Eric Le Cam

The Srs2 helicase prevents recombination by disrupting Rad51 nucleoprotein filaments

Homologous recombination is a ubiquitous process with key functions in meiotic and vegetative cells for the repair of DNA breaks. It is initiated by the formation of single-stranded DNA on which recombination proteins bind to form a nucleoprotein filament that is active in searching for homology, in the formation of joint molecules and in the exchange of DNA strands. This process contributes to genome stability but it is also potentially dangerous to cells if intermediates are formed that cannot be processed normally and thus are toxic or generate genomic rearrangements. Cells must therefore have developed strategies to survey recombination and to prevent the occurrence of such deleterious events. In Saccharomyces cerevisiae, genetic data have shown that the Srs2 helicase negatively modulates recombination, and later experiments suggested that it reverses intermediate recombination structures. Here we show that DNA strand exchange mediated in vitro by Rad51 is inhibited by Srs2, and that Srs2 disrupts Rad51 filaments formed on single-stranded DNA. These data provide an explanation for the anti-recombinogenic role of Srs2 in vivo and highlight a previously unknown mechanism for recombination control.

UvrD helicase, unlike Rep helicase, dismantles RecA nucleoprotein filaments in Escherichia coli

The roles of UvrD and Rep DNA helicases of Escherichia coli are not yet fully understood. In particular, the reason for rep uvrD double mutant lethality remains obscure. We reported earlier that mutations in recF, recO or recR genes suppress the lethality of uvrD rep, and proposed that an essential activity common to UvrD and Rep is either to participate in the removal of toxic recombination intermediates or to favour the proper progression of replication. Here, we show that UvrD, but not Rep, directly prevents homologous recombination in vivo. In addition to RecFOR, we provide evidence that RecA contributes to toxicity in the rep uvrD mutant. In vitro, UvrD dismantles the RecA nucleoprotein filament, while Rep has only a marginal activity. We conclude that UvrD and Rep do not share a common activity that is essential in vivo: while Rep appears to act at the replication stage, UvrD plays a role of RecA nucleoprotein filament remover. This activity of UvrD is similar to that of the yeast Srs2 helicase.

A Key Presynaptic Role in Transformation for a Widespread Bacterial Protein: DprA Conveys Incoming ssDNA to RecA

Natural transformation is a mechanism for genetic exchange in many bacterial genera. It proceeds through the uptake of exogenous DNA and subsequent homology-dependent integration into the genome. In Streptococcus pneumoniae, this integration requires the ubiquitous recombinase, RecA, and DprA, a protein of unknown function widely conserved in bacteria. To unravel the role of DprA, we have studied the properties of the purified S. pneumoniae protein and its Bacillus subtilis ortholog (Smf). We report that DprA and Smf bind cooperatively to single-stranded DNA (ssDNA) and that these proteins both self-interact and interact with RecA. We demonstrate that DprA-RecA-ssDNA filaments are produced and that these filaments catalyze the homology-dependent formation of joint molecules. Finally, we show that while the Escherichia coli ssDNA-binding protein SSB limits access of RecA to ssDNA, DprA lowers this barrier. We propose that DprA is a new member of the recombination-mediator protein family, dedicated to natural bacterial transformation.

Adsorption of DNA to Mica Mediated by Divalent Counterions: A Theoretical and Experimental Study

The adsorption of DNA molecules onto a flat mica surface is a necessary step to perform atomic force microscopy studies of DNA conformation and observe DNA-protein interactions in physiological environment. However, the phenomenon that pulls DNA molecules onto the surface is still not understood. This is a crucial issue because the DNA/surface interactions could affect the DNA biological functions. In this paper we develop a model that can explain the mechanism of the DNA adsorption onto mica. This model suggests that DNA attraction is due to the sharing of the DNA and mica counterions. The correlations between divalent counterions on both the negatively charged DNA and the mica surface can generate a net attraction force whereas the correlations between monovalent counterions are ineffective in the DNA attraction. DNA binding is then dependent on the fractional surface densities of the divalent and monovalent cations, which can compete for the mica surface and DNA neutralizations. In addition, the attraction can be enhanced when the mica has been pretreated by transition metal cations (Ni(2+), Zn(2+)). Mica pretreatment simultaneously enhances the DNA attraction and reduces the repulsive contribution due to the electrical double-layer force. We also perform end-to-end distance measurement of DNA chains to study the binding strength. The DNA binding strength appears to be constant for a fixed fractional surface density of the divalent cations at low ionic strength (I < 0.1 M) as predicted by the model. However, at higher ionic strength, the binding is weakened by the screening effect of the ions. Then, some equations were derived to describe the binding of a polyelectrolyte onto a charged surface. The electrostatic attraction due to the sharing of counterions is particularly effective if the polyelectrolyte and the surface have nearly the same surface charge density. This characteristic of the attraction force can explain the success of mica for performing single DNA molecule observation by AFM. In addition, we explain how a reversible binding of the DNA molecules can be obtained with a pretreated mica surface.

