Homologous Recombination, NHEJ and Maintenance of Genomic Integrity

At a glance

Defects of the mechanisms of detection and repair of DNA double-strand breaks can lead to cancer. In order to better characterize this process, our team investigates the molecular mechanisms of two repair pathways of genotoxic lesions: homologous recombination and non-homologous end joining (NHEJ). These repair pathways also allow cancer cells to resist radiotherapy- or chemotherapy-based treatment involving molecules such as mitomycin C or cisplatin. These treatments are indeed based on the capacity of these drugs to induce DNA double-strand breaks in cancer cells in order to kill them. Targeting factors involved in homologous recombination and NHEJ is thus a sensible strategy to improve cancer therapy.

Our team focuses on understanding the mechanisms of DNA double-strand break repair systems at the molecular level, including repair by Homologous Recombination and Non-Homologous End Joining. We exploit classical ensemble biochemical assays and cell biology to investigate these mechanisms. In addition, we also use “single-molecule” methods that allow visualization and monitoring of the dynamic behavior of repair proteins acting on single DNA molecules. To this purpose we are using optical tweezers to tether and manipulate single DNA molecules, and fluorescence microscopy to observe in real-time fluorescently labeled proteins interacting with the DNA substrate.


DNA molecules are subject to damage. If these lesions are left unrepaired or ill-repaired, mutations can appear and lead to genomic instability. Cells presenting such a situation can either start proliferating in an uncontrolled manner and lead to the formation of tumors, or die. The efficient repair of such lesions is thus essential for living organisms. Amongst such lesions, DNA double-strand breaks are particularly dangerous as they can lead to chromosomal rearrangements such as translocations or loss of chromosome fragments. 

In order to tackle the potential hazards of DNA double-strand breaks, our cells have developed two repair systems. The first one uses ‘Homologous Recombination’ which involves an exchange of strands between two identical or quasi-identical DNA molecules catalyzed by the RAD51 recombinase, the human ortholog of the bacterial recA protein. This system is a powerful means to maintain genome integrity, but when it operates incorrectly, or when it is defective or deregulated, the consequences can lead to the onset of cancer as is the case in breast cancer when the regulatory protein ErbB2 is defective. The second DNA double-strand break repair pathway is Non-Homologous End Joining (NHEJ). This pathway involves a distinct a group of effector proteins, in particular DNA ligase 4 and its cofactors XRCC4 and XLF which we study in detail in the team. This NHEJ system is essential for the repair of DNA double-strand breaks induced by ionizing radiations and protects our genome possible translocations. 

Our work aims to better characterize these two repair pathways at the molecular level. We study DNA repair proteins in vitro using biochemistry techniques, but also in vivo using cell biology approaches. We also use “single-molecule” methods that allow visualization and monitoring of the dynamic behavior of repair proteins acting on single DNA molecules.

Our ultimate goal is to provide a better understanding of these pathways in order to identify new therapeutic strategies for cancer.

Figure 1

Our latest work in collaboration with Murray Junop (McMaster University, Canada) and Kathy Meek (Michigan State University, USA) indicates an unexpected role for XRCC4 (blue) and XLF (orange) at the first stage of NHEJ by the formation of a higher order filamentous structure of XRCC4/XLF alternates able to bridge DNA double-strand break repairs (Andres SN et al, 2012, Nucleic Acids Res. 40(4):1868-1878 et Roy et al, 2012 Nucleic Acids Res. 40(4):1684-1694).

About the team leader

Mauro Modesti


  • 2009 - Present: DR2 CNRS section 21
  • 2007 - 2009: Associated Researcher, IGC, CNRS, Marseille, France
  • 2004 - 2007: Scientist, Department of Cell Biology and Genetics Erasmus Medical Center, Rotterdam, The Netherlands
  • 1999 - 2004: Senior Postdoctoral fellow, Department of Cell Biology and Genetics, Erasmus Medical Center, Rotterdam, The Netherlands (Supervisors: Roland Kanaar and Claire Wyman)
  • 1996 - 1999: Postdoctoral fellow, Laboratory of Molecular Biology National Institutes of Health, Bethesda, MD, USA(Supervisor: Martin Gellert)
  • 1991 - 1996: Ph.D. in Molecular Biology and Genetics, Wayne State University, Detroit, MI, USA (Supervisor: Craig N. Giroux)


    Main achievements

    In the field of NHEJ

    Mauro Modesti’s work on NHEJ contributed to the clarification of the roles and regulation of the human NHEJ repair proteins XRCC4, XLF and DNA Ligase IV.

