Dynamics of Ku and bacterial non-homologous end-joining characterized using single DNA molecule analysis

We use single-molecule techniques to characterize the dynamics of prokaryotic DNA repair by non-homologous end-joining (NHEJ), a system comprised only of the dimeric Ku and Ligase D (LigD). The Ku homodimer alone forms a ∼2 s synapsis between blunt DNA ends that is increased to ∼18 s upon addition of LigD, in a manner dependent on the C-terminal arms of Ku. The synapsis lifetime increases drastically for 4 nt complementary DNA overhangs, independently of the C-terminal arms of Ku. These observations are in contrast to human Ku, which is unable to bridge either of the two DNA substrates. We also demonstrate that bacterial Ku binds the DNA ends in a cooperative manner for synapsis initiation and remains stably bound at DNA junctions for several hours after ligation is completed, indicating that a system for removal of the proteins is active in vivo. Together these experiments shed light on the dynamics of bacterial NHEJ in DNA end recognition and processing. We speculate on the evolutionary similarities between bacterial and eukaryotic NHEJ and discuss how an increased understanding of bacterial NHEJ can open the door for future antibiotic therapies targeting this mechanism

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

International audienceDNA 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

Pressure and Chemical Unfolding of an α-Helical Bundle Protein: The GH2 Domain of the Protein Adaptor GIPC1.

When combined with NMR spectroscopy, high hydrostatic pressure is an alternative perturbation method used to destabilize globular proteins that has proven to be particularly well suited for exploring the unfolding energy landscape of small single-domain proteins. To date, investigations of the unfolding landscape of all-β or mixed-α/β protein scaffolds are well documented, whereas such data are lacking for all-α protein domains. Here we report the NMR study of the unfolding pathways of GIPC1-GH2, a small α-helical bundle domain made of four antiparallel α-helices. High-pressure perturbation was combined with NMR spectroscopy to unravel the unfolding landscape at three different temperatures. The results were compared to those obtained from classical chemical denaturation. Whatever the perturbation used, the loss of secondary and tertiary contacts within the protein scaffold is almost simultaneous. The unfolding transition appeared very cooperative when using high pressure at high temperature, as was the case for chemical denaturation, whereas it was found more progressive at low temperature, suggesting the existence of a complex folding pathway

FAN1-MLH1 interaction affects repair of DNA interstrand cross-links and slipped-CAG/CTG repeats

FAN1, a DNA structure-specific nuclease, interacts with MLH1, but the repair pathways in which this complex acts are unknown. FAN1 processes DNA interstrand crosslinks (ICLs) and FAN1 variants are modifiers of the neurodegenerative Huntington's disease (HD), presumably by regulating HD-causing CAG repeat expansions. Here, we identify specific amino acid residues in two adjacent FAN1 motifs that are critical for MLH1 binding. Disruption of the FAN1-MLH1 interaction confers cellular hypersensitivity to ICL damage and defective repair of CAG/CTG slip-outs, intermediates of repeat expansion mutations. FAN1-S126 phosphorylation, which hinders FAN1-MLH1 association, is cell cycle-regulated by cyclin-dependent kinase activity and attenuated upon ICL induction. Our data highlight the FAN1-MLH1 complex as a phosphorylation-regulated determinant of ICL response and repeat stability, opening novel paths to modify cancer and neurodegeneration

BRCA2-HSF2BP oligomeric ring disassembly by BRME1 promotes homologous recombination

In meiotic homologous recombination (HR), BRCA2 facilitates loading of the recombinases RAD51 and DMC1 at the sites of double-strand breaks (DSBs). The HSF2BP-BRME1 complex interacts with BRCA2. Its absence causes a severe reduction in recombinase loading at meiotic DSB. We previously showed that, in somatic cancer cells ectopically producing HSF2BP, DNA damage can trigger HSF2BP-dependent degradation of BRCA2, which prevents HR. Here, we report that, upon binding to BRCA2, HSF2BP forms octameric rings that are able to interlock into a large ring-shaped 24-nucleotide oligomer. Addition of BRME1 leads to dissociation of both of these ring structures and cancels the disruptive effect of HSF2BP on cancer cell resistance to DNA damage. It also prevents BRCA2 degradation during interstrand DNA crosslink repair in Xenopus egg extracts. We propose that, during meiosis, the control of HSF2BP-BRCA2 oligomerization by BRME1 ensures timely assembly of the ring complex that concentrates BRCA2 and controls its turnover, thus promoting HR.</p

