| 1. |
- Cristovao, Michele, et al.
(författare)
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Single-molecule multiparameter fluorescence spectroscopy reveals directional MutS binding to mismatched bases in DNA
- 2012
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 40:12, s. 5448-5464
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Tidskriftsartikel (refereegranskat)abstract
- Mismatch repair (MMR) corrects replication errors such as mismatched bases and loops in DNA. The evolutionarily conserved dimeric MMR protein MutS recognizes mismatches by stacking a phenylalanine of one subunit against one base of the mismatched pair. In all crystal structures of G:T mismatch-bound MutS, phenylalanine is stacked against thymine. To explore whether these structures reflect directional mismatch recognition by MutS, we monitored the orientation of Escherichia coli MutS binding to mismatches by FRET and anisotropy with steady state, pre-steady state and single-molecule multiparameter fluorescence measurements in a solution. The results confirm that specifically bound MutS bends DNA at the mismatch. We found additional MutS-mismatch complexes with distinct conformations that may have functional relevance in MMR. The analysis of individual binding events reveal significant bias in MutS orientation on asymmetric mismatches (G:T versus T:G, A:C versus C:A), but not on symmetric mismatches (G:G). When MutS is blocked from binding a mismatch in the preferred orientation by positioning asymmetric mismatches near the ends of linear DNA substrates, its ability to authorize subsequent steps of MMR, such as MutH endonuclease activation, is almost abolished. These findings shed light on prerequisites for MutS interactions with other MMR proteins for repairing the appropriate DNA strand.
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| 2. |
- Sisamakis, Evangelos
(författare)
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Förster Resonance Energy Transfer - from single molecule spectroscopy to imaging
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- During the last fifteen years several methods have been developed for probing biomolecules (DNA, RNA, proteins) one at a time. Among these methods fluorescence spectroscopy and in particular its many implementations for monitoring Förster Resonance Energy Transfer (FRET), have attracted much interest.This thesis deals mainly with high-precision single molecule FRET (smFRET) studies between a donor and an acceptor fluorophore attached to a biomolecule. Methodologies like multi-parameter fluorescence detection (MFD) and Probability Distribution Analysis (PDA) are used. We investigate, how and in which occasions; complex photophysical properties of the acceptor could influence the experimentally obtained FRET efficiency distributions. The value of smFRET experiments in enzymology is exemplified by presenting studies on DNA-related enzymes. Three structural conformations (Open, Closed, and Nucleotide-Binding) of Klentaq1, a DNA polymerase, have been resolved by measurements on freely diffusing molecules. We observe that the levels of occupancy of these conformations and the transitions among them, are dependent on the nature of the incoming dNTP, shedding more light into how conformational selection controls the incorporation cycle. Additionally, smFRET studies on MutS, a protein responsible for the initiation of the DNA mismatch repair machinery, have identified the existence of a preferred orientation of binding of the protein to asymmetric mismatches of DNA strands. Inhibiting MutS from binding in this preferred orientation has negative implications on the efficiency of the initiation of the overall DNA repair process.Shifting from spectroscopy to microscopy, we use FRET imaging for monitoring interactions between the Human Epidermal Growth Receptors, HER1 and HER2, and the Insulin Growth Factor 1 Receptor, IGF1R, in fixed cells obtained from patients with suspect breast cancer lesions. While working on FRET imaging, the need for developing methodologies for the objective evaluation of the sensitivity of confocal laser scanning microscopes (CLSM) was identified. In order to provide figure of merits for the sensitivity of a microscope, we use Fluorescence Correlation Spectroscopy (FCS) and Transient State (TRAST) imaging measurements on aqueous solutions of Rhodamine 110. Our results suggest that TRAST imaging measurements could serve as a fast and easy test for the day-to-day maintenance of a CLSM and could provide reference standards for comparing images obtained by different microscope systems.
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