Maintenance of genomic stability and DNA repair in human
Co-workers: Eric Liang, Alicja Bulsiewicz, Anna Motnenko, Anna Socha, David Lopez Martinez, Marian Kupculak, Ganesh Pitchai.
One of the most critical biological processes is protection and maintenance of the genome. Human chromosomes are constantly exposed to a number of insults and challenges, originating from both intra- and extra-cellular sources. Rapid and effective cellular responses to these diverse genotoxic stresses are essential to ensure genomic stability.
Mammalian cells have evolved multiple protective mechanisms, or DNA repair pathways, to deal with the different types of DNA damage. Each pathway is rapidly activated in response to specific kinds of DNA damage. This highly specific response often involves enzymatic reactions, such as phosphorylation of the histone variant H2AX by ATM, and deubiquitination of the FANCD2 protein by the newly discovered USP1/UAF1 deubiquitinating enzyme complex.
Failure in these DNA repair pathways is detrimental and often underlies severe genetic diseases and syndromes in human. For example, an interference with a DNA double strand break (DSB) repair pathway is associated with Nijmegen breakage syndrome, Werner's syndrome and familial breast cancer susceptibility, whereas abrogation of DNA Trans Lesion Synthesis by DNA polymerases is directly linked to the skin cancer syndrome Xeroderma Pigmentosum variant.
Our laboratory is interested in understanding how the various DNA repair pathways function in human cells. Our previous work has lead to the discovery of novel proteins participating in DNA repair pathways such as the Fanconi Anemia DNA repair pathway. Interestingly, our findings demonstrate that these proteins often function as part of large multisubunit protein complexes. Using a combination of molecular biology, cell biology with live-cell imaging, biochemisty and CRISPR/Cas9 mediated genome engineering, we are continuing to identify and study new proteins playing key roles in human DNA repair pathways.
Liang, C-C., Li, Z., Lopez-Martinez, D., Nicholson, W.V., Vénien-Bryan, C. and Cohn, M.A. (2016). The FANCD2-FANCI complex is recruited to DNA interstrand crosslinks before monoubiquitination of FANCD2. Nat Commun. 2016 Jul 13;7:12124.
Liang, C-C., Zhan, B., Yoshikawa, Y., Haas, W., Gygi, S.P. and Cohn, M.A. (2015). UHRF1 Is a Sensor for DNA Interstrand Crosslinks and Recruits FANCD2 to Initiate the Fanconi Anemia Pathway. Cell Rep. 10:1947-56
Zhang, J., Dewar, J.M., Budzowska, M., Motnenko, A., Cohn, M.A. and Walter, J.C. (2015). DNA interstrand cross-link repair requires replication-fork convergence. Nat Struct Mol Biol. 22:242-7
Cohn, M.A. and D'Andrea, A.D. (2008). Chromatin recruitment of DNA repair proteins: Lessons from the Fanconi Anemia and Double Strand Break repair pathways. Mol. Cell 32:306-12
Cohn, M.A., Kowal, P., Yang, K., Haas, W., Huang, T.T., Gygi, S.P. and D'Andrea, A.D. (2007). A UAF1-containing multisubunit protein complex regulates the Fanconi Anemia Pathway. Mol. Cell 28:786-797
Figure 1: A) Silver stain of glycerol gradient fractionation of native USP1/UAF1, USP12/UAF1 and USP46/UAF1 deubiquitinating enzyme complexes purified from human HeLa cells. B) In vitro assay measuring the deubiquitinating enzyme activity of the fractions in (A)
Figure 3: Live-cell imaging. EGFP-FANCD2 is recruited to DNA interstrand crosslinks (ICLs). Scale bar: 20 μm
Figure 2: Immunofluoresence staining of various DNA repair proteins in human
cells before and after DNA damage. Observe how the proteins translocate to
damaged chromatin and form the characteristic nuclear foci upon DNA damage
Figure 5: Cryo-EM structure of the human FANCD2/FANCI complex
Figure 4: Cryo-EM structure of human FANCD2/FANCI complex showing the Tower
domain of FANCD2 and the main body
Figure : Model showing the serial recruitment of DNA repair proteins to damaged chromatin. Reproduced with permission from Molecular Cell (Cohn, M.A. and D'Andrea, A.D. (2008). Mol. Cell 32:306-12)
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