Site-Specific Investigation of DNA Holliday Junction Dynamics and Structure with 6-Methylisoxanthopterin, a Fluorescent Guanine Analog.

DNA Holliday Junction (HJ) formation and resolution is requisite for maintaining genomic stability in processes such as replication fork reversal and double-strand break repair. If HJs are not resolved, chromosome disjunction and aneuploidy result, hallmarks of tumor cells. To understand the structural features that lead to processing of these four-stranded joint molecule structures, we seek to identify structural and dynamic features unique to the central junction core. We incorporate the fluorescent guanine analog 6-methylisoxanthopterin (6-MI) at ten different locations throughout a model HJ structure to obtain site-specific information regarding the structure and dynamics of bases relative to those in a comparable sequence context in duplex DNA. These comparisons were accomplished through measuring fluorescence lifetime, relative brightness, fluorescence anisotropy, and thermodynamic stability, along with fluorescence quenching assays. These time-resolved and steady-state fluorescence measurements demonstrate that the structural distortions imposed by strand crossing result in increased solvent exposure, less stacking of bases and greater extrahelical nature of bases within the junction core. The 6-MI base analogs in the junction reflect these structural changes through an increase in intensity relative to those in the duplex. Molecular dynamics simulations performed using a model HJ indicate the primary sources of deformation are in the shift and twist parameters of the bases at the central junction step. These results suggest that junction-binding proteins may use the unique structure and dynamics of the bases at the core for recognition.

SUMO fosters assembly and functionality of the MutSγ complex to facilitate meiotic crossing over.

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.

Mismatch Recognition by Saccharomyces cerevisiae Msh2-Msh6: Role of Structure and Dynamics.

The mismatch repair (MMR) pathway maintains genome integrity by correcting errors such as mismatched base pairs formed during DNA replication. In MMR, Msh2-Msh6, a heterodimeric protein, targets single base mismatches and small insertion/deletion loops for repair. By incorporating the fluorescent nucleoside base analog 6-methylisoxanthopterin (6-MI) at or adjacent to a mismatch site to probe the structural and dynamic elements of the mismatch, we address how Msh2-Msh6 recognizes these mismatches for repair within the context of matched DNA. Fluorescence quantum yield and rotational correlation time measurements indicate that local base dynamics linearly correlate with Saccharomyces cerevisiae Msh2-Msh6 binding affinity where the protein exhibits a higher affinity (KD ≤ 25 nM) for mismatches that have a significant amount of dynamic motion. Energy transfer measurements measuring global DNA bending find that mismatches that are both well and poorly recognized by Msh2-Msh6 experience the same amount of protein-induced bending. Finally, base-specific dynamics coupled with protein-induced blue shifts in peak emission strongly support the crystallographic model of directional binding, in which Phe 432 of Msh6 intercalates 3' of the mismatch. These results imply an important role for local base dynamics in the initial recognition step of MMR.