Biology subjects

Bochtler, M.

Publications and source records attributed to Bochtler, M..

8 recordsLinked to original sources

bioRxiv2026

Csx15 is a broad specificity ring nuclease bridging CRISPR-Cas and CBASS cyclic nucleotide signaling systems

Prokaryotic antiviral type III CRISPR-Cas and CBASS systems use cyclic nucleotides as second messengers to activate diverse effector proteins. Whereas CBASS signaling typically triggers abortive infection, type III CRISPR-Cas signaling is tightly regulated through controlled signal production and degradation. Here, we define the molecular mechanism of the CRISPR-associated Csx15 protein family and demonstrate that Csx15 functions as a broad specificity ring nuclease capable of degrading cyclic nucleotide signals from both CRISPR-Cas and CBASS pathways. Structural and biochemical analyses reveal that Csx15 adopts diverse oligomeric states and undergoes ligand-induced conformational transitions that promote filament assembly required for catalytic activity, analogous to filament-forming SAVED-domain effectors. Finally, we provide evidence that Csx15 is a close relative of SAVED domains suggesting that Csx15-like proteins may have also contributed to the evolution of CBASS cyclic nucleotide signaling.

biochemistry↗

bioRxiv2023

Prokaryotic winged helix domains as dsDNA adenine methylation sensors

Winged helix (wH) domains, also termed winged helix-turn-helix (wHTH) domains, are widespread in all kingdoms of life, and have diverse roles. In the context of DNA binding and DNA modification sensing, some eukaryotic wH domains are known as sensors of non-methylated CpG. In contrast, the prokaryotic wH domains in DpnI and phi.HhiV4I act as sensors of adenine methylation in 6mApT (6mA = N6mA) context. DNA binding modes and interactions with the probed dinucleotide are vastly different in the two cases. Here, we show that the role of the wH domain as a sensor of adenine methylation is widespread in prokaryotes. We present previously uncharacterized examples of PD-(D/E)XK--wH (FcyTI, Psp4BI), PUA--wH--HNH (HtuIII, Hsa13891I), wH--GIY-YIG (Ahi29725I, Apa233I) and PLD--wH (Aba4572I, CbaI) fusion endonucleases that sense adenine methylation in the Dam G6mATC, and possibly other, slightly more relaxed contexts. Representatives of the wH domain endonuclease fusion families with the exception of the PLD--wH family could be purified, and an in vitro preference for adenine methylation in the Dam context could be demonstrated. Like most other MDREs, the new fusion endonucleases except those in the PD-(D/E)XK--wH family cleave close to, but outside the recognition sequence. Taken together, our data illustrate the widespread combinatorial use of prokaryotic wH domains as adenine methylation sensors.

molecular biology↗

bioRxiv2023

Modification of DNA by a viral enzyme and charged tRNA

Bacteriophage enzymes synthesize varied and complex DNA hypermodifications. The enzyme encoded by the phage Mu gene mom is necessary for post-replicative carbamoylmethyl addition to the exocyclic amine of deoxyadenosine in DNA during the lytic phase of the viral life-cycle. The molecular details of this modification reaction, including the molecular origins of the modification itself, have long eluded understanding. Here, we demonstrate that Mom co-opts the translational machinery of the host by harvesting activated glycine from charged tRNAGly to hypermodify adenine. Based on this insight, we report the first in vitro reconstitution of the Mu hypermodification from purified components. Using isotope labeling, we demonstrate that the carbamoyl nitrogen of the Mom modification is derived from the N6 of adenine, indicating an on-base rearrangement of the N6 aminoacylation product, possibly via a cyclic intermediate. Informed by the X-ray crystal structure of Mom, we have probed the location of the active site, identified a novel insertion, and established substrate specificities of the Mom enzyme.

biochemistry↗

bioRxiv2022

Bicarbonate Activation of Monomeric Photosystem II-PsbS/Psb27 Complex

In thylakoid membranes, Photosystem II monomers from the stromal lamellae contain the subunits PsbS and Psb27 (PSIIm-S/27), while Photosystem II monomers from granal regions (PSIIm) lack these subunits. Here, we have isolated and characterised these two types of Photosystem II complexes. The PSIIm-S/27 showed enhanced fluorescence, the near-absence of oxygen evolution, as well as limited and slow electron transfer from QA to QB compared to the near-normal activities in the granal PSIIm. However, when bicarbonate was added to the PSIIm-S/27, water splitting and QA to QB electron transfer rates were comparable to those in granal PSIIm. The findings suggest that the binding of PsbS and/or Psb27 inhibits forward electron transfer and lowers the binding affinity for the bicarbonate. This can be rationalized in terms of the recently discovered photoprotection role played by bicarbonate binding via the redox tuning of the QA/QA*- couple, which controls the charge recombination route, and this limits chlorophyll triplet mediated 1O2 formation (Brinkert K et al. (2016) Proc Natl Acad Sci U S A. 113(43):12144-12149). These findings suggest that PSIIm-S/27 is an intermediate in the assembly of PSII in which PsbS and/or Psb27 restrict PSII activity while in transit, by using a bicarbonate-mediated switch and protective mechanism. One sentence summaryA photosystem II monomer with PsbS and Psb27 as additional subunits, is inactive as isolated but activated by bicarbonate, and is attributed to be a late-stage intermediate in photoassembly.

