MutL traps MutS at a DNA mismatch

DNA mismatch repair is the process by which errors generated during DNA replication are corrected. Mutations in the proteins that initiate mismatch repair, MutS and MutL, are associated with greater than 80% of hereditary nonpolyposis colorectal cancer (HNPCC) and many sporadic cancers. The assembly of MutS and MutL at a mismatch is an essential step for initiating repair; however, the nature of these interactions is poorly understood. Here, we have discovered that MutL fundamentally changes the properties of mismatch-bound MutS by preventing it from sliding away from the mismatch, which it normally does when isolated. This finding suggests a mechanism for localizing the activity of repair proteins near the mismatch

Single-Molecule Analysis Reveals the Kinetics and Physiological Relevance of MutL-ssDNA Binding

DNA binding by MutL homologs (MLH/PMS) during mismatch repair (MMR) has been considered based on biochemical and genetic studies. Bulk studies with MutL and its yeast homologs Mlh1-Pms1 have suggested an integral role for a single-stranded DNA (ssDNA) binding activity during MMR. We have developed single-molecule Förster resonance energy transfer (smFRET) and a single-molecule DNA flow-extension assays to examine MutL interaction with ssDNA in real time. The smFRET assay allowed us to observe MutL-ssDNA association and dissociation. We determined that MutL-ssDNA binding required ATP and was the greatest at ionic strength below 25 mM (KD = 29 nM) while it dramatically decreases above 100 mM (KD>2 µM). Single-molecule DNA flow-extension analysis suggests that multiple MutL proteins may bind ssDNA at low ionic strength but this activity does not enhance stability at elevated ionic strengths. These studies are consistent with the conclusion that a stable MutL-ssDNA interaction is unlikely to occur at physiological salt eliminating a number of MMR models. However, the activity may infer some related dynamic DNA transaction process during MMR

High-Resolution Vessel Wall Magnetic Resonance Imaging in Angiogram-Negative Non-Perimesencephalic Subarachnoid Hemorrhage

Standard magnetic resonance imaging (MRI) rarely identifies the cause of hemorrhage in patients with an angiogram-negative, non-perimesencephalic subarachnoid hemorrhage (SAH). Yet up to 10 % of these patients have recurrent hemorrhage. The aim of the study was to explore the potential role of high-resolution contrast-enhanced 3-Tesla vessel wall-MRI in patients with angiogram-negative SAH. We performed intracranial vessel wall-MRI of the circle of Willis using a 3-Tesla scanner in consecutive patients presenting with a spontaneous, angiogram-negative, non-perimesencephalic SAH. Vessel wall-MRI included T1-, T2-, and gadolinium-enhanced T1-weighted two-dimensional black-blood sequences in multiple planes (voxel size 0.4 x 0.4 x 2.0 mm). Two neuroradiologists independently scored abnormalities of the arterial wall. In all, 11 patients (mean age 59 years) underwent vessel wall-MRI. A total of seven patients had vessel wall abnormalities despite normal catheter angiography. Two patients had focal abnormalities contiguous with the outer margin of the basilar artery wall for which we considered a differential of ruptured blood blister aneurysm, thrombosed aneurysm, and loculated extramural blood from elsewhere. Two patients had arterial wall enhancement involving multiple arteries, possibly secondary to SAH. Three patients had arterial wall enhancement at sites of dural penetration, remote from the SAH, likely related to age and atherosclerotic risk factors. Vessel wall-MRI did not alter patient management in this cohort. Vessel wall-MRI showed abnormalities in seven patients with angiogram-negative SAH. These findings did not alter patient management, but the findings may be useful for other physicians who choose to perform vessel wall-MRI in this patient populatio

MutL traps MutS at a DNA mismatch

DNA mismatch repair (MMR) identifies and corrects errors made during replication. In all organisms except those expressing MutH, interactions between a DNA mismatch, MutS, MutL, and the replication processivity factor (β-clamp or PCNA) activate the latent MutL endonuclease to nick the error-containing daughter strand. This nick provides an entry point for downstream repair proteins. Despite the well-established significance of strand-specific nicking in MMR, the mechanism(s) by which MutS and MutL assemble on mismatch DNA to allow the subsequent activation of MutL’s endonuclease activity by β-clamp/PCNA remains elusive. In both prokaryotes and eukaryotes, MutS homologs undergo conformational changes to a mobile clamp state that can move away from the mismatch. However, the function of this MutS mobile clamp is unknown. Furthermore, whether the interaction with MutL leads to a mobile MutS–MutL complex or a mismatch-localized complex is hotly debated. We used single molecule FRET to determine that Thermus aquaticus MutL traps MutS at a DNA mismatch after recognition but before its conversion to a sliding clamp. Rather than a clamp, a conformationally dynamic protein assembly typically containing more MutL than MutS is formed at the mismatch. This complex provides a local marker where interaction with β-clamp/PCNA could distinguish parent/daughter strand identity. Our finding that MutL fundamentally changes MutS actions following mismatch detection reframes current thinking on MMR signaling processes critical for genomic stability

Dřevostavba pro rekreační účely

The bachelor thesis deals with the design of a wooden building, which will be used for recreational purposes, and it will also be inhabitable for the whole year. The introductory part focuses on already realized wood buildings for recreational purposes. The architectural design of the recreational building is devised in the theoretical part, including a detailed description of the selected structural system and a description of materials, which will be used for the aforementioned building. For the layers of perimeter construction, there is an assessment of the thermal protection aspect. At the end of the thesis, economic aspects are shown. The thesis also contains a technical report, an accompanying report and basic drawings with added drawings of specified structural details