    1. Modesti, M., Hesse, J.E., and Gellert, M. (1999). DNA binding of Xrcc4 protein is associated with V(D)J recombination but not with stimulation of DNA ligase IV activity. The EMBO Journal 18, 2008-2018.
    2. Junop, M.S., Modesti, M., Guarne, A., Ghirlando, R., Gellert, M., and Yang, W. (2000). Crystal structure of the Xrcc4 DNA repair protein and implications for end joining. The EMBO Journal 19, 5962-5970.
    3. Modesti, M., Junop, M.S., Ghirlando, R., van de Rakt, M., Gellert, M., Yang, W., and Kanaar, R. (2003). Tetramerization and DNA ligase IV interaction of the DNA double-strand break repair protein XRCC4 are mutually exclusive. Journal of Molecular Biology 334, 215-228.
    4. Wang, Y.G., Nnakwe, C., Lane, W.S., Modesti, M., and Frank, K.M. (2004). Phosphorylation and regulation of DNA ligase IV stability by DNA-dependent protein kinase. The Journal of Biological Chemistry 279, 37282-37290.
    5. Mari, P.O., Florea, B.I., Persengiev, S.P., Verkaik, N.S., Bruggenwirth, H.T., Modesti, M., Giglia-Mari, G., Bezstarosti, K., Demmers, J.A., Luider, T.M., Houtsmuller AB, van Gent DC.. (2006). Dynamic assembly of end-joining complexes requires interaction between Ku70/80 and XRCC4. Proceedings of the National Academy of Sciences of the United States of America 103, 18597-18602.
    6. Andres, S.N., Modesti, M., Tsai, C.J., Chu, G., and Junop, M.S. (2007). Crystal Structure of Human XLF: A Twist in Nonhomologous DNA End-Joining. Molecular Cell 28, 1093-1101.
    7. Wu PY, Frit P, Meesala S, Dauvillier S, Modesti M, Andres SN, Huang Y, Sekiguchi J, Calsou P, Salles B, Junop MS. Structural and functional interaction between the human DNA repair proteins DNA ligase IV and XRCC4. Mol Cell Biol. 2009 Jun;29(11):3163-72.
    8. Andres SN, Vergnes A, Ristic D, Wyman C, Modesti M# and Junop M#. A human XRCC4-XLF complex bridges DNA. Nucleic Acids Res. 2012 Feb;40(4):1868-1878 #Corresponding authors
    9. Roy S, Andres SN, Vergnes A, Neal JA, Xu Y, Yu Y, Lees-Miller SP, Junop M, Modesti M#, Meek K#. XRCC4's interaction with XLF is required for coding (but not signal) end joining. Nucleic Acids Res. 2012 Feb;40(4):1684-1694. #Corresponding authors
    10. Cottarel J, Frit P, Bombarde O, Salles B, Négrel A, Bernard S, Jeggo PA, Lieber MR, Modesti M and Calsou P. A Noncatalytic Function of the Ligation Complex during Nonhomologous End Joining. JCB 2012, In press


    In the field of Homologous Recombination mechanism

    Mauro Modesti’s work on Homologous Recombination contributed studies on the human RAD51, RAD52, MRE11, RAD52, MUS81-EME1, BRCA2 and RAD51AP1 proteins.

