Single-molecule multiparameter fluorescence spectroscopy reveals directional MutS binding to mismatched bases in DNA

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

Identification of endogenous gibberellins in inflorescence of Ornithogalum thyrsoides

Endogenous gibberellins (GAs) were extracted from inflorescence of Ornithogalum thyrsoides and identified by using combined gas-chromatography / mass spectrometry (GC/MS). Three 13-hydroxylated GAs, GA19, GA20 and GA53, and thirteen 13-non-hydroxylated GAs, GA4, GA7, GA9, GA12, GA15, GA24, GA25, GA51, GA61, GA112, GA115, 1,2-didehydro GA9 (which is a novel GA, and has been assigned as GA120), and GA120 - isolactone were detected. The presence of these GAs suggests that both the early-13-hydroxylation GA biosynthesis pathway and the early-13-non-hydroxylated GA biosynthesis pathway were operating in the inflorescence of Ornithogalum. The presence of GA7, GA9 and GA120 suggests that GA120 could be considered as a metabolic intermediate in the conversion of GA9 into GA7 in O. thyrsoides

Endogenous gibberellins in Lolium perenne and influence of defoliation on their contents in elongating leaf bases and in leaf sheats

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