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Crossing over is essential for chromosome segregation during meiosis. Protein modification by SUMO is implicated in crossover control, but pertinent targets have remained elusive. Here we identify Msh4 as a target of SUMO-mediated crossover regulation. Msh4 and Msh5 constitute the MutSγ complex, which stabilizes joint-molecule (JM) recombination intermediates and facilitates their resolution into crossovers. Msh4 SUMOylation enhances these processes to ensure that each chromosome pair acquires at least one crossover. Msh4 is directly targeted by E2 conjugase Ubc9, initially becoming mono-SUMOylated in response to DNA double-strand breaks, then multi/poly-SUMOylated forms arise as homologs fully engage. Mechanistically, SUMOylation fosters interaction between Msh4 and Msh5. We infer that initial SUMOylation of Msh4 enhances assembly of MutSγ in anticipation of JM formation, while secondary SUMOylation may promote downstream functions. Regulation of Msh4 by SUMO is distinct and independent of its previously described stabilization by phosphorylation, defining MutSγ as a hub for crossover control.

Integration host factor (IHF) is a nucleoid-associated protein involved in DNA packaging, integration of viral DNA and recombination. IHF binds with nanomolar affinity to duplex DNA containing a 13 bp consensus sequence, inducing a bend of ~160° upon binding. We determined that IHF binds to DNA Four-way or Holliday junctions (HJ) with high affinity regardless of the presence of the consensus sequence, signifying a structure-based mechanism of recognition. Junctions, important intermediates in DNA repair and homologous recombination, are dynamic and can adopt either an open or stacked conformation, where the open conformation facilitates branch migration and strand exchange. Using ensemble and single molecule Förster resonance energy transfer (FRET) methods, we investigated IHF-induced changes in the population distribution of junction conformations and determined that IHF binding shifts the population to the open conformation. Further analysis of smFRET dynamics revealed that even in the presence of protein, the junctions remain dynamic as fast transitions are observed for the protein-bound open state. Protein binding alters junction conformational dynamics, as cross correlation analyses reveal the protein slows the transition rate at 1 mM Mg2+ but accelerates the transition rate at 10 mM Mg2+. Stopped flow kinetic experiments provide evidence for two binding steps, a rapid, initial binding step followed by a slower step potentially associated with a conformational change. These measurements also confirm that the protein remains bound to the junction during the conformer transitions and further suggest that the protein forms a partially dissociated state that allows junction arms to be dynamic. These findings, which demonstrate that IHF binds HJs with high affinity and stabilizes junctions in the open conformation, suggest that IHF may play multiple roles in the processes of integration and recombination in addition to stabilizing bacterial biofilms. [ABSTRACT FROM AUTHOR]

Raman spectroscopy is a sensitive method for monitoring the self-assembly of biological macromolecules. Amyloidogenic peptides have been shown to form peptide nanotubes often with a central diphenylalanine core. In this [...]

The fluorescence intensity of 4-amino-8-(2-deoxy-beta-D-ribofuranosyl)-5'-O-dimethoxy-trityl-6-methyl-7(8H)-pteridone (6MAP), which is very sensitive to base stacking interactions, is used to monitor base-base interactions during the premelting transition. Fluorescence melting curves indicate that 6MAP is a more sensitive reporter of the premelting transition than UV absorption spectroscopy.

Research has been conducted on the DNA dodecamer duplexes A (sub)3 T (sub)3 and [(ST) (sub)3]. The results of the UV resonance Raman and circular dichroism studies of these duplexes are reported.

Data are presented to show that the binding of HU to DNA involves local bending or unwinding of the DNA. HU is a histone-like DNA binding protein involved in replications, transcription, recombination, and DNA packaging in E. coli.

Results show that out of the seven tryptophan residues in the C-domain of SecA protein, three residues exhibit fluorescence signal and different degrees of solvent accessibility. Data also indicate that the residues contribute to the spectoscopic changes due to endothermic transition or associated phospholipids or signal peptide binding.

The structure of sickle cell hemoglobin (Hb S) fibers was probed using UV resonance Raman spectroscopy. For these studies a functional analogue of Hb S, was used to study structural changes that accompany fiber formation.

A study was conducted to analyze a reaction coordinate for the allosteric transition in human hemoglobin based on the static and time-resolved Raman spectroscopy of hemoglobin(sub A) and of the mutant hemoglobin(sub K)(D-alpha-99N). The heme was held between the proximal and distal helices that were tethered using H-bonds. Results indicated that cooperativity in hemoglobin is influenced by interhelical H-bonds that promote the helices to act as levers.

