Biology subjects

Minakhin, L.

Publications and source records attributed to Minakhin, L..

4 recordsLinked to original sources

bioRxiv2024

Structural Basis for Pol theta-Helicase DNA Binding and Microhomology-Mediated End-Joining

DNA double-strand breaks (DSBs) present a critical threat to genomic integrity, often precipitating genomic instability and oncogenesis. Repair of DSBs predominantly occurs through homologous recombination (HR) and non-homologous end joining (NHEJ). In HR-deficient cells, DNA polymerase theta (Pol{theta}) becomes critical for DSB repair via microhomology-mediated end joining (MMEJ), also termed theta-mediated end joining (TMEJ). Thus, Pol{theta} is synthetically lethal with BRCA1/2 and other HR factors, underscoring its potential as a therapeutic target in HR-deficient cancers. However, the molecular mechanisms governing Pol{theta}-mediated MMEJ remain poorly understood. Here we present a series of cryo-electron microscopy structures of the Pol{theta} helicase domain (Pol{theta}-hel) in complex with DNA containing 3'-overhang. The structures reveal the sequential conformations adopted by Pol{theta}-hel during the critical phases of DNA binding, microhomology searching, and microhomology annealing. The stepwise conformational changes within the Pol{theta}-hel subdomains and its functional dimeric state are pivotal for aligning the 3'-overhangs, facilitating the microhomology search and subsequent annealing necessary for DSB repair via MMEJ. Our findings illustrate the essential molecular switches within Pol{theta}-hel that orchestrate the MMEJ process in DSB repair, laying the groundwork for the development of targeted therapies against the Pol{theta}-hel.

molecular biology↗

bioRxiv2022

Tail-tape-fused virion and non-virion RNA polymerases of a thermophilic virus with an extremely long tail

Thermus thermophilus bacteriophage P23-45 encodes a giant 5,002-residue tail tape measure protein (TMP)1 that defines the length of its extraordinarily long 800 nm tail2,3. We found that the N-terminal portion of P23-45 TMP is an unusual RNA polymerase (RNAP) homologous to cellular and viral two-barrel RNAPs. The TMP-fused virion RNAP transcribes pre-early phage genes, including a gene that encodes another, non-virion RNAP, that transcribes early and some middle phage genes. We determined the crystal structures of both P23-45 RNAPs. The non-virion RNAP has a crab claw-like architecture similar to previously reported two-barrel RNAPs. The virion RNAP adopts a unique flat structure without a clamp, which likely reflects the requirement for its extrusion through the narrow channel in the phage tail for delivery into the cell. Structure and sequence comparisons of the P23-45 RNAPs with other phage and cellular RNAPs suggest that, despite the extensive functional differences, the two P23-45 RNAPs originate from an ancient gene duplication in an ancestral phage. Our findings demonstrate remarkable adaptability of two-barrel RNAPs that can be attained within a single virus species.

molecular biology↗

bioRxiv2021

tRNA anticodon cleavage by target-activated CRISPR-Cas13a effector

Type VI CRISPR-Cas systems are the only CRISPR variety that cleaves exclusively RNA1,2. In addition to the CRISPR RNA (crRNA)-guided, sequence-specific binding and cleavage of target RNAs, such as phage transcripts, the type VI effector, Cas13, causes collateral RNA cleavage, which induces bacterial cell dormancy, thus protecting the host population from phage spread3,4. We show here that the principal form of collateral RNA degradation elicited by Cas13a protein from Leptotrichia shahii upon target RNA recognition is the cleavage of anticodons of multiple tRNA species, primarily those with anticodons containing uridines. This tRNA cleavage is necessary and sufficient for bacterial dormancy induction by Cas13a. In addition, Cas13a activates the RNases of bacterial toxin-antitoxin modules, thus indirectly causing mRNA and rRNA cleavage, which could provide a back-up defense mechanism. The identified mode of action of Cas13a resembles that of bacterial anticodon nucleases involved in antiphage defense5, which is compatible with the hypothesis that type VI effectors evolved from an abortive infection module6,7 encompassing an anticodon nuclease.

microbiology↗

bioRxiv2020

Structure and function of virion RNA polymerase of crAss-like phage

CrAss-like phages are a recently described family-level group of viruses that includes the most abundant virus in the human gut1,2. Genomes of all crAss-like phages encode a large virion-packaged protein2,3 that contains a DFDxD sequence motif, which forms the catalytic site in cellular multisubunit RNA polymerases (RNAPs)4. Using Cellulophaga baltica crAss-like phage phi14:2 as a model system, we show that this protein is a novel DNA-dependent RNAP that is translocated into the host cell along with the phage DNA and transcribes early phage genes. We determined the crystal structure of this 2,180-residue enzyme in a self-inhibited, likely pre-virion-packaged state. This conformation is attained with the help of a Cleft-blocking domain that interacts with the active site motif and occupies the RNA-DNA hybrid binding grove. Structurally, phi14:2 RNAP is most similar to eukaryotic RNAPs involved in RNA interference5,6, although most of phi14:2 RNAP structure (nearly 1,600 residues) maps to a new region of protein folding space. Considering the structural similarity, we propose that eukaryal RNA interference polymerases take their origin in a phage, which parallels the emergence of the mitochondrial transcription apparatus7.

molecular biology↗