SLX4 Assembles a Telomere Maintenance Toolkit by Bridging Multiple Endonucleases with Telomeres

SummarySLX4 interacts with several endonucleases to resolve structural barriers in DNA metabolism. SLX4 also interacts with telomeric protein TRF2 in human cells. The molecular mechanism of these interactions at telomeres remains unknown. Here, we report the crystal structure of the TRF2-binding motif of SLX4 (SLX4TBM) in complex with the TRFH domain of TRF2 (TRF2TRFH) and map the interactions of SLX4 with endonucleases SLX1, XPF, and MUS81. TRF2 recognizes a unique HxLxP motif on SLX4 via the peptide-binding site in its TRFH domain. Telomeric localization of SLX4 and associated nucleases depend on the SLX4-endonuclease and SLX4-TRF2 interactions and the protein levels of SLX4 and TRF2. SLX4 assembles an endonuclease toolkit that negatively regulates telomere length via SLX1-catalyzed nucleolytic resolution of telomere DNA structures. We propose that the SLX4-TRF2 complex serves as a double-layer scaffold bridging multiple endonucleases with telomeres for recombination-based telomere maintenance

CP1 domain of leucyl-tRNA synthetase: dissecting its dual roles in amino acid editing and RNA splicing

The essential family of aminoacyl-tRNA synthetase (AARS) enzymes catalyzes the attachment of an amino acid to its cognate tRNA during ribosome-based translation of mRNA. Leucyl-tRNA synthetase (LeuRS) ensures fidelity in protein synthesis via proofreading or editing mechanisms. The editing that hydrolyzes noncognate amino acids mischarged onto tRNALeu is called post-transfer editing. The hydrolytic post-transfer editing active site is located in a discretely folded polypeptide insertion called connective polypeptide 1 (CP1) that is linked to the enzyme’s main body by two flexible β-strand linkers. Disruption of the CP1 domain-based editing function in LeuRS results in amino acid toxicities that compromise cell viability. A fluorescence-based in vivo assay was designed to quantify the effects of editing defects and hence assess the limits of mistranslation that can be borne by the cell. Sequence enabled reassembly of N and C-terminal fragments of the green fluorescence protein (GFP) were studied in vivo in the presence of editing defective LeuRS and noncognate amino acids. In the yeast cytoplasmic LeuRS (ycLeuRS), the conserved post-transfer editing pocket is the target binding site for a novel class of benzoxaborole-based antimicrobials that trap tRNALeu and halt protein synthesis. Resistance mutations (D487G and D487N) to the antimicrobial compound AN2690 lie outside the drug binding pocket and provided a unique opportunity to study editing mechanisms in the ycLeuRS. The Asp487 residue is located in a CP1 domain-based eukaryote-specific flexible insert called I4 that forms a ‘cap’ over the benzoxaborole-AMP adduct bound in the CP1 domain editing active site. Mutational and biochemical analysis at Asp487 identified a salt bridge between Asp487 and Arg316 in the hinge region of the I4 cap that is critical to tRNA deacylation. Thus, this electrostatic interaction stabilizes the cap during binding of the editing substrate for hydrolysis in the ycLeuRS. An alternative pre-transfer editing pathway has also been identified in LeuRS and cleaves the noncognate amino acid before it is transferred to tRNALeu, at the stage of aminoacyl-AMP. Co-existence of both pre- and post-transfer editing pathways was highlighted in the ycLeuRS, as has also been shown earlier for E. coli LeuRS. Detailed biochemical investigations on the editing activity of this enzyme revealed that ycLeuRS shifts between the two editing pathways and this shift is dictated by the chemical identity of the noncognate amino acid misactivated by the enzyme. While isoleucine is mainly cleared via the post-transfer editing route that targets Ile-tRNALeu, methionine is edited via the pre-transfer pathway by hydrolysis of methionyl-adenylate in ycLeuRS. The yeast mitochondrial LeuRS (ymLeuRS) was recruited to perform an alternate cellular role of mRNA splicing. Splicing-sensitive sites have been located within and and in close proximity to the CP1 domain. Remarkably, E. coli LeuRS supports splicing in vivo, although its CP1 domain appears to lack the finer adaptations for efficient splicing compared to its counterpart from the ymLeuRS. In vitro and in vivo analysis dissected functional divergences of the ymLeuRS CP1 domain that accommodate an alternate cellular role for RNA splicing at the expense of its housekeeping aminoacylation and editing function. A close look at the connecting β-strands between the CP1 domain and main body highlighted that these β-strands, as well short extensions into the enzyme main body, are indispensable to not only LeuRS’s editing function, but also to its splicing activity

Ion-pair extraction of uranium(VI) using N-cinnamoyl-<i>o</i>-tolyl hydroxylamine in presence of tri-<i>n</i>-octyl phosphine oxide in chloroform and hexafluorophosphoric acid

244-246N-cinnamoyl-o-tolylbhydroxylamine (CT0HA.HL) forms extractable binary complex with uranium. The extracted complex, UO2L2(0) when shaken with tri-n-octylphosphine oxide (TOPO R) in chloroform, is gradually back-extracted into aqueous phase due to formation of the cationic complex, UO2LR +. The gradual substitution has been followed spectrophotometrically by the decrease in absorbance of UO2L2(0) Uranium(VI) can be extracted into the chloroform phase by formation of ion-pair [UO2LR + .PF)0 of UO2LR + with the anion hexafluorophosphate. Extraction of the ion-pair complex is followed by enhanced absorbance values. The substitution constant. Ka and ion-pair constant, KI, have been evaluated to be 0.075 and 2.05 x 103 respectively

To Disclose or Not to Disclose? Reasons for HIV Serostatus Disclosure and Non-disclosure by People Living with HIV/AIDS in Eastern India

Background: Understanding the reasons for HIV serostatus disclosure and nondisclosure and how these reasons differ by certain characteristics of the people living with HIV/AIDS (PLWHA) is important for effective HIV prevention intervention strategies. Objectives: This study was undertaken to investigate/identify the reasons for disclosure and nondisclosure of HIV serostatus by PLWHA, and to find out any association between the reasons for disclosure or nondisclosure and certain demographic and clinical characteristics, i.e. age, gender, WHO clinical stage and CD4 count of the study population. Methods: A cross-sectional study was conducted among all patients of 18-49 years with confirmed HIV infection registered at the antiretroviral therapy (ART) center of a tertiary care hospital in eastern Uttar Pradesh, India for one year, from July 2017 to June 2018. Results: The most common reason for disclosure of HIV serostatus was the presence of any family member at the time of collection of HIV test report (68.5%), and the most common reason for not disclosing the serostatus was stigmatization (68%). The reasons for disclosure was found to be associated with the WHO clinical stage of the respondents (p &lt;0.05). &nbsp;Conclusions: The findings of this study highlights the need for tailoring intervention strategies for improving disclosure decision making according to the specific needs of PLWHA. There is also a need to address the concerns of those who are reluctant to disclose. More emphasis should be given on creating awareness about HIV stigma, on the importance of serostatus disclosure and secondary HIV prevention in the community