Dynamics and Conformations of a Full-Length CRESS-DNA Replicase

Circular Rep-encoding single-stranded DNA (CRESS-DNA) viruses encode for a Replicase (Rep) that is essential for viral replication. Rep is a helicase with three domains: an endonuclease, an oligomeric, and an ATPase domain (ED, OD, and AD). Our recent cryo-EM structure of the porcine circovirus 2 (PCV2) Rep provided the first structure of a CRESS-DNA Rep. The structure visualized the ED to be highly mobile, Rep to form a homo-hexamer, bound ssDNA and nucleotides, and the AD to adopt a staircase arrangement around the ssDNA. We proposed a hand-over-hand mechanism by the ADs for ssDNA translocation. The hand-over-hand mechanism requires extensive movement of the AD. Here, we scrutinize this mechanism using all-atom Molecular Dynamics (MD) simulation of Rep in three states: (1) Rep bound to ssDNA and ADP, (2) Rep bound to ssDNA, and (3) Rep by itself. Each of the 700 nsec simulations converges within 200 nsec and provides important insight into the dynamics of Rep, the dynamics of Rep in the presence of these biomolecules, and the importance of ssDNA and ADP in driving the AD to adopt the staircase arrangement around the ssDNA. To the best of our knowledge, this is the first example of an all-atom MD simulation of a CRESS-DNA Rep. This study sets the basis of further MD studies aimed at obtaining a chemical understanding of how Rep uses nucleotide binding and hydrolysis to translocate ssDNA.