Tpp1 is a DNA 3-phosphatase in Saccharomyces cerevisiae that is believed to act during strand break repair. It is homologous to one domain of mammalian polynucleotide kinase/3-phosphatase. Unlike in yeast, we found that Tpp1 could confer resistance to methylmethane sulfonate when expressed in bacteria that lack abasic endonuclease/3-phosphodiesterase function. This species difference was due to the absence of ␦-lyase activity in S. cerevisiae, since expression of bacterial Fpg conferred Tpp1-dependent resistance to methylmethane sulfonate in yeast lacking the abasic endonucleases Apn1 and Apn2. In contrast, -only lyases increased methylmethane sulfonate sensitivity independently of Tpp1, which was explained by the inability of Tpp1 to cleave 3 ␣,-unsaturated aldehydes. In parallel experiments, mutations of TPP1 and RAD1, encoding part of the Rad1/Rad10 3-flap endonuclease, caused synthetic growth defects in yeast strains lacking Apn1. In contrast, Fpg expression led to a partial rescue of apn1 apn2 rad1 synthetic lethality by converting lesions into Tpp1-cleavable 3-phosphates. The collected experiments reveal a profound toxicity of strand breaks with irreparable 3 blocking lesions, and extend the function of the Rad1/Rad10 salvage pathway to 3-phosphates. They further demonstrate a role for Tpp1 in repairing endogenously created 3-phosphates. The source of these phosphates remains enigmatic, however, because apn1 tpp1 rad1 slow growth could be correlated with neither the presence of a yeast ␦-lyase, the activity of the 3-phosphate-generating enzyme Tdp1, nor levels of endogenous oxidation.
In budding yeast, Apn1, Apn2, Tpp1, and Rad1/Rad10 are important enzymes in the removal of spontaneous DNA lesions. apn1 apn2 rad1 yeast are inviable due to accumulation of abasic sites and strand breaks with 3Ј blocking lesions. We found that tpp1 apn1 rad1 yeast exhibited slow growth but frequently gave rise to spontaneous slow growth suppressors that segregated as single-gene mutations. Using a candidate gene approach, we identified several tpp1 apn1 rad1 suppressors. Deleting uracil glycosylase suppressed both tpp1 apn1 rad1 and apn1 apn2 rad1 growth defects by reducing the abasic site burden. Mutants affecting the Chk1-Pds1 metaphase-anaphase checkpoint only suppressed tpp1 apn1 rad1 slow growth. In contrast, most S-phase checkpoint mutants were synthetically lethal in a tpp1 apn1 rad1 background. Epistasis analyses showed an additive effect between chk1 and ung1, indicating different mechanisms of suppression. Loss of Chk1 partially restored cell-growth parameters in tpp1 apn1 rad1 yeast, but at the same time exacerbated chromosome instability. We propose a model in which recombinational repair during S phase coupled with failure of the metaphase-anaphase checkpoint allows for tolerance of persistent single-strand breaks at the expense of genome stability. ous lesion leading to toxicity in these strains.gene replacement was also confirmed by the loss of product in PCR using primers in the coding region of the targeted gene.We have found that a haploid strain lacking Apn1, et al. 1994Sanchez et al. 1999). There is the samples were then measured at 15-to 30-min intervals by using a GENESYS20 spectrophotometer for at least two considerable overlap in the proteins required for these from at least six different isolates were analyzed. As with theIn this study we found that tpp1 apn1 rad1 mutant tetrad scores, this was done prior to genotyping, so that all yeast spontaneously gave rise to single-gene slow-growth data were recorded blind to the strain genotype.suppressors at a high frequency. Using a candidate gene Plasmid loss assay: Plasmid loss rates were determined using the method described by Huang and Koshland (2003).approach, we observed that disruption of the M-A checkBriefly, YW1495, a diploid strain carrying the pRS412 plasmid point conferred suppression of tpp1 apn1 rad1 slow with the CEN/ARS and the ADE2 marker gene, was dissected growth. In contrast, deletion of S-phase checkpoint on selective media plates to get the strains with appropriate genes either had no effect or conferred synthetic lethalgenotypes and containing the plasmid. Either a 6-day tetrad ity in a tpp1 apn1 rad1 mutant background. The implicacolony or a wild-type strain (YW465) carrying the plasmid was used to inoculate the initial culture and allowed to grow in tions of these findings are discussed.YPAD for approximately six to seven doublings. In the case of slow-growing strains, three or more colonies were pooled to form the initial inoculum. The plasmid loss rate was calculated MATERIALS AND METHODS using the formula: l...
