Search

DNA mismatch repair (MMR) maintains genome stability through repair of DNA replication errors. InEscherichia coli, initiation of MMR involves recognition of the mismatch by MutS, recruitment of MutL, activation of endonuclease MutH and DNA strand incision at a hemimethylated GATC site. Here we studied the mechanism of communication that couples mismatch recognition to daughter strand incision. We investigated the effect of catalytically-deficient Cas9 as well as stalled RNA polymerase as roadblocks placed on DNA in between the mismatch and GATC site in ensemble and single molecule nanomanipulation incision assays. The MMR proteins were observed to incise GATC sites beyond a roadblock, albeit with reduced efficiency. This residual incision is completely abolished upon shortening the disordered linker regions of MutL. These results indicate that roadblock bypass can be fully attributed to the long, disordered linker regions in MutL and establish that communication during MMR initiation occurs along the DNA backbone.

In this issue of Structure, Cho and colleagues provide intriguing insight into the first steps of the DNA mismatch repair process. By using single-molecule techniques, they show that the protein MutS undergoes two different types of diffusion on error-containing DNA in an ATP-dependent way.

In E. coli the error rate of replication (10-6) is lowered another thousand-fold by the MutS, MutL and MutH system of proteins responsible for directing repair of base insertions, deletions, and mismatches. We provide here a study using magnetic trapping of single DNA molecules to analyze the interactions between these proteins and DNA containing a single indel mutation. MutS, albeit at relatively high concentrations, can be observed to stabilize small thermal loops of DNA on an indel-containing substrate, and this in an ATP-dependent manner; addition of MutL reduces the concentration of MutS required for looping to be observed. Loop size distributes roughly according to a J-factor distribution, and decreases as force increases, consistent with thermal loop stabilization by MutSL complexes. The reaction is allowed to proceed to the stage of DNA incision by the addition of MutH. With the further addition of T4 DNA ligase the steady-state rate of incision can be studied as a function of different parameters including DNA supercoiling, extending force, length of substrate DNA, or relative positioning between indel and incision sites. We compare these results to biophysical models for the protein-DNA interactions involved as well as stochastic simulations of the incision reaction occurring on linear DNA with a centrally-located indel and two symmetric proximal incision sites. Experimental results indicate that DNA incision can occur even in the absence of looping, and comparison to simulations supports the view that communication from DNA mismatch to DNA incision site takes place via thermal diffusion.

This thesis presents the development of a new generation of optical frequency standards using trapped atoms. The clock stability will be first limited by the local oscillator noise. In the first part, different parameters such as the duty cycle are discussed to reduce these noise effects: the clock cycle time has to be optimized. With our ultra-stable laser used as a local oscillator, the clock stability could be 10^-16 tau^-1/2 which is two order of magnitude better than the best current atomic clocks. The second part describes an efficient cold strontium atoms source, which is a determinant step to shorten the atoms preparation time during the clock cycle. We also reported the frequency measurement of the clock transition 1S0-3P0 of the ^87 Sr within 15 kHz accuracy., Ce mémoire présente le développement d'une nouvelle génération d'étalons de fréquence optique utilisant des atomes piégés. La stabilité de fréquence d'une telle horloge sera limitée dans un premier temps par le bruit de l'oscillateur local. Nous discutons dans la première partie du mémoire de différents paramètres, comme le rapport cyclique ou la méthode d'interrogation, pouvant réduire les effets de ce bruit: la séquence temporelle du cycle d'horloge doit être optimisée. Avec l'oscillateur local que nous avons réalisé, un laser ultra-stable, la stabilité attendue est de quelques 10^-16 tau^-1/2 soit près de deux ordres de grandeurs mieux que les fontaines atomiques actuelles. La deuxième partie du mémoire décrit une source d'atomes froids de strontium performante, étape essentielle pour réduire le temps de préparation des atomes dans le cycle. Enfin, nous rapportons la mesure de la transition d'horloge 1S0-3P0 du ^87 Sr avec une résolution de 15 kHz.

Single-molecule experiments show that the RSC chromatin remodeling complex, a member of the SNF2 ATPase family, induces formation of a negatively supercoiled DNA loop by active translocation.

