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Cryo-EM Structure of AAV2 Rep68 bound to integration site AAVS1: Insights into the mechanism of DNA melting.
Structural highlights
FunctionREP68_AAV2S Plays an essential role in the initiation of viral DNA synthesis. Binds specifically to an inverted terminal repeat element (ITR) on the 3' and 5' ends of the viral DNA, where it cleaves a site specifically to generate a priming site for initiation of the synthesis of a complementary strand. Plays also a role as transcriptional regulator, DNA helicase and as key factor in site-specific integration of the viral genome. Inhibits the host cell cycle G1/S and G2/M transitions. These arrests may provide essential cellular factors for viral DNA replication.[1] Publication Abstract from PubMedThe Rep68 protein from Adeno-Associated Virus (AAV) is a multifunctional SF3 helicase that performs most of the DNA transactions necessary for the viral life cycle. During AAV DNA replication, Rep68 assembles at the origin of replication, catalyzing the DNA melting and nicking reactions during the hairpin rolling replication process to complete the second-strand synthesis of the AAV genome. We report the cryo-electron microscopy structures of Rep68 bound to the adeno-associated virus integration site 1 in different nucleotide-bound states. In the nucleotide-free state, Rep68 forms a heptameric complex around DNA, with three origin-binding domains (OBDs) bound to the Rep-binding element sequence, while three remaining OBDs form transient dimers with them. The AAA+ domains form an open ring without interactions between subunits and DNA. We hypothesize that the heptameric structure is crucial for loading Rep68 onto double-stranded DNA. The ATPgammaS complex shows that only three subunits associate with the nucleotide, leading to a conformational change that promotes the formation of both intersubunit and DNA interactions. Moreover, three phenylalanine residues in the AAA+ domain induce a steric distortion in the DNA. Our study provides insights into how an SF3 helicase assembles on DNA and provides insights into the DNA melting process. Cryo-EM structure of AAV2 Rep68 bound to integration site AAVS1: insights into the mechanism of DNA melting.,Jaiswal R, Braud B, Hernandez-Ramirez KC, Santosh V, Washington A, Escalante CR Nucleic Acids Res. 2025 Jan 24;53(3):gkaf033. doi: 10.1093/nar/gkaf033. PMID:39883011[2] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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