Virginie Ropars

Structural characterization of filaments formed by human Xrcc4–Cernunnos/XLF complex involved in nonhomologous DNA end-joining

Cernunnos/XLF is a core protein of the nonhomologous DNA end-joining (NHEJ) pathway that processes the majority of DNA double-strand breaks in mammals. Cernunnos stimulates the final ligation step catalyzed by the complex between DNA ligase IV and Xrcc4 (X4). Here we present the crystal structure of the X41–157-Cernunnos1–224 complex at 5.5-Å resolution and identify the relative positions of the two factors and their binding sites. The X-ray structure reveals a filament arrangement for X41–157 and Cernunnos1–224 homodimers mediated by repeated interactions through their N-terminal head domains. A filament arrangement of the X4–Cernunnos complex was confirmed by transmission electron microscopy analyses both with truncated and full-length proteins. We further modeled the interface and used structure-based site-directed mutagenesis and calorimetry to characterize the roles of various residues at the X4–Cernunnos interface. We identified four X4 residues (Glu55, Asp58, Met61, and Phe106) essential for the interaction with Cernunnos. These findings provide new insights into the molecular bases for stimulatory and bridging roles of Cernunnos in the final DNA ligation step.

Proto-oncogene TCL1: more than just a coactivator for Akt

Serine threonine kinase Akt, also called PKB (protein kinase B), plays a central role in regulating intracellular survival. Deregulation of this Akt signaling pathway underlies various human neoplastic diseases. Recently, the proto‐oncogene TCL1 (T cell leukemia 1), with a previously unknown physiological function, was shown to interact with the Akt pleckstrin homology domain, enhancing Akt kinase activity; hence, it functions as an Akt kinase coactivator. In contrast to pathological conditions in which the TCL1 gene is highly activated in various human neoplasmic diseases, the physiological expression of TCL1 is tightly limited to early developmental cells as well as various developmental stages of immune cells. The NBRE (nerve growth factor‐responsive element) of the proximal TCL1 promoter sequences can regulate the restricted physiological expression of TCL1 in a negative feedback mechanism. Further, based on the NMR structural studies of Akt‐TCL1 protein complexes, an inhibitory peptide, “Akt‐in,” consisting of the βA strand of TCL1, has been identified and has therapeutic potential. This review article summarizes and discusses recent advances in the understanding of TCL1‐Akt functional interaction in order to clarify the biological action of the proto‐oncogene TCL1 family and the development avenues for a suppressive drug specific for Akt, a core intracellular survival regulator.—Noguchi, M., Ropars, V., Roumestand, C., Suizu, F. Proto‐oncogene TCL1: more than just a coactivator for Akt. FASEB J. 21, 2273–2284 (2007)

Delineation of the Xrcc4-interacting Region in the Globular Head Domain of Cernunnos/XLF

In mammals, the majority of DNA double-strand breaks are processed by the nonhomologous end-joining (NHEJ) pathway, composed of seven factors: Ku70, Ku80, DNA-PKcs, Artemis, Xrcc4 (X4), DNA-ligase IV (L4), and Cernunnos/XLF. Cernunnos is part of the ligation complex, constituted by X4 and L4. To improve our knowledge on the structure and function of Cernunnos, we performed a systematic mutagenesis study on positions selected from an analysis of the recent three-dimensional structures of this factor. Ten of 27 screened mutants were nonfunctional in several DNA repair assays. Outside amino acids critical for the expression and stability of Cernunnos, we identified three amino acids (Arg64, Leu65, and Leu115) essential for the interaction with X4 and the proper function of Cernunnos. Docking the crystal structures of the two factors further validated this probable interaction surface of Cernunnos with X4.

