Biology subjects

Wenzel, S.

Publications and source records attributed to Wenzel, S..

5 recordsLinked to original sources

bioRxiv2021

Structure and RNA template requirements of Arabidopsis RNA-DEPENDENT RNA POLYMERASE 2

RNA-dependent RNA polymerases play essential roles in RNA-mediated gene silencing in eukaryotes. In Arabidopsis, RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) physically interacts with DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) and their activities are tightly coupled, with Pol IV transcriptional arrest or termination, involving the nontemplate DNA strand, somehow enabling RDR2 to engage Pol IV transcripts and generate double-stranded RNAs. The dsRNAs are then released from the Pol IV-RDR2 complex and diced into siRNAs that guide RNA-directed DNA methylation and silencing. Here we report the structure of full-length RDR2, at an overall resolution of 3.1 [A], determined by cryo-electron microscopy. The N-terminal region contains an RNA-recognition motif (RRM) adjacent to a positively charged channel that leads to a catalytic center with striking structural homology to the catalytic centers of multisubunit DNA-dependent RNA polymerases. We show that RDR2 initiates 1-2 nucleotides (nt) internal to the 3 ends of its templates and can transcribe the RNA of an RNA-DNA hybrid provided that 9 or more nucleotides at the RNAs 3 end is unpaired. Using a nucleic acid configuration that mimics the arrangement of RNA and DNA strands upon Pol IV transcriptional arrest, we show that displacement of the RNA 3 end occurs as the DNA template and non-template strands reanneal, enabling RDR2 transcription. These results suggest a model in which Pol IV arrest and backtracking displaces the RNA 3 end as the DNA strands reanneal, allowing RDR2 to engage the RNA and transcribe the second strand. SignificanceRDR2 is critical for siRNA-directed DNA methylation in Arabidopsis, functioning in physical association with DNA-dependent Pol IV to synthesize the second strands of double-stranded siRNA precursors. Basepairing between the DNA template strand transcribed by Pol IV and the nontemplate DNA strand is known to induce Pol IV arrest and Pol IV-RDR2 transcriptional coupling, but how this occurs is unknown. We report the structure of RDR2 and experimental evidence for how RDR2 engages its RNA templates and initiates transcription. RDR2 engages the ends of RNAs displaced from RNA-DNA hybrids, suggesting a model in which Pol IV arrest and backtracking, accompanied by DNA strand reannealing, extrudes the 3 end of the Pol IV transcript, allowing RNA engagement and second-strand synthesis.

molecular biology↗

bioRxiv2019

The Hrq1 helicase stimulates Pso2 translesion nuclease activity to promote DNA inter-strand crosslink repair

DNA inter-strand crosslink (ICL) repair requires a complicated network of DNA damage response pathways. Removal of these lesions is vital as they are physical barriers to essential DNA processes that require the separation of duplex DNA, such as replication and transcription. The Fanconi anemia (FA) pathway is the principle mechanism for ICL repair in metazoans and is coupled to replication. In Saccharomyces cerevisiae, a degenerate FA pathway is present, but ICLs are predominantly repaired by a pathway involving the Pso2 nuclease that is hypothesized to digest through the lesion to provide access for translesion polymerases. However, Pso2 lacks translesion nuclease activity in vitro, and mechanistic details of this pathway are lacking, especially relative to FA. We recently identified the Hrq1 helicase, a homolog of the disease-linked RECQL4, as a novel component of Pso2- mediated ICL repair. Here, we show that Hrq1 stimulates the Pso2 nuclease in a mechanism that requires Hrq1 catalytic activity. Importantly, Hrq1 also stimulates Pso2 translesion nuclease activity through a site- specific ICL in vitro. Stimulation of Pso2 nuclease activity is specific to eukaryotic RecQ4 subfamily helicases, and Hrq1 likely interacts with Pso2 through their N-terminal domains. These results advance our understanding of FA-independent ICL repair and establish a role for the RecQ4 helicases in the repair of these dangerous lesions.

biochemistry↗

bioRxiv2018

A practical method for efficient and optimal production of selenomethionine-labeled recombinant protein complexes in the insect cells

The use of Selenomethionine (SeMet) incorporated protein crystals for single or multiwavelength anomalous diffraction (SAD or MAD) to facilitate phasing has become almost synonymous with modern X-ray crystallography. The anomalous signals from SeMets can be used for phasing as well as sequence markers for subsequent model building. The production of large quantities of SeMet incorporated recombinant proteins is relatively straightforward when expressed in E. coli. In contrast, production of SeMet substituted recombinant proteins expressed in the insect cells is not as robust due to the toxicity of SeMet in eukaryotic systems. Previous protocols for SeMet-incorporation in the insect cells are laborious, and more suited for secreted proteins. In addition, these protocols have generally not addressed the SeMet toxicity issue, and typically result in low recovery of the labeled proteins. Here we report that SeMet toxicity can be circumvented by fully infecting insect cells with baculovirus. Quantitatively controlling infection levels using our Titer Estimation of Quality Control (TEQC) method allows for incorporation of substantial amounts of SeMet, resulting in an efficient and optimal production of labeled recombinant protein complexes. With the method described here, we were able to consistently reach incorporation levels of about 75% and protein yield of 60-90% compared to native protein expression.

biochemistry↗

bioRxiv2018

Molecular basis of human asparagine synthetase inhibitor specificity

Expression of the enzyme human asparagine synthetase (ASNS) promotes metastatic progression in breast cancer, which affects L-asparagine levels and tumor cell invasiveness. Human ASNS has therefore emerged as a bona fide drug target for cancer therapy. We have reported a slow-onset, tight binding ASNS inhibitor with nanomolar affinity, but our compound exhibits poor cell permeability. On the other hand, we show here that this inhibitor exhibits remarkable selectivity for the human ASNS in HCT-116 cell lysates. By determining the first high-resolution (1.85 [A]) X-ray crystal structure for human ASNS, we have built a computational model of the enzyme complexed to our inhibitor, which provides the first insights into the intermolecular interactions mediating specificity. These findings should facilitate the development of a second generation of ASNS inhibitors, leading to the discovery of drugs to prevent metastasis.

biochemistry↗

bioRxiv2018

Titer estimation for quality control (TEQC) method: a practical approach for optimal production of protein complexes using the baculovirus expression vector system

The baculovirus expression vector system (BEVS) is becoming the method of choice for expression of many eukaryotic proteins and protein complexes for biochemical, structural and pharmaceutical studies. Significant technological advancement has made generation of recombinant baculoviruses easy, efficient and user-friendly. However, there is a tremendous variability in the amount of proteins made using the BEVS, including different batches of virus made to express the same proteins. Yet, what influences the overall production of proteins or protein complexes remains largely unclear. Many downstream applications, particularly protein structure determination, require purification of large quantities of proteins in a repetitive manner, calling for a reliable experimental set-up to obtain the protein or protein complexes of interest consistently. During our investigation of optimizing the expression of the Mediator Head module, we discovered that the initial infectivity was an excellent indicator of overall production of protein complexes. Further, we show that this initial infectivity can be mathematically described as a function of multiplicity of infection (MOI), correlating recombinant protein yield and virus titer. All these findings led us to develop the Titer Estimation for Quality Control (TEQC) method, which enables researchers to estimate initial infectivity, titer/MOI values in a simple and affordable way, and to use these values to quantitatively optimize protein expressions utilizing BEVS in a highly reproducible fashion.

molecular biology↗