Structural dissection of sequence recognition and catalytic mechanism of human LINE-1 endonuclease

Long interspersed nuclear element-1 (L1) is an autonomous non-LTR retrotransposon comprising ∼20% of the human genome. L1 self-propagation causes genomic instability and is strongly associated with aging, cancer and other diseases. The endonuclease domain of L1’s ORFp2 protein (L1-EN) initiates de novo L1 integration by nicking the consensus sequence 5′-TTTTT/AA-3′. In contrast, related nucleases including structurally conserved apurinic/apyrimidinic endonuclease 1 (APE1) are non-sequence specific. To investigate mechanisms underlying sequence recognition and catalysis by L1-EN, we solved crystal structures of L1-EN complexed with DNA substrates. This showed that conformational properties of the preferred sequence drive L1-EN’s sequence-specificity and catalysis. Unlike APE1, L1-EN does not bend the DNA helix, but rather causes ‘compression’ near the cleavage site. This provides multiple advantages for L1-EN’s role in retrotransposition including facilitating use of the nicked poly-T DNA strand as a primer for reverse transcription. We also observed two alternative conformations of the scissile bond phosphate, which allowed us to model distinct conformations for a nucleophilic attack and a transition state that are likely applicable to the entire family of nucleases. This work adds to our mechanistic understanding of L1-EN and related nucleases and should facilitate development of L1-EN inhibitors as potential anticancer and antiaging therapeutics.

Coordination of phage genome degradation versus host genome protection by a bifunctional restriction-modification enzyme visualized by CryoEM

ABSTRACTRestriction enzymes that combine DNA methylation and cleavage activities into a single polypeptide or protein assemblage and that modify just one DNA strand for host protection are capable of more efficient adaptation towards novel target sites. However, they must solve the problem of discrimination between newly replicated and unmodified host sites (needing methylation) and invasive foreign site (needing to lead to cleavage). One solution to this problem might be that the activity that occurs at any given site is dictated by the oligomeric state of the bound enzyme. Methylation requires just a single bound site and is relatively slow, while cleavage requires that multiple unmethylated target sites (often found in incoming, foreign DNA) be brought together into an enzyme-DNA complex to license rapid cleavage. To validate and visualize the basis for such a mechanism, we have determined the catalytic behavior of a bifunctional Type IIL restriction-modification (‘RM’) enzyme (DrdV) and determined its high-resolution structure at several different stages of assembly and coordination with multiple bound DNA targets using CryoEM. The structures demonstrate a mechanism of cleavage by which an initial dimer is formed between two DNA-bound enzyme molecules, positioning the single endonuclease domain from each enzyme against the other’s DNA and requiring further oligomerization through differing protein-protein contacts of additional DNA-bound enzyme molecules to enable cleavage. The analysis explains how endonuclease activity is licensed by the presence of multiple target-containing DNA duplexes and provides a clear view of the assembly through 3D space of a DNA-bound RM enzyme ‘synapse’ that leads to rapid cleavage of foreign DNA.

Inteins in Science: Evolution to Application

Inteins are mobile genetic elements that apply standard enzymatic strategies to excise themselves post-translationally from the precursor protein via protein splicing. Since their discovery in the 1990s, recent advances in intein technology allow for them to be implemented as a modern biotechnological contrivance. Radical improvement in the structure and catalytic framework of cis- and trans-splicing inteins devised the development of engineered inteins that contribute to various efficient downstream techniques. Previous literature indicates that implementation of intein-mediated splicing has been extended to in vivo systems. Besides, the homing endonuclease domain also acts as a versatile biotechnological tool involving genetic manipulation and control of monogenic diseases. This review orients the understanding of inteins by sequentially studying the distribution and evolution pattern of intein, thereby highlighting a role in genetic mobility. Further, we include an in-depth summary of specific applications branching from protein purification using self-cleaving tags to protein modification, post-translational processing and labelling, followed by the development of intein-based biosensors. These engineered inteins offer a disruptive approach towards research avenues like biomaterial construction, metabolic engineering and synthetic biology. Therefore, this linear perspective allows for a more comprehensive understanding of intein function and its diverse applications.

Somatic CAG expansion in Huntington’s disease is dependent on the MLH3 endonuclease domain, which can be excluded via MLH3 splice redirection to suppress expansion

Somatic expansion of the CAG repeat tract that causes Huntington’s disease (HD) is thought to contribute to the rate of disease pathogenesis. Therefore, factors influencing repeat expansion are potential therapeutic targets. Genes in the DNA mismatch repair pathway are critical drivers of somatic expansion in HD mouse models. Here, we have tested, using genetic and pharmacological approaches, the role of the endonuclease domain of the mismatch repair protein MLH3 in somatic CAG expansion in HD mice and patient cells. A point mutation in the MLH3 endonuclease domain completely eliminated CAG expansion in the brain and peripheral tissues of a HD knock-in mouse model (HttQ111). To test whether the MLH3 endonuclease could be manipulated pharmacologically, we delivered splice switching oligonucleotides in mice to redirect Mlh3 splicing to exclude the endonuclease domain. Splice redirection to an isoform lacking the endonuclease domain was associated with reduced CAG expansion. Finally, CAG expansion in HD patient-derived primary fibroblasts was also significantly reduced by redirecting MLH3 splicing to the endogenous endonuclease domain-lacking isoform. These data indicate the potential of targeting the MLH3 endonuclease domain to slow somatic CAG repeat expansion in HD, a therapeutic strategy that may be applicable across multiple repeat expansion disorders.