The checkpoint Saccharomyces cerevisiae Rad9 protein contains a tandem tudor domain that recognizes DNA

DNA damage checkpoints are signal transduction pathways that are activated after genotoxic insults to protect genomic integrity. At the site of DNA damage, ‘mediator’ proteins are in charge of recruiting ‘signal transducers’ to molecules ‘sensing’ the damage. Budding yeast Rad9, fission yeast Crb2 and metazoan 53BP1 are presented as mediators involved in the activation of checkpoint kinases. Here we show that, despite low sequence conservation, Rad9 exhibits a tandem tudor domain structurally close to those found in human/mouse 53BP1 and fission yeast Crb2. Moreover, this region is important for the resistance of Saccharomyces cerevisiae to different genotoxic stresses. It does not mediate direct binding to a histone H3 peptide dimethylated on K79, nor to a histone H4 peptide dimethylated on lysine 20, as was demonstrated for 53BP1. However, the tandem tudor region of Rad9 directly interacts with single-stranded DNA and double-stranded DNAs of various lengths and sequences through a positively charged region absent from 53BP1 and Crb2 but present in several yeast Rad9 homologs. Our results argue that the tandem tudor domains of Rad9, Crb2 and 53BP1 mediate chromatin binding next to double-strand breaks. However, their modes of chromatin recognition are different, suggesting that the corresponding interactions are differently regulated.

The TCL1A Oncoprotein Interacts Directly with the NF-κB Inhibitor IκB

The T cell leukaemia/lymphoma 1A (TCL1A) oncoprotein plays key roles in several B and T cell malignancies. Lacking enzymatic activity, TCL1As transforming action was linked to its capacity to co-activate the protein kinase AKT via binding to its pleckstrin homology (PH) domain. However, perturbation of AKT signalling alone was recently shown insufficient to explain TCL1A oncogenesis, suggesting that TCL1A has additional cellular partners. Searching for such additional targets, we found that TCL1A binds specifically and directly to the ankyrin domain of IκB, the inhibitor of the NF-κB transcription factors. Through binding assays and a structural analysis by small angle X-ray scattering, we show that TCL1A and IκB interact in yeast-two-hybrid systems, when transiently overexpressed in 293 cells, and as recombinant proteins in vitro. We further establish that the association between TCL1A and IκB is compatible with AKT binding to TCL1A, but incompatible with IκB binding to NF-κB. By interfering with the inhibition of NF-κB by IκB, TCL1A may increase the concentration of free NF-κB molecules sufficiently to trigger expression of anti-apoptotic genes. Thus our data suggest an additional route by which TCL1A might cause cancer.

Autocyclized and oxidized forms of SCR7 induce cancer cell death by inhibiting nonhomologous DNA end joining in a Ligase IV dependent manner

Nonhomologous DNA end joining (NHEJ) is the major DNA double‐strand break (DSB) repair pathway in mammals. Previously, we have described a small molecule inhibitor, SCR7, which can inhibit NHEJ in a Ligase IV‐dependent manner. Administration of SCR7 within the cells resulted in the accumulation of DNA breaks, cell death, and inhibition of tumor growth in mice. In the present study, we report that parental SCR7, which is unstable, can be autocyclized into a stable form. Both parental SCR7 and cyclized SCR7 possess the same molecular weight (334.09) and molecular formula (C18H14N4OS), whereas its oxidized form, SCR7‐pyrazine, possesses a different molecular formula (C18H12N4OS), molecular weight (332.07), and structure. While cyclized form of SCR7 showed robust inhibition of NHEJ in vitro, both forms exhibited efficient cytotoxicity. Cyclized and oxidized forms of SCR7 inhibited DNA end joining catalyzed by Ligase IV, whereas their impact was minimal on Ligase III, Ligase I, and T4 DNA Ligase‐mediated joining. Importantly, both forms inhibited V(D)J recombination, although the effect was more pronounced for SCR7‐cyclized. Both forms blocked NHEJ in a Ligase IV‐dependent manner leading to the accumulation of DSBs within the cells. Although cytotoxicity due to SCR7‐cyclized was Ligase IV specific, the pyrazine form exhibited nonspecific cytotoxicity at higher concentrations in Ligase IV‐null cells. Finally, we demonstrate that both forms can potentiate the effect of radiation. Thus, we report that cyclized and oxidized forms of SCR7 can inhibit NHEJ in a Ligase IV‐dependent manner, although SCR7‐pyrazine is less specific to Ligase IV inside the cell.