Use of Single-Cysteine Variants for Trapping Transient States in DNA Mismatch Repair.

DNA mismatch repair (MMR) is necessary to prevent incorporation of polymerase errors into the newly synthesized DNA strand, as they would be mutagenic. In humans, errors in MMR cause a predisposition to cancer, called Lynch syndrome. The MMR process is performed by a set of ATPases that transmit, validate, and couple information to identify which DNA strand requires repair. To understand the individual steps in the repair process, it is useful to be able to study these large molecular machines structurally and functionally. However, the steps and states are highly transient; therefore, the methods to capture and enrich them are essential. Here, we describe how single-cysteine variants can be used for specific cross-linking and labeling approaches that allow trapping of relevant transient states. Analysis of these defined states in functional and structural studies is instrumental to elucidate the molecular mechanism of this important DNA MMR process.

MutS/MutL crystal structure reveals that the MutS sliding clamp loads MutL onto DNA.

To avoid mutations in the genome, DNA replication is generally followed by DNA mismatch repair (MMR). MMR starts when a MutS homolog recognizes a mismatch and undergoes an ATP-dependent transformation to an elusive sliding clamp state. How this transient state promotes MutL homolog recruitment and activation of repair is unclear. Here we present a crystal structure of the MutS/MutL complex using a site-specifically crosslinked complex and examine how large conformational changes lead to activation of MutL. The structure captures MutS in the sliding clamp conformation, where tilting of the MutS subunits across each other pushes DNA into a new channel, and reorientation of the connector domain creates an interface for MutL with both MutS subunits. Our work explains how the sliding clamp promotes loading of MutL onto DNA, to activate downstream effectors. We thus elucidate a crucial mechanism that ensures that MMR is initiated only after detection of a DNA mismatch.

Covalently trapping MutS on DNA to study DNA mismatch recognition and signaling.

The DNA repair protein MutS forms clamp-like structures on DNA that search for and recognize base mismatches leading to ATP-transformed signaling clamps. In this study, the mobile MutS clamps were trapped on DNA in a functional state using single-cysteine variants of MutS and thiol-modified homoduplex or heteroduplex DNA. This approach allows stabilization of various transient MutS-DNA complexes and will enable their structural and functional analysis.

Generation of DNA nanocircles containing mismatched bases.

The DNA mismatch repair (MMR) system recognizes and repairs errors that escaped the proofreading function of DNA polymerases. To study molecular details of the MMR mechanism, in vitro biochemical assays require specific DNA substrates carrying mismatches and strand discrimination signals. Current approaches used to generate MMR substrates are time-consuming and/or not very flexible with respect to sequence context. Here we report an approach to generate small circular DNA containing a mismatch (nanocircles). Our method is based on the nicking of PCR products resulting in single-stranded 3' overhangs, which form DNA circles after annealing and ligation. Depending on the DNA template, one can generate mismatched circles containing a single hemimethylated GATC site (for use with the bacterial system) and/or nicking sites to generate DNA circles nicked in the top or bottom strand (for assays with the bacterial or eukaryotic MMR system). The size of the circles varied (323 to 1100 bp), their sequence was determined by the template DNA, and purification of the circles was achieved by ExoI/ExoIII digestion and/or gel extraction. The quality of the nanocircles was assessed by scanning-force microscopy and their suitability for in vitro repair initiation was examined using recombinant Escherichia coli MMR proteins.

Crosslinking of (cytosine-5)-DNA methyltransferase SsoII and its complexes with specific DNA duplexes provides an insight into their structures.

(Cytosine-5)-DNA methyltransferase SsoII (M.SsoII) functions as a methyltransferase and also as a transcription factor. Chemical and photochemical crosslinking was used for exploring the structure of M.SsoII-DNA complexes and M.SsoII in the absence of DNA. Photocrosslinking with 4-(N-maleimido)benzophenone demonstrated that in the M.SsoII complex with DNA containing the regulatory site, the M.SsoII region responsible for methylation was bound to DNA flanking the regulatory site, which contained no methylation sequence. This required high flexibility of the linker connecting the M.SsoII N-terminal domain and the M.SsoII region responsible for methylation. The flexibility was demonstrated by crosslinking with bis-maleimidoethane and 1,11-bis-maleimidotetraethyleneglycol.

Chemical trapping of the dynamic MutS-MutL complex formed in DNA mismatch repair in Escherichia coli.

