Free Energy Landscape and Conformational Kinetics of Hoogsteen Base-Pairing in DNA vs RNA

Abstract

Genetic information is encoded in the DNA double helix which, in its physiological milieu, is characterized by the iconical Watson-Crick nucleobase pairing. Recent NMR relaxation experiments revealed the transient presence of an alternative, Hoogsteen base pairing pattern in naked DNA duplexes and estimated its relative stability and lifetime. In contrast, HG transitions in RNA were not observed. Understanding Hoogsteen (HG) base pairing is important because the underlying "breathing" can modulate significantly DNA/RNA recognition by proteins. However, a detailed mechanistic insight into the transition pathways and kinetics is still missing. We performed enhanced sampling simulation (with combined metadynamics and adaptive force bias method) and Markov State modeling to obtain accurate free energy, kinetics and the intermediates in the transition pathway between WC and HG base pair for both naked B-DNA and A-RNA duplexes. The Markov state model constructed from our unbiased MD simulation data revealed previously unknown complex extra-helical intermediates in this seemingly simple process of base pair conformation switching in B-DNA. Extending our calculation to A-RNA, for which HG base pair is not observed experimentally, resulted in relatively unstable single hydrogen bonded distorted Hoogsteen like base pair. Unlike B-DNA the transition pathway primarily involved base paired and intra-helical intermediates with transition timescales much higher than that of B-DNA. The seemingly obvious flip-over reaction coordinate, i.e., the glycosidic torsion angle is unable to resolve the intermediates; so a multidimensional picture, involving backbone dihedral angles and distance between atoms participating in hydrogen bonds, is required to gain insight into the molecular mechanism. SIGNIFICANCE Formation of unconventional Hoogsteen (HG) base pairing is an important problem in DNA biophysics owing to its key role in facilitating the binding of DNA repairing enzymes, proteins and drugs to damaged DNA. X-ray crystallography and NMR relaxation experiments revealed the presence of HG base pair in naked DNA duplex and protein-DNA complex but no HG base pair was observed in RNA. Molecular dynamics simulations could reproduce the experimental free energy cost of HG base pairing in DNA although a detailed mechanistic insight is still missing. We performed enhanced sampling simulation and Markov state modeling to obtain accurate free energy, kinetics and the intermediates in the transition pathway between WC and HG base pair for both B-DNA and A-RNA.Manuscript submitted to Biophysical Journal 1 D. Ray and I. Andricioaei duplex DNA beyond what can be achieved by Watson-Crick base-pairing alone. For example, HG base pairs are recognized by DNA repairing enzymes resulting in selective binding in the damaged regions of DNA, rich in syn-anti configuration over the anti-anti WC base pair rich normal DNA. Thus, apart from providing structural integrity to the damaged and deformed DNA, HG base pairs assist in the DNA repair mechanism (13).A...

Sequence dependence of transient Hoogsteen base-pairing in DNA

“…2). This dominant outside pathway agrees with our previous results using TPS [25], as well as with published reports using Markov state models [20]. Our result is likely to settle the debate arising for previous simulations that showed favored inside pathways, but either with no explicit solvent [5], or with a modified force field and restrained neighboring bases upon initialization [16].…”

“…2C). Such a prevalent outside mechanism agrees with our previous results for the CAA sequence using TPS [25], as well as Markov state modeling by Ray and Andricioaei [20]. Most of the outside 3' rotations also show a transition state closer to HG (s ≈ 0) than to WCF, which agrees with experiments [7].…”

“…A predominance of outside pathways could explain why sequence variation does not seem to impact significantly the WCF-to-HG free-energy barrier, according to Alvey and coworkers' NMR results [7]. Markov state modeling has also confirmed a dominant outside pathway [20].…”

“…A few years later, Yang and coworkers reported another (χ, θ) free-energy surface, this time for a base pair with an N1-methylated adenine [19]. Similarly, Ray and Andricioaei calculated a (χ, θ) free-energy surface for DNA and RNA [20]. While such 2D free-energy landscapes are rich in insight, they also demand significant computational expense to obtain several µs-long MD runs; hindering the systematic study of multiple DNA variations.…”