Structure and dynamics of RNase H and their substrate complexes

RNase H is an endoribonuclease that hydrolyzes the RNA strand in a RNA/DNA hybrid. It is widely present in bacteria, archaea and eukaryotes. Their significance has become clear with its role in biological processes such as removal of RNA primers from Okazaki fragments, processing R-loops to modulate replication and to restore DNA topology. RNase H activity is required for the proliferation of retroviruses and HIV-1 RNase H is therefore a target for AIDS therapy. Recently, it is found that elimination of RNase HI causes embryonic lethality in mice, implicating their role in mitochondrial DNA replication. These enzymes are also found to be associated with a neuro-degenerative disease Acardi Goutiéres Syndrome, underlying their links to infectious and genetic diseases. RNase H is broadly classified into two major families, RNase HI and RNase HII. RNase HI shares homology with reverse transcriptase RNase H. Both RNase HI and RNase HII share a common ‘RNase H fold’ despite lack of sequence resemblance and dissimilar substrate specificity. While biochemical and structural information of the RNase H enzymes and DNA.RNA duplexes are quite well-known, mechanistic insights into its substrate recognition and specificity is still far from clear. Structural and biochemical aspects about RNase HII as well its substrate recognition and specificity are much less explored. With the aid of modelling and molecular dynamics simulations, we aim to fill in these critical gaps by delineating intrinsic conformational flexibility of RNases HI and RNase HII as well as their substrate complexes and their role in the recognition process. It is expected that such studies in combination with X-ray and NMR structures would go a long way in providing vital information that will be extremely critical in devising better inhibitors and therapeutic agents.

Structure and dynamics of nucleic acid triplexes

Ever since the finding of the ability of nucleic acid duplex to accommodate a Triplex Forming Oligonucleotide (TFO) along its major groove, a variety of biological roles for nucleic acid triplexes have been demonstrated. Triplex formation has shown to play a role in regulating gene expression, replication, site-directed mutagenesis, cleavage, non-enzymatic ligation and cross-linking. There are increasing evidences for their participation in cellular processes as well. Triplex formation relies on the sequence specific recognition of the target DNA duplex by TFO. TFO interacts primarily with the purine rich strand of the target DNA duplex, either in parallel or in antiparallel orientation by forming hydrogen bonds. Number of base triplets such as T*AT, C+*GC, G*GC and A*AT are a few examples . Among them, some base triplets are unique by being isosteric (equivalent structural geometry), while the others are non-isosteric (non-equivalent structural geometry). It is extremely important to understand the nature and extent of non-isostericity as they strongly influence triplex formation and its stability. Recently, we suggested a simple scheme to provide a measure of non-isostericity between any two triplets in terms of residual twist and radial difference. We have also demonstrated through MD simulations their efficacy in assessing the impact of base triplet non-isostericity on the structure of anti-parallel DNA triplexes, comprising the non-isomorphic G*GC & T*AT and G*GC & A*AT triplets. Current work is focused to towards deciphering the impact of juxtaposition of various non-isosteric triplets to form triplexes, their energetics and on sequence dependency. RNA and RNA .DNA hybrid triplexes are also understudy. The results are expected to provide a comprehensive understanding of the structure and dynamics of nucleic acid triplexes.

Consequent to success of these approaches, efforts are also underway to extend such concepts to Watson and Crick base pairs, Crick’s Wobble and several non-Watson & Crick base pairs that dominate RNA structures with a view to explore the impact of non-isosteric base pairs on RNA structure and interaction.