HIV-1 causes diverse immunomodulatory responses in the host, including the down-regulation of co-stimulatory proteins CD80/86, mediated by HIV-1 protein Nef, blunting T-cell activation. Using a screening cascade of biochemical and cell-based assays, we identified potent small molecules representing three chemical scaffolds namely amino pyrimidine, phenoxy acetamide and bi-aryl heteroaryl carbamate which target the protein-protein interaction interface of CD80/86 and Nef with sub-micromolar potency. These molecules restore CD80/86 surface levels in HIV-1-Nef infected antigen presenting cells and T-cell activation. Nef-CD80 interface and small molecule binding sites were mapped by using computational docking and structural studies, followed by validation by mutational analysis. This analysis resulted in the identification of two key residues, K99 and R111, which were associated with down-modulation of CD80 surface levels by Nef and important for small molecule binding. Targeting these interacting residues disabled Nef-mediated down-modulation of CD80 surface levels, consequently restoring T-cell activation. Thus, we validate a new target, the Nef-CD80/86 protein-protein interaction interface, with a potential to develop new inhibitors to counteract the immunomodulatory consequences of HIV-1.
Background There are limited global data on head-to-head comparisons of vaccine platforms assessing both humoral and cellular immune responses, stratified by pre-vaccination serostatus. The COVID-19 vaccination drive for the Indian population in the 18 to 45-year age-group began in April 2021 when seropositivity rates in the general population were rising due to the Delta wave in April-May 2021. Methods Between 30 June 2021 and 28 January 2022, we enrolled 691 participants in the 18-45 age group across 4 clinical sites in India. In this non-randomized and laboratory blinded study, participants received either two doses of Covaxin® 4 weeks apart or two doses of Covishield™ 12 weeks apart per the national vaccination policy. The primary outcome was the seroconversion rate and the geometric mean titer (GMT) of antibodies against the SARS-CoV-2 spike and nucleocapsid proteins. The secondary outcome was the frequency of cellular immune responses pre- and post-vaccination. Findings When compared to pre-vaccination baseline, both vaccines elicited statistically significant seroconversion and binding antibody levels in both seronegative and seropositive individuals. In the per-protocol cohort, Covishield™ elicited higher antibody responses than Covaxin® as measured by seroconversion rate (98.3% vs 74.4%, p<0.0001 in seronegative individuals; 91.7% vs 66.9%, p<0.0001 in seropositive individuals) as well as by anti-spike antibody levels against the ancestral strain (GMT 1272.1 vs 75.4 BAU/ml, p<0.0001 in seronegative individuals; 2089.07 vs 585.7 BAU/ml, p<0.0001 in seropositive individuals). Not all sites recruited at the same time, therefore site-specific immunogenicity was impacted by the timing of vaccination relative to the Delta and Omicron waves. Surrogate neutralizing antibody responses against variants-of-concern were higher in Covishield™ recipients than in Covaxin® recipients and in seropositive than in seronegative individuals after both vaccination and asymptomatic Omicron infection. T cell responses are reported from only one of the four site cohorts where the vaccination schedule preceded the Omicron wave. In seronegative individuals, Covishield™ elicited both CD4+ and CD8+ spike-specific cytokine-producing T cells whereas Covaxin® elicited mainly CD4+ spike-specific T cells. Neither vaccine showed significant post-vaccination expansion of spike-specific T cells in seropositive individuals. Interpretation Covishield™ elicited immune responses of higher magnitude and breadth than Covaxin® in both seronegative individuals and seropositive individuals, across cohorts representing the pre-vaccination immune history of the majority of the vaccinated Indian population.
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