TOPOVIBL-REC114 interaction regulates meiotic DNA double-strand breaks

Meiosis requires the formation of programmed DNA double strand breaks (DSBs), essential for fertility and for generating genetic diversity. In male and female meiotic cells, DSBs are induced by the catalytic activity of the TOPOVIL complex formed by SPO11 and TOPOVIBL. To ensure genomic integrity, DNA cleavage activity is tightly regulated, and several accessory factors (REC114, MEI4, IHO1, and MEI1) are needed for DSB formation in mice. How and when these proteins act is not understood. Here, we show that REC114 is a direct partner of TOPOVIBL, and identified their conserved interacting domains by structural analysis. We then analysed the role of this interaction by monitoring meiotic DSBs in female and male mice carrying point mutations in TOPOVIBL that decrease or disrupt its binding to REC114. In these mutants, DSB activity was strongly reduced genome-wide in oocytes, but only in sub-telomeric regions in spermatocytes. In addition, in mutant spermatocytes, DSB activity was delayed in autosomes. These results provide evidence that REC114 is a key member of the TOPOVIL catalytic complex, and that the REC114/TOPOVIBL interaction ensures the efficiency and timing of DSB activity by integrating specific chromosomal features.

Spectral analysis of radical metalloenzyme using EPR in Hydrolases

One of the categories of hydrolase is acid phosphatase which is isolated from sweet potato (spAP). It contains metal ions such as Zn, Mn, Fe etc. with molecular weight 110kDa. An aqueous epr spectrum in buffer (pH 5.6) exhibits class II mixed valence nature of Fe+3 together with Fe+2, Mn+2, Zn+2 and Cu+2 metallobiosites. It indicates reduced form of enzyme with gMn(II)= 2.0084 and grad= 2.0041 signals with radical signal. The localized electron interaction with 55Mn+2 nucleus is evidenced by six-hyperfine splitted spin allowed transitions flanked by ten satellite lines. Allowed and forbidden doublets are computed with A (55Mn) coupling constant~96 G and Zfs parameter D=0.029 cm-1. The spin integration of Tyrradical signal is shown in lyophilized sample spectrum (1.029 x 1015 spins /gm). It also shows DNA cleavage activity with pUC 19 plasmid DNA.

The synthesis and evaluation of the antitumor and antibacterial activity of two novel oxovanadium complexes

Two novel oxovanadium(IV) complexes ([VO(hntdtsc)(BPIP)] and [VO(hntdtsc)(MOPIP)] (hntdtsc = 2-hydroxy-1-naphthaldehydethiosemicarbazone, BPIP = 2-(4-bromophenyl)-imidazo[4,5- f]-1,10-phenanthroline, MOPIP = 2-(4-methoxyphenyl)-imidazo[4,5- f]1,10-phenanthroline), are synthesized and characterized. Subsequently, the Methyl Thiazolyl Tetrazolium (MTT) assay is used to investigate the antitumor activity of the ligand and two complexes in vitro.The results indicate that both complexes could significantly inhibit selected tumor cells (SH-SY5Y, MCF-7, and SK-N-SH). In addition, the antibacterial activity of VO(hntdtsc)(BPIP) against Staphylococcus aureus is further investigated. Interestingly, VO(hntdtsc)(BPIP) can efficiently attenuate S. aureus growth and abrogate α-hemolysin secretion and biofilm formation. The plasmid DNA cleavage activity of both complexes is also investigated. The results suggest that supercoiled plasmid DNA is efficiently cleaved after treatment with each complex, which might contribute to the biological activity of these oxovanadium(IV) complexes.

Structural and mechanistic insight into CRISPR-Cas9 inhibition by anti-CRISPR protein AcrIIC4

Phages, plasmids, and other mobile genetic elements express inhibitors of CRISPR-Cas immune systems, known as anti-CRISPR proteins, to protect themselves from targeted destruction. These anti-CRISPRs have been shown to function through very diverse mechanisms. In this work we investigate the activity of an anti-CRISPR isolated from a prophage in Haemophilus parainfluenzae that blocks CRISPR-Cas9 DNA cleavage activity. We determine the three-dimensional crystal struture of AcrIIC4 and show that it binds to the Cas9 Recognition Domain. This binding does not prevent the Cas9-anti-CRISPR complex from interacting with target DNA but does inhibit DNA cleavage. AcrIIC4 likely acts by blocking the conformational changes that allow the HNH and RuvC endonuclease domains to contact the DNA sites to be nicked.