XLF and APLF bind Ku80 at two remote sites to ensure DNA repair by non-homologous end joining

International audienceThe Ku70-Ku80 (Ku) heterodimer binds rapidly and tightly to the ends of DNA double-strand breaks and recruits factors of the non-homologous end-joining (NHEJ) repair pathway through molecular interactions that remain unclear. We have determined crystal structures of the Ku-binding motifs (KBM) of the NHEJ proteins APLF (A-KBM) and XLF (X-KBM) bound to a Ku-DNA complex. The two KBM motifs bind remote sites of the Ku80 alpha/beta domain. The X-KBM occupies an internal pocket formed by an unprecedented large outward rotation of the Ku80 alpha/beta domain. We observe independent recruitment of the APLF-interacting protein XRCC4 and of XLF to laser-irradiated sites via binding of A- and X-KBMs, respectively, to Ku80. Finally, we show that mutation of the X-KBM and A-KBM binding sites in Ku80 compromises both the efficiency and accuracy of end joining and cellular radiosensitivity. A- and X-KBMs may represent two initial anchor points to build the intricate interaction network required for NHEJ

Les protéines oncogènes TCL1 (plus que des co-activateurs d'AKT)

Des translocations récurrentes corrélées à l apparition d un certain nombre de cancers du sang et notamment de leucémies prolymphocytaires de cellules T, ont permis de mettre en lumière les oncogènes MTCP1, TCL1A et TCL1B. L oncogène TCL1A est une protéine qui interagit avec la kinase Akt et fonctionne comme un co-activateur. Cette protéine Akt joue un rôle essentiel dans la régulation de l apoptose cellulaire et se retrouve de ce fait impliquée dans un grand nombre de processus néoplasique. Dans une étude précédente, des méthodes RMN et de diffusion des rayons X aux petits angles (SAXS) ont été combinées pour déterminer les affinités, les interfaces moléculaires et la structure à basse résolution du complexe formé entre le domaine d'homologie à la pleckstrine (PHD) d Akt2 et TCL1A. Nous avons prouvé que TCL1A se lie à Akt2-PHD à un emplacement qui n'a pas encore été observé dans les interactions entre les domaines PH et les protéines en général. Nous avons également observé que l'affinité d Akt2-PHD pour MTCP1, un membre de la famille TCL1, est sensiblement inférieure (deux ordres de grandeur) que celle pour TCL1A. MTCP1 est impliqué dans les maladies semblables à TCL1A, et ses concentrations intracellulaires sont comparables. Mais, contrairement à TCL1A, MTCP1 est monomérique en solution. Nous avons utilisé la mutagenèse dirigée pour identifier les résidus critiques sur MTCP1, qui peuvent reconstituer la pleine affinité de TCL1A pour Akt, afin d'obtenir une structure RMN de haute résolution du complexe Akt2 PHD:MTCP1. Utilisant ces résultats, nous avons essayé de concevoir des inhibiteurs peptidiques (comme Akt-in) qui pourraient moduler l'activité d'Akt, ouvrant la voie à une nouvelle classe de drogues antitumorales. Par ailleurs, nous avons recherché d'autres cibles potentielles pour les protéines de la famille TCL1. Nous avons découvert que la protéine TCL1A se lie à IkBa avec une affinité comparable à celle d'Akt. IkBa est l'inhibiteur du facteur de transcription NF-kB, dont l activité est régulée en aval par Akt. L'affinité d IkBa pour MTCP1 est également inférieure à celle de TCL1A. Nous discuterons d un modèle à basse résolution du complexe TCL1A: IkBa établi à partir de données SAXS.MONTPELLIER-BU Pharmacie (341722105) / SudocSudocFranceF