found that it possesses endonuclease activity that functions cooperatively with MRX5. We are further characterizing Sae2 as well as CtIP and investigating how Sae2 and CtIP are regulated by cell cycle progression. Our overall goal is to decipher the functions of each of these factors at a molecular level in order to understand how they cooperate to guard cells against genetic rearrangements and transformation.

ATM:

The MRN complex works in concert with the Ataxia-Telangiectasia-Mutated (ATM) protein kinase that phosphorylates many downstream targets responsible for checkpoint activation and DNA damage signaling in eukaryotes. We have previously shown that MRN recruits ATM to broken DNA ends and activates its kinase activity at these sites 6, 7. Loss of ATM function in humans is the cause of the genetic disorder Ataxia-Telangiectasia, which results in a diverse set of clinical symptoms that include progressive loss of cerebellum function, immunodeficiency, diabetes, and cancer predisposition. Cells from A-T patients exhibit chromosomal instability, loss of DNA damage checkpoints, and radiation sensitivity. Patients with A-T-like Disorder and Nijmegen Breakage Syndrome show a related subset of symptoms but are caused by mutations in the genes that encode MRN components - another indication of the overlap in function between MRN and the ATM protein. We are currently investigating the mechanisms through which ATM is activated, how post-translational modifications affect this process, and how other ATM-interacting factors influence its regulation8.

We have also recently found that ATM can be activated in an MRN-independent manner through direct oxidation 9, 10. This pathway is important for cellular control of antioxidant functions and for global responses of human cells to reactive oxygen species. Cells from A-T patients exhibit signs of chronic oxidative stress and some of the tissues that are most affected by ATM loss in mouse models of A-T can be protected from damage through exposure to antioxidants. The targets of ATM activation through this pathway are currently being investigated, as well as the relationship between the DNA damage and oxidation pathways of ATM regulation in human cells.

research diagram

Dual modes of ATM activation: through DNA damage and through oxidative stress (top). In the absence of ATM, unidentified components of the antioxidant system are non-functional, leading to high ROS and increases in damage to cellular components, including DNA (bottom)9.

1. Hopkins, B., and Paull, T.T. (2008) The P. furiosus Mre11/Rad50 complex promotes 5' strand resection at a DNA double-strand break. Cell 135, 250-260 [pubmed]

2. Nicolette, M.L., et al. (2010) Mre11-Rad50-Xrs2 and Sae2 promote 5' strand resection of DNA double-strand breaks. Nat Struct Mol Biol 17, 1478-1485 [pubmed]

3. Shim, E.Y., et al. (2010) Saccharomyces cerevisiae Mre11/Rad50/Xrs2 and Ku proteins regulate association of Exo1 and Dna2 with DNA breaks. EMBO J 29, 3370-3380 [pubmed]

4. Paull, T.T. (2010) Making the best of the loose ends: Mre11/Rad50 complexes and Sae2 promote DNA double-strand break resection. DNA Repair (Amst) 9, 1283-1291. [pubmed]

5. Lengsfeld, B.M., et al. (2007) Sae2 Is an Endonuclease that Processes Hairpin DNA Cooperatively with the Mre11/Rad50/Xrs2 Complex. Mol Cell 28, 638-651 [pubmed]

6. Lee, J.H., and Paull, T.T. (2004) Direct activation of the ATM protein kinase by the Mre11/Rad50/Nbs1 complex. Science 304, 93-96 [pubmed]

7. Lee, J.H., and Paull, T.T. (2005) ATM activation by DNA double-strand breaks through the Mre11-Rad50-Nbs1 complex. Science 308, 551-554 [pubmed]

8. Lee, J.H., and Paull, T.T. (2007) Activation and regulation of ATM kinase activity in response to DNA double-strand breaks. Oncogene 26, 7741-7748 [pubmed]

9. Guo, Z., et al. (2010) ATM activation in the presence of oxidative stress. Cell Cycle 9, 4805-4811 [pubmed]

10. Guo, Z., et al. (2010) ATM activation by oxidative stress. Science 330, 517-521 [pubmed]