The ternary complex comprising MutS, MutL, and DNA is a key intermediate in DNA mismatch repair. We used chemical cross-linking and fluorescence resonance energy transfer (FRET) to study the interaction between MutS and MutL and to shed light onto the structure of this complex. Via chemical cross-linking, we could stabilize this dynamic complex and identify the structural features of key events in DNA mismatch repair. We could show that in the complex between MutS and MutL the mismatch-binding and connector domains of MutS are in proximity to the N-terminal ATPase domain of MutL. The DNA- and nucleotide-dependent complex formation could be monitored by FRET using single cysteine variants labeled in the connector domain of MutS and the transducer domain of MutL, respectively. In addition, we could trap MutS after an ATP-induced conformational change by an intramolecular cross-link between Cys-93 of the mismatch-binding domain and Cys-239 of the connector domain.

[The film festival "Ausnahme|Zustand" ("State of Emergency")--a strategy against stigmatization of the mentally ill?]. / Das Filmfestival "AUSNAHME|ZUSTAND"--Eine Strategie gegen die Stigmatisierung psychisch kranker Menschen?

OBJECTIVE: From April until November 2006, the association Irrsinnig Menschlich e. V. from Leipzig carried out the film festival "Ausnahme|zustand" about the topic Mental Illnesses all over Germany. The festival was on tour in more than 70 cities. The aim of the festival was to give the topic mental illness a podium, to inform and to entertain. METHOD: A Pre-Post-Test was carried out to look for the effect of the documentaries shown on social distance of the audience towards mentally ill people. A total of 405 visitors to the film festival in Leipzig could be questioned. In addition, 104 other viewers could be questioned. RESULTS: As the results show, the effect on the viewers' social distance strongly depends on the content of the shown documentaries: A reduction as well as an increase of social distance were identified. For two of the documentaries no significant changes were found. It is noticeable that a highly selective group of people has been reached with this activity; with the main proportion of the visitors suffering from a mental illness themselves or knowing a mentally ill person. This is also reflected in the significantly lower social distance of the film festival viewers compared with the other viewers. DISCUSSION: It has to be taken into consideration that documentaries about mental illness do not automatically reduce stigma, and that these kind of activities will mainly attract people who are already sensitised for this topic.

A prospective study of Quality of life in schizophrenia in three European countries.

Only a small number of studies have tried to identify factors influencing the subjective QoL of patients suffering from schizophrenia in a longitudinal design. These studies suffer from small clinical samples or compare baseline data only with a single follow-up. The European Schizophrenia Cohort Study overcomes these shortcomings by providing data from five time points on 1208 patients in psychiatric treatment in three European countries over a period of 2 years. QoL was measured with the brief version of Lehman's Quality of Life Interview. Random effects, between-effects and within-effects regression models were computed in order to measure the influence on subjective QoL of patients' socio-demographic and clinical characteristics and objective QoL. Objective QoL scores were generally found to be related to the equivalent subjective QoL scores. People's financial situation, and depressive and positive symptoms had a general effect on almost all subjective domains. The significant effects of objective finances on subjective domains like health and social relations raise interesting possibilities for intervention. Sufficient financial resources appear to be a necessary condition for achieving satisfactory QoL in schizophrenia patients. However, changes in individual's characteristics and circumstances did not relate as strongly as expected to changes in QoL, suggesting effective intervention may be difficult.

Issues in conducting cross-cultural research: implementation of an agreed international protocol [corrected] designed by the WHOQOL Group for the conduct of focus groups eliciting the quality of life of older adults.

Multi-centre and cross-cultural research require the use of common protocols if the results are to be either pooled or compared. All too often adherence to protocols is not discussed in reports and where it is reported poor adherence is frequently noted. This paper discusses the use of international guidelines developed by WHOQOL Field Centres to conduct and report focus groups aimed at eliciting key concepts of quality of life among older adults. This was the first step in the development of the WHOQOL-OLD instrument. Although there was overall adherence to the agreed guidelines, there were some differences in the level of reporting, even after participating Field Centres had the opportunity to explain their reports. The reasons for these discrepancies are reported. It is concluded that because of local situations, it is difficult to achieve identical implementation of multi-centre cross-cultural protocols and that the highest standards of auditing are required if findings are to be compared. Suggestions for how such protocols can be improved are given.

[The WHOQOL-OLD]. / Der WHOQOL-OLD -- Ein Fragebogen zur interkulturellen Erfassung der Lebensqualität im Alter.

The WHOQOL-OLD is an international cooperation of 22 research centres under the sponsorship of the WHO for the development of an intercultural instrument for the assessment of quality of life in old age. This article presents the results of the empirical investigations (field trial) for the German version of WHOQOL-OLD. Based on the results of the focus groups which were conducted in the first step of the project new items were generated and the dimensional structure was determined. Items were then tested and their number reduced using both classical and modern psychometric methods. A field trial was then carried out testing psychometric characteristics and standardization of the questionnaire. The outcome of this study is a 24-item 6-facet add-on module which can be used with the WHOQOL-BREF or the WHOQOL-100 for assessment of quality of life in older adults.