    1. de Jager, M., Dronkert, M.L., Modesti, M., Beerens, C.E., Kanaar, R., and van Gent, D.C. (2001). DNA-binding and strand-annealing activities of human Mre11: implications for its roles in DNA double-strand break repair pathways. Nucleic Acids Research 29, 1317-1325.
    2. Ristic, D., Modesti, M., Kanaar, R., and Wyman, C. (2003). Rad52 and Ku bind to different DNA structures produced early in double-strand break repair. Nucleic Acids Research 31, 5229-5237.
    3. Ristic, D., Modesti, M., van der Heijden, T., van Noort, J., Dekker, C., Kanaar, R., and Wyman, C. (2005). Human Rad51 filaments on double- and single-stranded DNA: correlating regular and irregular forms with recombination function. Nucleic Acids Research 33, 3292-3302.
    4. Hanada, K., Budzowska, M., Modesti, M., Maas, A., Wyman, C., Essers, J., and Kanaar, R. (2006). The structure-specific endonuclease Mus81-Eme1 promotes conversion of interstrand DNA crosslinks into double-strands breaks. The EMBO Journal 25, 4921-4932.
    5. Modesti, M. (#), Budzowska, M., Baldeyron, C., Demmers, J.A., Ghirlando, R., and Kanaar, R. (2007). RAD51AP1 is a structure-specific DNA binding protein that stimulates joint molecule formation during RAD51-mediated homologous recombination. Molecular Cell 28, 468-481. (#) Mauro Modesti corresponding author and organize “Recommended” article by the Faculty of 1000 Biology
    6. Holthausen JT, van Loenhout MT, Sanchez H, Ristic D, van Rossum-Fikkert SE, Modesti M, Dekker C, Kanaar R, Wyman C. Effect of the BRCA2 CTRD domain on RAD51 filaments analyzed by an ensemble of single molecule techniques. Nucleic Acids Res. 2011 Aug;39(15):6558-6567.


    In the field of Homologous “Single molecule” approaches to study DNA repair transactions

    Together with Biophysicists Erwin Peterman and Gijs Wuite (VU Amsterdam, The Netherlands) and Cees Dekker (TU Delft, The Netherlands), Mauro Modesti participated in the development of “single molecule” approaches to study DNA repair mechanisms.

    1. van Mameren J, Modesti M, Kanaar R, Wyman C, Wuite GJ, Peterman EJ. Dissecting elastic heterogeneity along DNA molecules coated partly with Rad51 using concurrent fluorescence microscopy and optical tweezers. Biophys J. 2006 Oct 15;91(8):L78-80
    2. Modesti M#, Ristic D, van der Heijden T, Dekker C, van Mameren J, Peterman EJ, Wuite GJ, Kanaar R, Wyman C. Fluorescent human RAD51 reveals multiple nucleation sites and filament segments tightly associated along a single DNA molecule. Structure. 2007 May;15(5):599-609.#Correponding author
    3. van der Heijden T, Seidel R, Modesti M, Kanaar R, Wyman C, Dekker C. Real-time assembly and disassembly of human RAD51 filaments on individual DNA molecules. Nucleic Acids Res. 2007;35(17):5646-57.
    4. van der Heijden T, Modesti M, Hage S, Kanaar R, Wyman C, Dekker C. Homologous recombination in real time: DNA strand exchange by RecA. Mol Cell. 2008 May 23;30(4):530-8.
    5. van Mameren J, Modesti M, Kanaar R, Wyman C, Peterman EJ, Wuite GJ. Counting RAD51 proteins disassembling from nucleoprotein filaments under tension. Nature. 2009 Feb 5;457(7230):745-8.
    6. van Mameren J, Gross P, Farge G, Hooijman P, Modesti M, Falkenberg M, Wuite GJ, Peterman EJ. Unraveling the structure of DNA during overstretching by using multicolor, single-molecule fluorescence imaging. Proc Natl Acad Sci U S A. 2009 Oct 27;106(43):18231-6.
    7. Modesti, M. Fluorescent labeling of proteins. Methods Mol Biol. 2011;783:101-120.
    8. Holthausen JT, van Loenhout MT, Sanchez H, Ristic D, van Rossum-Fikkert SE, Modesti M, Dekker C, Kanaar R, Wyman C. Effect of the BRCA2 CTRD domain on RAD51 filaments analyzed by an ensemble of single molecule techniques. Nucleic Acids Res. 2011 Aug;39(15):6558-6567.

    Join the Team "Homologous Recombination, NHEJ and Maintenance of Genomic Integrity"

    Our general interest is in deciphering the protein-DNA transactions at the heart of genomic integrity maintenance mechanisms – precisely, at the molecular level. We are addressing challenging questions about biological systems that can only be answered by multidisciplinary approaches.

    Whether your background is in biological or medical sciences, physics, nanotechnologies, materials engineering or computer science - whether you are a Master student (M2), a prospective Ph.D. student, a CR (Chargé de Recherche), IR (Ingénieur de Recherche) or are looking for a post-doctoral training, your creativity and ideas are of interest to us.

    We are applying for new research grants that could support you but we can also work together to try to get your own Fellowships at ARC, EMBO, HFSP, La Ligue contre le Cancer, European Commission or any other sponsor.