Genomic DNA is subject to damage at such high frequencies that only with efficient DNA-repair pathways genomic stability is maintained. Research efforts of many groups over several decades have revealed the salient properties of the various DNA-repair mechanisms, but it has largely remained unclear how damage sites are localized in genomic DNA. In PNAS, Gorman et al. (1) unveil insights into the DNA-repair process called mismatch repair (MMR), which is responsible for the repair of incorrectly paired DNA bases. They use a combination of single-molecule fluorescence imaging and nanofabrication to study how different search mechanisms are used in eukaryotic MMR to find mismatches.

Recent additions to the helicase family include motor proteins that do not actually unwind DNA, but rather translocate it. By sensing short polar sequences that orient the bacterial chromosome, the FtsK helicase translocates DNA so as to align the termini of replicated chromosomes with each other, facilitating the late stages of chromosome segregation.

This thesis presents the development of a new generation of optical frequency standards using trapped atoms. The clock stability will be first limited by the local oscillator noise. In the first part, different parameters such as the duty cycle are discussed to reduce these noise effects: the clock cycle time has to be optimized. With our ultra-stable laser used as a local oscillator, the clock stability could be 10^-16 tau^-1/2 which is two order of magnitude better than the best current atomic clocks. The second part describes an efficient cold strontium atoms source, which is a determinant step to shorten the atoms preparation time during the clock cycle. We also reported the frequency measurement of the clock transition 1S0-3P0 of the ^87 Sr within 15 kHz accuracy.; Ce mémoire présente le développement d'une nouvelle génération d'étalons de fréquence optique utilisant des atomes piégés. La stabilité de fréquence d'une telle horloge sera limitée dans un premier temps par le bruit de l'oscillateur local. Nous discutons dans la première partie du mémoire de différents paramètres, comme le rapport cyclique ou la méthode d'interrogation, pouvant réduire les effets de ce bruit: la séquence temporelle du cycle d'horloge doit être optimisée. Avec l'oscillateur local que nous avons réalisé, un laser ultra-stable, la stabilité attendue est de quelques 10^-16 tau^-1/2 soit près de deux ordres de grandeurs mieux que les fontaines atomiques actuelles. La deuxième partie du mémoire décrit une source d'atomes froids de strontium performante, étape essentielle pour réduire le temps de préparation des atomes dans le cycle. Enfin, nous rapportons la mesure de la transition d'horloge 1S0-3P0 du ^87 Sr avec une résolution de 15 kHz.

We report the frequency measurement with an accuracy in the 100 kHz range of several optical transitions of atomic Sr : $^1S_0- ^3P_1$ at 689 nm, $^3P_1- ^3S_1$ at 688 nm and $^3P_0- ^3S_1$ at 679 nm. Measurements are performed with a frequency chain based on a femtosecond laser referenced to primary frequency standards. They allowed the indirect determination with a 70 kHz uncertainty of the frequency of the doubly forbidden $5s^2^1S_0- 5s5p^3P_0$ transition of $^{87}$Sr at 698 nm and in a second step its direct observation. Frequency measurements are performed for $^{88}$Sr and $^{87}$Sr, allowing the determination of $^3P_0$, $^3P_1$ and $^3S_1$ isotope shifts, as well as the $^3S_1$ hyperfine constants., Comment: 12 pages, 16 figures

This paper reports the frequency measurement with an accuracy in the 100 kHz range of several optical transitions of atomic Sr. Measurements are performed with a frequency chain based on a femto-second laser and referenced to a hydrogen maser. They allowed the determination with a 70 kHz uncertainty of the energy of the 5s5p 3 Po state and the direct observation and frequency measurement of the doubly forbidden 1S0 rarr3 P0 transition at 698 nm. This line has a natural width of 10 - Hz and can be used for a new generation of optical frequency standards using atoms trapped in a light shift free dipole trap

Nat. Struct. Mol. Biol. 13, 948–950 (2006). In the version of this News and Views initially published, the authors present a model to explain the low degree of positive DNA supercoiling generated during DNA translocation through the FtsK helicase. The authors' conclusion, that in a rotary inchworm model for DNA translocation the helicase should generate positive supercoils ahead of itself when taking steps of less than 1.