SignificanceThe assembly mechanism of RNA, vital to describing its functions, depends on both the sequence and the metal ion concentration. How the latter influences the folding trajectories remains an important unsolved problem. Here, we examine the folding pathways of an RNA pseudoknot (PK) with key functional roles in transcription and translation, using a combination of experiments and simulations. We demonstrate that the PK, consisting of two hairpins with differing stabilities, folds by parallel pathways. Surprisingly, the flux between them is modulated by monovalent salt concentration. Our work shows that the order of assembly of PKs is determined by the relative stability of the hairpins, implying that the folding landscape can be controlled by sequence and ion concentration.

Rad4/XPC DNA damage sensor protein specifically binds to a photocleavable NPOM-DNA adduct, and this recognition is abolished upon photo-cleavage of NPOM.

XPC protein recognizes diverse DNA lesions including ultravioletphotolesions and carcinogen-DNA adducts, initiating nucleotide excision repair. Structural studies showed that Rad4 (yeast ortholog) bound specifically to DNA flips out damaged nucleotides away from the protein, indicating that it relies on indirect readout for damage recognition. However, characterizing intrinsic DNA deformability has been a significant challenge. Using fluorescence lifetime measurements on DNA containing model lesions sandwiched by tC o and tC nitro -cytosine-analog FRET pair exquisitely sensitive to local distortions, we unveiled the conformational heterogeneities of DNA with varying Rad4-binding specificities and revealed a direct connection between intrinsic DNA distortions/deformability and Rad4 recognition: high-specificity CCC/ CCC mismatch sampled conformations that deviated significantly from B-DNA-like, even in the absence of Rad4; nonspecific TAT/TAT mismatch was largely homogeneous and B-DNA-like. We employed laser temperaturejump perturbation to measure the rates of these distortional dynamics and found that these rates in CCC/CCC-containing DNA remained essentially unchanged with and without Rad4, pointing to a conformational capture mechanism for Rad4. These initial studies were done with short, linear DNA oligomers, while DNA in our cells is typically bent and supercoiled, which is expected to have a profound impact on DNA damage recognition. We examined the effect of DNA bending strain on the intrinsic deformability at the CCC/CCC site by extending the fluorescence lifetime studies in the context of 126-bp DNA minicircles. These minicircles amplified DNA distortions and exhibited >100-fold binding affinity for Rad4 compared with linear DNA. Taken together, these results show that lesion-containing DNA has the propensity to undergo spontaneous unwinding fluctuations to adopt pre-distorted conformations that Rad4 recognizes, and bending deformations amplify these effects.

The MutSg homologs, Msh4 and Msh5, play a significant role in meiotic recombination by assisting in the proper segregation of chromosomes through binding to DNA Holliday Junctions (HJ). We have employed fluorescent measurements with molecular dynamic simulations to generate a homologymodeled MD-refined structure of the protein-junction interaction. We have used this model coupled with our experimental results to examine Msh4-Msh5 conformational dynamics in DNA binding and identify the features of the Msh4-Msh5-junction interaction that lead to specific recognition. Our studies show that Msh4-Msh5 preferentially binds HJ DNA relative to duplex DNA and our model of the Msh4-Msh5-HJ interaction illustrates how Msh4-Msh5 stabilizes the stacked form of the junction. Stabilization of this junction conformation is generally refractory to branch migration, which is consistent with a potential role for Msh4-Msh5 (MutSg) in trapping HJ until they are resolved by Mlh1-Mlh3 (MutLg). Our homology modeled MD-refined structure of Msh4-Msh5 also revealed a putative DNA-binding region, in which the Msh4 subunit interacts more directly with either junction or duplex DNA relative to the Msh5 subunit. The model also indicates that the protein complex contains a second cavity, which could be important for binding other DNA substrates. Consequently, we have examined Msh4-Msh5 binding to six other recombination intermediates including DNA overhangs, forks, D-loop with single end strand invasion, pre-HJ and open junctions. Although nanomolar binding affinities are observed, the junction is the preferred substrate of Msh4-Msh5. Steady-state ATPase assays in the presence and absence of these different recombination intermediates elucidate the role of ATP hydrolysis in binding and specificity. The ability of the Msh4-Msh5 complex to specifically recognize these different recombination intermediates will be presented in terms of binding affinity, conformational dynamics, and utilization of ATP.

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