Double stranded DNA formed via Watson-Crick basepairing needs to be separated into single strands for genome duplication or repair to occur. Likewise, double stranded RNA are frequently separated into single strands during transcription, splicing, ribosome biogenesis and translation. Such nucleic acids unwinding should not occur indiscriminately. Using advanced single molecule measurement technologies we discovered a novel mechanism of regulating helicase activities through a conformational switch. This led to the development of a superhelicase that can unwind thousands of base pairs processively even against a strong opposing force. A mirror process to nucleic acids unwinding is protein-dependent annealing of two stands of nucleic acids. Examples include Rec/Rad51-mediated DNA recombination, non-coding RNA-based gene regulation and CRISPR-based DNA degradation. All of these processes rely on basepairing interactions above the threshold number of bp for specificity. How the target DNA or RNA can be both rapidly and accurately identified in the presence of other sequences in large excess is an unanswered question. `Nucleic acids remodeling' mediated by proteins either in the direction of unwinding or in the direction of annealing is the overarching theme of this project. The premise here is that a balance between the basepair breaking and formation is critical in normal functions of these proteins inside the cell and if the balance is not properly maintained, it leads to mis-regulation and diseased states. The key questions to address are: (1) What is the in vivo role of various helicase conformations? (2) Can we detect all reaction intermediates during helicase function? (3) Can we mimic co-transcriptional RNA folding and ribosome assembly using superhelicases? (4) How do basepairing interactions control replication fork reversal and restoration by annealing helicases? (5) How do basepairing interactions determine in vivo kinetics of sRNA-based gene regulation? (6) How does the balance between heteroduplex extension and reversal control CRISPR- Cas9/Cpf1 target verification and cleavage activation? (7) How does the balance between heteroduplex extension and reversal control Cas3 helicase-nuclease recruitment?

Public Health Relevance

Defective or mis-regulated nucleic acids unwinding can cause severe genetic disorders. Rapid and accurate annealing between matching sequences of DNA and RNA is crucial for many processes required for cell survival. Single molecule studies of these fundamental processes will help us understand the normal functions of the proteins involved with potential implications in therapies targeting them.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Unknown (R35)
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Special Emphasis Panel (ZGM1)
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Reddy, Michael K
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Johns Hopkins University
Schools of Medicine
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