Nucleosome chiral transition under positive torsional stress in single chromatin fibers

Using magnetic tweezers to investigate the mechanical response of single chromatin fibers, we show that fibers submitted to large positive torsion transiently trap positive turns at a rate of one turn per nucleosome. A comparison with the response of fibers of tetrasomes (the [H3-H4](2) tetramer bound with approximately 50 bp of DNA) obtained by depletion of H2A-H2B dimers suggests that the trapping reflects a nucleosome chiral transition to a metastable form built on the previously documented right-handed tetrasome. In view of its low energy, <8 kT, we propose that this transition is physiologically relevant and serves to break the docking of the dimers on the tetramer that in the absence of other factors exerts a strong block against elongation of transcription by the main RNA polymerase.

The Srs2 Helicase Activity Is Stimulated by Rad51 Filaments on dsDNA: Implications for Crossover Incidence during Mitotic Recombination

Saccharomyces cerevisiae Srs2 helicase was shown to displace Rad51 in vitro upon translocation on single-stranded DNA. This activity is sufficient to account for its antirecombination effect and for the elimination of otherwise dead-end recombination intermediates. Roles for the helicase activity are yet unknown. Because cells lacking Srs2 show increased incidence of mitotic crossovers, it was postulated that Srs2 promotes synthesis-dependent strand annealing (SDSA) by unwinding the elongating invading strand from the donor strand. We report here that synthetic DNA structures that mimic D loops are good substrates for the Srs2 helicase activity, that Srs2 translocates on RPA-coated ssDNA, and, furthermore, that the helicase activity is largely stimulated by the presence of Rad51 nucleoprotein filaments on double-stranded DNA. These properties strongly support the idea that Srs2 actively prevents crossovers by promoting SDSA.

High-resolution AFM imaging of single-stranded DNA-binding (SSB) protein—DNA complexes

DNA in living cells is generally processed via the generation and the protection of single-stranded DNA involving the binding of ssDNA-binding proteins (SSBs). The studies of SSB-binding mode transition and cooperativity are therefore critical to many cellular processes like DNA repair and replication. However, only a few atomic force microscopy (AFM) investigations of ssDNA nucleoprotein filaments have been conducted so far. The point is that adsorption of ssDN A–SSB complexes on mica, necessary for AFM imaging, is not an easy task. Here, we addressed this issue by using spermidine as a binding agent. This trivalent cation induces a stronger adsorption on mica than divalent cations, which are commonly used by AFM users but are ineffective in the adsorption of ssDNA–SSB complexes. At low spermidine concentration (<0.3 mM), we obtained AFM images of ssDNA–SSB complexes (E. coli SSB, gp32 and yRPA) on mica at both low and high ionic strengths. In addition, partially or fully saturated nucleoprotein filaments were studied at various monovalent salt concentrations thus allowing the observation of SSB-binding mode transition. In association with conventional biochemical techniques, this work should make it possible to study the dynamics of DNA processes involving DNA–SSB complexes as intermediates by AFM.

Properties and growth mechanism of the ordered aggregation of a model RNA by the HIV-1 nucleocapsid protein: An electron microscopy investigation

NCp7, the nucleocapsid protein of the human immunodeficiency virus type 1, induces an ordered aggregation of RNAs, a mechanism that is thought to be involved in the NCp7-induced promotion of nucleic acid annealing. To further investigate this aggregation the morphology and the properties of the NCp7-induced aggregates of the model RNA homoribopolymer, polyA, were investigated by electron microscopy in various conditions. In almost all the tested conditions, the aggregates were spherical and consisted of a central dense core surrounded by a less dense halo made of NCp7-covered polyA molecules. The formation of these aggregates with a narrow distribution of sizes constitutes a distinctive feature of NCp7 over other single-stranded nucleic acid binding proteins. In most conditions, at the shortest times that can be reached experimentally, all the polyA molecules were already incorporated in small aggregates, suggesting that the nucleation step and the first aggregation events took place rapidly. The aggregates then orderly grew with time by fusion of the smaller aggregates to give larger ones. The aggregate halo was important in the fusion process by initiating the bridging between the colliding aggregates. In the presence of an excess of protein, the aggregates grew rapidly but were loosely packed and dissociated easily, suggesting adverse protein-protein interactions in the aggregates obtained in these conditions. In the presence of an excess of nucleotides, the presence of both amorphous nonspherical and slowly growing spherical aggregates suggested some changes in the mechanism of aggregate growth due to an incomplete covering of polyA molecules by NCp7. Finally, we showed that in the absence of added salt, the aggregate fusions were unfavored but not the initial events giving the first aggregates, the reverse being true in the presence of high salt concentrations (> or = 300 mM).

Contribution of DNA Conformation and Topology in Right-handed DNA Wrapping by the Bacillus subtilis LrpC Protein

The Bacillus subtilis LrpC protein belongs to the Lrp/AsnC family of transcriptional regulators. It binds the upstream region of the lrpC gene and autoregulates its expression. In this study, we have dissected the mechanisms that govern the interaction of LrpC with DNA by electrophoretic mobility shift assay, electron microscopy, and atomic force microscopy. LrpC is a structure-specific DNA binding protein that forms stable complexes with curved sequences containing phased A tracts and wraps DNA to form spherical, nucleosome-like structures. Formation of such wraps, initiated by cooperative binding of LrpC to DNA, results from optimal protein/protein interactions specified by the DNA conformation. In addition, we have demonstrated that LrpC constrains positive supercoils by wrapping the DNA in a right-handed superhelix, as visualized by electron microscopy.

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.