Droge, P. | bioRxiv Science

bioRxiv2025

Cryo-EM structures of anti Z-DNA antibodies in complex with antigen reveal distinct recognition modes of a left-handed geometry

Double-stranded nucleic acids can undergo transitions from canonical B/A-forms to alternate left-handed Z-DNA/Z-RNA (Z-NAs). Z-NAs are implicated in processes such as neuroinflammation in Alzheimers disease, Lupus Erythematosus, microbial biofilms, and type I interferon-mediated human pathologies. Since endogenous Z-NA sensors like the Z domain can induce B-to-Z transitions, monoclonal antibodies (mAbs) Z-D11 and Z22 have been regarded as conformation-specific tools to confirm Z-NA in situ, although high-resolution structural information is missing. Here, we employed single-particle cryo-electron microscopy to solve structures of Z-D11 and Z22 bound to synthetic d(CG)6 12mer Z-DNA duplex. Both mAbs form filamentous trimers around the Z-DNA axis, further stabilized by Fab-Fab interactions. The mAbs achieve specificity through extensive contacts to both Z-form backbone strands and the exposed guanine/cytosine bases in the major groove. This mode of recognition is dictated by shape complementarity rather than sequence specificity, sensing the alternating syn/anti backbone torsions and the phosphate zig-zag geometry unique to Z-DNA. Our data also suggest that these mAbs are not inducing B-to-Z transitions under normal physiological conditions. Finally, comparison to other double-stranded NA-binding mAbs defines a similar structural logic adapted to different helical geometry recognition patterns, thus providing a framework for engineering highly specific nucleic acid probes.

biochemistry↗

bioRxiv2023

Expanding the DNA editing toolbox: novel lambda integrase variants targeting microalgal and human genome sequences.

Recombinase enzymes are extremely efficient at integrating very large DNA fragments into target genomes. However, intrinsic sequence specificities curtail their use to DNA sequences with sufficient homology to endogenous target motifs. Extensive engineering is therefore required to broaden applicability and robustness. Here, we describe the directed evolution of novel lambda integrase variants capable of editing exogenous target sequences identified in the diatom Phaeodactylum tricornutum and the algae Nannochloropsis oceanica. These microorganisms hold great promise as conduits for green biomanufacturing and carbon sequestration. The evolved enzyme variants show >1000-fold switch in specificity towards the non-natural target sites when assayed in vitro. A single-copy target motif in the human genome with homology to the Nannochloropsis oceanica site can also be efficiently targeted using an engineered integrase, both in vitro and in human cells. The developed integrase variants represent useful additions to the DNA editing toolbox, with particular application for targeted genomic insertion of large DNA cargos.

bioengineering↗

bioRxiv2019

The chromatin structuring protein HMGA2 influences human subtelomere stability and cancer chemosensitivity

The transient build-up of DNA supercoiling during the translocation of replication forks threatens genome stability and is controlled by DNA topoisomerases (TOPs). This crucial process has been exploited with TOP poisons for cancer chemotherapy. However, pinpointing cellular determinants of the best clinical response to TOP poisons still remains enigmatic. Here, we present an integrated approach and demonstrate that endogenous and exogenous expression of the oncofetal high-mobility group AT-hook 2 (HMGA2) protein exhibited broad protection against the formation of hydroxyurea-induced DNA breaks in various cancer cells, thus corroborating our previously proposed model in which HMGA2 functions as a replication fork chaperone that forms a protective DNA scaffold at or close to stalled replication forks. We now further demonstrate that high levels of HMGA2 also protected cancer cells against DNA breaks triggered by the clinically important TOP1 poison irinotecan. This protection is most likely due to the recently identified DNA supercoil constraining function of HMGA2 in combination with exclusion of TOP1 from binding to supercoiled substrate DNA. In contrast, low to moderate HMGA2 protein levels surprisingly potentiated the formation of irinotecan-induced genotoxic covalent TOP1-DNA cleavage complexes. Our data from cell-based and several in vitro assays indicate that, mechanistically, this potentiating role involves enhanced drug-target interactions mediated by HMGA2 in ternary complexes with supercoiled DNA. Subtelomeric regions were found to be extraordinarily vulnerable to these genotoxic challenges induced by TOP1 poisoning, pointing at strong DNA topological barriers located at human telomeres. These findings were corroborated by an increased irinotecan sensitivity of patient-derived xenografts of colorectal cancers exhibiting low to moderate HMGA2 levels. Collectively, we uncovered a therapeutically important control mechanism of transient changes in chromosomal DNA topology that ultimately leads to enhanced human subtelomere stability. Author SummaryDNA replication fork stability in rapidly dividing cancer cells is of utmost importance for the maintenance of genome stability and cancer cell viability. Cancer cells efficiently prevent fork collapse into lethal double strand breaks as a first line of defense during replication stress, but the corresponding protective mechanisms often remain elusive. Uncontrolled high levels of DNA supercoiling that are generally regulated by topoisomerases can cause replication stress and are major threats to fork stability. Using a multidisciplinary approach, we identified a possible regulatory mechanism of replication stress, which appears to involve mitigating the consequences of DNA topological changes by the oncofetal replication fork chaperone HMGA2. Our work provides mechanistic insights into the control of DNA damage triggered by clinically important anti-cancer drugs, which is mediated by the replication fork chaperone HMGA2. We thereby also identify HMGA2 expression as a predictive therapeutic marker, which could allow clinicians to take informed decisions to prevent tumor recurrence and improve survival.

cancer biology↗