Biology subjects

Korolev, S.

Publications and source records attributed to Korolev, S..

5 recordsLinked to original sources

bioRxiv2026

Chemical control of 2'-hydroxyl-dependent Cas9 target engagement enables CRISPR RNA ribose replacement

Advanced CRISPR-based therapies benefit from CRISPR RNA (crRNA) with high nuclease resistance and enhanced drug-like properties, which is primarily achieved through chemical replacement of the RNA ribose moiety. However, for gene editing enzymes like CRISPR-Cas9 a handful of residues cannot be replaced with chemical ribose analogues, limiting the scope of therapeutic strategies. The mechanism underlying this restriction has remained unclear. Here, using nucleic acid chemistry, biochemistry, cryo-EM, and molecular dynamics simulations, we show that the ribose 2-hydroxyl group at specific crRNA residues is required to achieve a conformational state competent for Cas9 target DNA binding. Based on the mechanistic principles uncovered, we combined site-specific phosphorothioate linkage chemistry with ribose replacement chemistry to restore binding and activity, resulting in high Cas9 editing efficiency and fidelity with a ribose-free crRNA. This study offers novel mechanistic insight and crRNAs with full chemical stabilization, making rational design of guide RNAs with complete nuclease protection for CRISPR-based medicines possible.

biochemistry↗

bioRxiv2025

A guide RNA repeat checkpoint steers CRISPR-Cas9 catalysis

A widely adopted CRISPR-Cas9 modification is fusion of the naturally occurring two-component dual guide RNA (dgRNA) to create an artificial single guide RNA (sgRNA). However, mechanistic and functional differences between dgRNA and sgRNA have not been systematically explored. By investigating the activity of these two guide architectures, we discover a guide RNA repeat checkpoint (GRC) that senses the structure and dynamics of the guide repeat region. The GRC coordinates with other checkpoint mechanisms known to recognize spacer-target R-loop fidelity, together licensing target cleavage. Based on these principles, dgRNA and sgRNA properties could be combined into guide repeat-truncated sgRNAs (grtRNAs) and paired with high-fidelity Cas9 variants to further reduce off target editing. A mechanism that helps govern Cas9 catalysis via guide RNA structure sensing and communicates with other checkpoints offers a previously unappreciated path for understanding and further improving gene editing outcomes.

biochemistry↗

bioRxiv2023

The PALB2 DNA binding domain is an intrinsically disordered recombinase.

The Partner and Localizer of BRCA2 (PALB2) is a scaffold protein that links BRCA1 with BRCA2 to initiate homologous recombination (HR). PALB2 interaction with DNA strongly enhances HR efficiency in cells. The PALB2 DNA-binding domain (PALB2-DBD) supports strand exchange, a complex multistep reaction conducted by only a few proteins such as RecA-like recombinases and Rad52. Using bioinformatics analysis, small-angle X-ray scattering, circular dichroism, and electron paramagnetic spectroscopy, we determined that PALB2-DBD is an intrinsically disordered region (IDR) forming compact molten globule-like dimer. IDRs contribute to oligomerization synergistically with the coiled-coil interaction. Using confocal single-molecule FRET we demonstrated that PALB2-DBD compacts single-stranded DNA even in the absence of DNA secondary structures. The compaction is bimodal, oligomerization-dependent, and is driven by IDRs, suggesting a novel strand exchange mechanism. Intrinsically disordered proteins (IDPs) are prevalent in the human proteome. Novel DNA binding properties of PALB2-DBD and the complexity of strand exchange mechanism significantly expands the functional repertoire of IDPs. Multivalent interactions and bioinformatics analysis suggest that PALB2 function is likely to depend on formation of protein-nucleic acids condensates. Similar intrinsically disordered DBDs may use chaperone-like mechanism to aid formation and resolution of DNA and RNA multichain intermediates during DNA replication, repair and recombination.

biophysics↗

bioRxiv2018

Novel strand exchange activity of the human PALB2 DNA Binding Domain and its critical role for DNA repair in cells

Breast cancer associated proteins 1 and 2 (BRCA1, -2) and partner and localizer of BRCA2 (PALB2) protein are tumor suppressors linked to a spectrum of malignancies, including breast cancer and Fanconi anemia. They stimulate RAD51 recombinase during homology-directed repair (HDR). Along with being a hub for a protein interaction network, PALB2 interacts with DNA. The mechanism of PALB2 DNA binding and its function are poorly understood. We identified a major DNA-binding site in PALB2, mutation of which reduces the RAD51 foci formation and the overall HDR efficiency in cells by 50%. PALB2 N-terminal DNA-binding domain (N-DBD) stimulates the RAD51 strand exchange reaction. Surprisingly, it promotes the strand exchange without RAD51. Moreover, N-DBD stimulates the inverse strand exchange and can use both DNA and RNA substrates. Our data reveal a versatile DNA interaction property of PALB2 and demonstrate a critical role of PALB2 DNA binding for chromosome repair in cells.

biochemistry↗

bioRxiv2017

A novel dimeric active site and regulation mechanism revealed by the crystal structure of iPLA2β

Calcium-independent phospholipase A2{beta} (iPLA2{beta}) regulates several physiological processes including inflammation, calcium homeostasis and apoptosis. It is linked genetically to neurodegenerative disorders including Parkinsons disease. Despite its known enzymatic activity, the mechanisms underlying pathologic phenotypes remain unknown. Here, we present the first crystal structure of iPLA2{beta} that significantly revises existing mechanistic models. The catalytic domains form a tight dimer. The ankyrin repeat domains wrap around the catalytic domains in an outwardly flared orientation, poised to interact with membrane proteins. The closely integrated active sites are positioned for cooperative activation and internal transacylation. A single calmodulin binds and allosterically inhibits both catalytic domains. These unique structural features identify the molecular interactions that can regulate iPLA2{beta} activity and its cellular localization, which can be targeted to identify novel inhibitors for therapeutic purposes. The structure provides a well-defined framework to investigate the role of neurodegenerative mutations and the function of iPLA2{beta} in the brain.

biochemistry↗