plant biology↗

bioRxiv2021

Pronounced sequence specificity of the TET enzyme catalytic domain guides its cellular function

TET (ten-eleven translocation) enzymes catalyze the oxidation of 5-methylcytosine bases in DNA, thus driving active and passive DNA demethylation. Here, we report that the catalytic cores of mammalian TET enzymes favor CpGs embedded within bHLH and bZIP transcription factor binding sites, with 250-fold preference in vitro. Crystal structures and molecular dynamics calculations show that sequence preference is caused by intra-substrate interactions and CpG flanking sequence indirectly affecting enzyme conformation. TET sequence preferences are physiologically relevant as they explain the rates of DNA demethylation in TET-rescue experiments in culture and in vivo within the zygote and germline. Most and least favorable TET motifs represent DNA sites that are bound by methylation-sensitive immediate-early transcription factors and OCT4, respectively, illuminating TET function in transcriptional responses and pluripotency support. One-Sentence SummaryThe catalytic domains of the enzymes that facilitate passive and drive active DNA demethylation have intrinsic sequence preferences that target DNA demethylation to bHLH and bZIP transcription factor binding sites.

molecular biology↗

bioRxiv2021

Clustered PHD domains in mixed lineage leukaemia proteins are attracted by acetylation-rich active promoters and enhancers

Histone lysine-specfic methyltransferase 2 (KMT2A-D) proteins, alternatively called mixed lineage leukaemia (MLL1-4) proteins, mediate positive transcriptional memory. As the catalytic subunits of human COMPASS-like complexes, they methylate H3K4 at promoters and enhancers. KMT2A-D contain understudied highly conserved triplets and a quartet of plant homeodomains (PHDs). Here, we show that all clustered PHDs localise to the well-defined loci of H3K4me3 and H3 acetylation-rich active promoters and enhancers. Surprisingly, we observe little difference in binding pattern between PHDs from promoter-specific KMT2A-B and enhancer-specific KMT2C-D. Fusion of the KMT2A CXXC domain to the PHDs drastically enhances their preference for promoters over enhancers. Hence, the presence of CXXC domains in KMT2A-B, but not KMT2C-D, may explain the promoter/enhancer preferences of the full-length proteins. Importantly, targets of PHDs overlap with KMT2A targets and are enriched in genes involved in the cancer pathways. We also observe that PHDs of KMT2A-D are mutated in cancer, especially within conserved folding motifs (Cys4HisCys2Cys/His), which cause a domain loss-of-function. Taken together, our data suggests that PHDs of KMT2A-D guide the full-length proteins to active promoters and enhancers, and thus play a role in positive transcriptional memory. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/462366v2_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1d62842org.highwire.dtl.DTLVardef@ed1041org.highwire.dtl.DTLVardef@1c62b43org.highwire.dtl.DTLVardef@1426bcd_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2021

Adar-mediated A-to-I editing is required for establishment of embryonic body axes in zebrafish

Adenosine deaminases (ADARs) catalyze the deamination of adenosine to inosine, also known as A-to-I editing, in RNA. Although A-to-I editing occurs widely across animals, and is well studied, new biological roles are still being discovered. Here, we study the role of A-to-I editing in early zebrafish development. We demonstrate that Adar, the zebrafish orthologue of mammalian ADAR1, is essential for establishing the antero-posterior and dorso-ventral axes and patterning. Genome-wide editing discovery revealed pervasive editing in maternal and the earliest zygotic transcripts, the majority of which occurred in the 3-UTR. Interestingly, transcripts implicated in gastrulation as well as dorso-ventral and antero-posterior patterning were found to contain multiple editing sites. Adar knockdown or overexpression affected gene expression and global editing patterns at 12 hpf, but not earlier. Our study established that RNA editing by Adar is necessary for the earliest steps of embryonic patterning along the zebrafish antero-posterior and dorso-ventral axes.

developmental biology↗

bioRxiv2017

Targeted RNA knockdown by crRNA guided Csm in zebrafish

RNA interference (RNAi) is a powerful experimental tool for RNA knockdown, but not all organisms are amenable. Here, we provide a \"proof of principle\" demonstration that CRISPR endoribonucleases can be used for programmable mRNA transcript degradation. Using zebrafish as the animal model and Csm(crRNA) complexes as the CRISPR endoribonucleases, we have targeted a transgenic EGFP transcript expressed from a variety of promoters. A drastic decrease of fluorescence was achieved in germ cells of the vasa:EGFP line. Weaker effects were also seen in fish lines that express EGFP zygotically. Knockdown was statistically significant in cmcl2:EGFP and fli1:EGFP zebrafish lines at 1 day post fertilization (dpf), but reduced to background levels at 2 dpf. The nkx2.5:EGFP fish line was least susceptible to Csm mediated EGFP knockdown. We conclude that at the present stage, Csm mediated knockdown is already efficient for maternal transcripts, and may compare favorably with morpholinos for such targets in zebrafish.

genetics↗