RNA-CLAMP Enables Photo-activated Control of CRISPR-Cas9 Gene Editing by Site-specific Intramolecular Cross-linking of the sgRNA

Here we introduce RNA-CLAMP, a technology which enables site-specific and enzymatic cross-linking (clamping) of two selected stem loops within an RNA of interest. Intramolecular clamping of the RNA can disrupt normal RNA function, whereas subsequent photo-cleavage of the crosslinker restores activity. We applied the RNA-CLAMP technique to the single guide RNA of the CRISPR-Cas9 gene editing system. By clamping two stem loops of the single-guide RNA (sgRNA) with a photo-cleavable cross-linker, gene editing was completely silenced. Visible light irradiation cleaved the crosslinker and restored gene editing with high spatiotemporal resolution. Furthermore, by designing two photo-cleavable linkers which are responsive to different wavelength of lights, we achieved multiplexed photo-activation of gene editing in mammalian cells. Notably, although the Cas9-sgRNA RNP is not capable of DNA cleavage activity upon clamping, it maintained the capability to bind to the target DNA. The RNA-CLAMP enabled photo-activated CRISPR-Cas9 gene editing platform offers clean background, free choice of activation wavelength and multiplexing capability. We believe that this technology to precisely and rapidly control gene editing will serve as a versatile tool in the future development of stimuli responsive gene editing technologies. Beyond gene editing, RNA-CLAMP provides a site-specific tool for manipulating the internal structure of functional RNAs.

A nucleotide-sensing endonuclease from the Gabija bacterial defense system

The arms race between bacteria and phages has led to the development of exquisite bacterial defense systems including a number of uncharacterized systems distinct from the well-known restriction-modification and CRISPR/Cas systems. Here, we report functional analyses of the GajA protein from the newly predicted Gabija system. The GajA protein is revealed as a sequence-specific DNA nicking endonuclease unique in that its activity is strictly regulated by nucleotide concentration. NTP and dNTP at physiological concentrations can fully inhibit the robust DNA cleavage activity of GajA. Interestingly, the nucleotide inhibition is mediated by an ATPase-like domain, which usually hydrolyzes ATP to stimulate the DNA cleavage when associated with other nucleases. These features suggest a mechanism of the Gabija defense in which an endonuclease activity is suppressed under normal conditions, while it is activated by the depletion of NTP and dNTP upon the replication and transcription of invading phages. This work highlights a concise strategy to utilize a DNA nicking endonuclease for phage resistance via nucleotide regulation.

A YoeB toxin cleaves both RNA and DNA

Type II toxin-antitoxin systems contain a toxin protein, which mediates diverse interactions within the bacterial cell when it is not bound by its cognate antitoxin protein. These toxins provide a rich source of evolutionarily-conserved tertiary folds that mediate diverse catalytic reactions. These properties make toxins of interest in biotechnology applications, and studies of the catalytic mechanisms continue to provide surprises. In the current work, our studies on a YoeB family toxin from Agrobacterium tumefaciens have revealed a conserved ribosome-independent non-specific nuclease activity. We have quantified the RNA and DNA cleavage activity, revealing they have essentially equivalent dose-dependence while differing in requirements for divalent cations and pH sensitivity. The DNA cleavage activity is as a nickase for any topology of double-stranded DNA, as well as cleaving single-stranded DNA. AtYoeB is able to bind to double-stranded DNA with mid-micromolar affinity. Comparison of the ribosome-dependent and -independent reactions demonstrates an approximate tenfold efficiency imparted by the ribosome. This demonstrates YoeB toxins can act as non-specific nucleases, cleaving both RNA and DNA, in the absence of being bound within the ribosome.