Biology subjects

Gulkis, M.

Publications and source records attributed to Gulkis, M..

3 recordsLinked to original sources

bioRxiv2026

AAV2 Crosslinks Actin Filaments: Implications for AAV Gene Therapy Vector Design

Adeno-associated virus (AAV) capsids are important gene therapy vectors, allowing for the one-time treatment of monogenetic disorders, with durable gene expression lasting for years. However, despite the clinical success of AAV usage, low transduction efficiencies require high dosage to achieve therapeutic efficacy, resulting in prohibitive costs and rare, but life-threatening immune responses. One key knowledge gap is how the capsids traffic to the nucleus following endocytosis. Here, we identify a direct interaction between AAV2 and actin filaments. Our results show that AAV2 bundles multiple actin filaments in a highly periodic hexagonal lattice. Therefore, we propose that AAV2 is the founding member of a new class of actin binding proteins, the actin bundling viruses (ABVs). Using cryogenic electron microscopy, we determined the structure of AAV2-actin filaments, identified the interaction interfaces, and engineered capsid variants that no longer bundle actin. Together, our results suggest that the AAV2-actin interaction may be responsible for trapping capsids and mediating peri-nuclear accumulation. Overall, this interaction expands the current understanding of AAV2 biology and offers new directions for capsid engineering.

biochemistry↗

bioRxiv2023

Structures of LIG1 active site mutants reveal the importance of DNA end rigidity for mismatch discrimination

ATP-dependent DNA ligases catalyze phosphodiester bond formation in the conserved three-step chemical reaction of nick sealing. Human DNA ligase I (LIG1) finalizes almost all DNA repair pathways following DNA polymerase-mediated nucleotide insertion. We previously reported that LIG1 discriminates mismatches depending on the architecture of the 3-terminus at a nick, however the contribution of conserved active site residues to faithful ligation remains unknown. Here, we comprehensively dissect the nick DNA substrate specificity of LIG1 active site mutants carrying Ala(A) and Leu(L) substitutions at Phe(F)635 and Phe(F)F872 residues and show completely abolished ligation of nick DNA substrates with all 12 non-canonical mismatches. LIG1EE/AA structures of F635A and F872A mutants in complex with nick DNA containing A:C and G:T mismatches demonstrate the importance of DNA end rigidity, as well as uncover a shift in a flexible loop near 5-end of the nick, which causes an increased barrier to adenylate transfer from LIG1 to the 5-end of the nick. Furthermore, LIG1EE/AA/8oxoG:A structures of both mutants demonstrated that F635 and F872 play critical roles during steps 1 or 2 of the ligation reaction depending on the position of the active site residue near the DNA ends. Overall, our study contributes towards a better understanding of the substrate discrimination mechanism of LIG1 against mutagenic repair intermediates with mismatched or damaged ends and reveals the importance of conserved ligase active site residues to maintain ligation fidelity.

biochemistry↗

bioRxiv2022

Structures of LIG1 uncover a lack of sugar discrimination against a ribonucleotide at the 3'-end of nick DNA

Human DNA ligase I (LIG1) is the main replicative ligase that seals Okazaki fragments and finalizes DNA repair pathways by joining canonical 3-OH and 5-P ends of the nick DNA in a three-step ligation reaction. Ribonucleotides can be misincorporated by DNA polymerases resulting in a nick with 3-ribonucleotide while RNase H2 mediated cleavage leaves a nick harboring 5-ribonucleotide during ribonucleotide excision repair. However, how LIG1 surveils DNA ends with a "wrong" sugar at atomic resolution is unknown. Here, we determine X-ray structures of LIG1/nick DNA complexes with 3- or 5-single ribonucleotide during different stages of the ligation reaction. Our LIG1/5-rG:C structure reveals a global conformational change, which discriminates against 5-RNA/DNA junctions at the initial step when the ligase-AMP intermediate is formed. Furthermore, we capture LIG1/3-RNA-DNA heteroduplexes that are tolerated at the active site where AMP is transferred to nick DNA (step 2) and final phosphodiester bond formation occurs (step 3). Finally, we demonstrate the mutagenic and defective ligation of the nick DNA with 3- and 5-ribonucleotide, respectively, in vitro. Together, these results uncover how LIG1 encounters ribonucleotides embedded into genome during nuclear replication and the last step of DNA repair pathways to maintain genome integrity.

biochemistry↗