Enhanced efficacy of endonuclease inhibitor baloxavir acid against orthobunyaviruses when used in combination with ribavirin

Objectives Baloxavir acid is an endonuclease inhibitor approved for use against influenza. We evaluated whether this compound also targets the endonuclease domain of orthobunyaviruses and therefore could potentially be used against orthobunyavirus infections. Methods We performed a thermal shift assay and a fluorescence resonance energy transfer (FRET)-based nuclease monitoring assay using the La Crosse virus (LACV) endonuclease and baloxavir acid to prove their interaction and identify an inhibitory effect. Their interaction was further studied in a docking simulation using Glide SP. We show that baloxavir acid inhibits the viral replication of Bunyamwera virus (BUNV)–mCherry in vitro using high-content imaging and virus yield assay. Lastly, we investigated the use of baloxavir acid in combination with ribavirin in vitro by implementing the Zero Interaction Potency response surface model. Results We show that baloxavir acid augments LACV enzyme’s melting temperature with ΔTm 9.5 ± 0.4°C and inhibited substrate cleavage with IC50 0.39 ± 0.03 μM. Moreover, our docking simulation suggests that baloxavir acid is able to establish an efficient binding with the LACV endonuclease. In the cell-based assay, we observed that baloxavir acid and ribavirin inhibited BUNV–mCherry with an EC50 of 0.7 ± 0.2 μM and 26.6 ± 8.9 μM, respectively. When used in combination, we found a maximum synergistic effect of 8.64. Conclusions The influenza endonuclease inhibitor baloxavir acid is able to bind to and interfere with the endonuclease domain of orthobunyaviruses and yields a more potent antiviral effect than ribavirin against BUNV–mCherry. The combination of both compounds results in a more potent antiviral effect, suggesting that these molecules could potentially be combined to treat orthobunyavirus-infected patients.

Off-targets in ZFN/TALEN would be potentially very large, similar to CRISPR, if monomeric FokI has endonuclease activity

In a seminal study in 1996 , Kim et, al [1] demonstrated that a fusion of the C-terminal endonuclease domain of the FokI protein to a single zinc finger protein induces DNA cuts (ZFNs). This also applied to transcription activator-like effector proteins (TALEN). Later studies suggested that FokI needs to dimerize in order to cleave, without ever repeating the simple experiment mentioned above. Dimerization might increase efficiency, but the single monomer of FokI contains the catalytic site to cleave. If the monomeric FokI cleaves, the off-target problems with ZFN/TALENS would be significant in a large genome (especially with mismatches), since each component (left and right ZFN/TALEN) are typically 18 nucleotides or less. Previously, I had shown an unreported off-target in hornless TALEN-edited cattle [2]. Here, I show multiple off-targets in an ZFN study that edited two loci (AAVS1 and IL2RG) in stem cells [3]. The plasmid integrates at a loci in the HBB gene, which was not edited, across multiple(11) samples, with significant number of reads corroborating this fact. This also highlights the problem of plasmid integration in such gene-therapies, including the bacterial nuclease, which ought to be unacceptable. I also provide information on pre-clinical studies using ZFN that are now in clinical trials. Thus, more studies are needed to demonstrate safety in ZFN/TALEN studies with respect to off-targets.

KHNYN is essential for the zinc finger antiviral protein (ZAP) to restrict HIV-1 containing clustered CpG dinucleotides

CpG dinucleotides are suppressed in most vertebrate RNA viruses, including HIV-1, and introducing CpGs into RNA virus genomes inhibits their replication. The zinc finger antiviral protein (ZAP) binds regions of viral RNA containing CpGs and targets them for degradation. ZAP does not have enzymatic activity and recruits other cellular proteins to inhibit viral replication. We found that KHNYN, a protein with no previously known function, interacts with ZAP. KHNYN overexpression selectively inhibits HIV-1 containing clustered CpG dinucleotides and this requires ZAP and its cofactor TRIM25. KHNYN requires both its KH-like domain and NYN endonuclease domain for antiviral activity. Crucially, depletion of KHNYN eliminated the deleterious effect of CpG dinucleotides on HIV-1 RNA abundance and infectious virus production and also enhanced the production of murine leukemia virus. Overall, we have identified KHNYN as a novel cofactor for ZAP to target CpG-containing retroviral RNA for degradation.