Research was conducted to examine the tertiary and quaternary structure of HbS by UV resonance Raman and fluorescence spectroscopic approaches. The study was conducted using the tetrameric form as the fluoromet derivative and compared with HbA under the same conditions. Combined spectroscopic experiments revealed an altered tertiary structure in HbS where the A-helix is displaced toward the E-helix as monitored by the strength of the beta-15 Trp***beta72 Ser H-bond.

An ultraviolet resonance Raman spectroscopic study shows that the hydrogen bonds in d(A+-G)10 nucleic acids involve exocyclic amino hydrogens of the d-adenine (dA). The d-guanine residues fail to show such behavior. The anti-residue conformation is probably the result of the glycosyl bond modes. The nitrogen at position 1 in dA is protonated. The bonding and protonation properties may improve the understanding of the secondary structure of these single-stranded nucleic acids.

Results of the effects of replacement of Ca by Sr on the structure of the Mn complex of photosystem (PS II) by using XAS spectroscopy showed that the structure of the Mn complex in PS II preparations depleted of Ca and reactivated with Ca was unchanged relative to that found in untreated PS II preparations. This confirms the structural proximity of Ca to the Mn cluster suggested before. The expected structural arrangements for a calcium-binding site are discussed.

Ultraviolet resonance Raman (UVRR) spectroscopic analysis of the quaternary and tertiary structures of symmetrical cyanomet valency hybrids indicates that the hybrids display distinct UVRR spectra in the absence of CO and that the R state perturbation in the spectra for unligated chains include E helix disruption. Inositol hexaphosphate introduction enhances the T state proportion from 40% to 53%, while IHP binding changes the R and T state frequencies and affects the hybrid structures. The R state perturbation elucidates the quaternary increase phenomenon in the hybrids.

Mn K-edge spectra and X-ray absorption fine structure spectroscopic studies of the orientation of oxygen evolving complex with respect to lipid bilayer in plant photosystem (PSII) reveal that PSII membranes exhibit high K-edge dichroism, confirming the presence of an asymmetric tetranuclear Mn cluster. The transition from S1 to S2 states of the Kok cycle leads to minor structural changes in the cluster with no changes in the orientation of Mn-Mn vectors with respect to membrane normal.

X-ray absorption fine structure spectroscopic studies are employed to analyze the Mn structures in the oxygen-evolving complexes (OEC) in photosystem II of spinach and Synechococcus sp. The mean radial distribution function of scatterers in the vicinity of the Mn atoms are calculated and their values enable the prediction of number, type and distances between Mn atoms. Mn units in OEC are bridged by binuclear bis(Mu-oxo) units and are not influence by Mn(III) to Mn(IV) oxidation.

Ultraviolet resonance Raman spectra were obtained for Fluoromethoglobin (metHbF) with and without inositol hexaphosphate (IHP). Addition of IHP to metHbF yielded difference spectra similar to that obtained between deoxyhemoglobin and HbCO, but its intensity was attenuated by around one-third. R and T state subunit interactions in metHbF and native Hb were almost identical, except that the T state population is lower than 100%. A new difference feature arising from the interior trytophans was detected and was attributed to an alteration in their H-bonding caused by antihelical forces created by a flouride in the T state.

Forster resonance energy transfer methodology was employed to map the location of the SecA signal peptide-binding domain using a collection of functional monocysteine SecA mutants and alkaline phosphatase signal peptides labeled with appropriate donor-acceptor fluorophores. The identified domain region by FRET was found to be located at a multidomain interface within the heart of SecA, surrounded by and potentially responsive to domains important for binding nucleotide, mature portions of the preprotein, and the SecYEG channel.

The UV resonance Raman spectroscopy is employed to monitor the formation of sickle cell hemoglobin (Hb S) fibers in real time through aromatic amino acid vibrational modes. The initial fiber formation and subsequent formation of larger assemblies due to turbidity or Phe signal intensity provide molecular insight into the interactions governing Hb S fiber formation.

Integration host factor (IHF) has a major impact on the morphology of DNA condensed by polyamines in vitro. The studies have concluded that the common function of nucleoid-associated proteins, IHF and HU, in bacterial cells is to provide DNA organization in the nucleoid by the introduction of sharp bends in chromosomal DNA.