Biology subjects

Mozafari, N.

Publications and source records attributed to Mozafari, N..

4 recordsLinked to original sources

bioRxiv2026

Mechanisms of fork stalling and reversal at pathogenic CANVAS (AAGGG)n repeats

Expansions of the pathogenic (AAGGG)n repeat within the RFC1 gene cause the hereditary disease CANVAS. We have shown that this repeat impedes DNA replication both in vitro and in vivo. Here, we investigated the mechanisms of replication fork stalling and reversal at (AAGGG)n repeats within an SV40-based episome in human cells. A comprehensive 2-dimensional electrophoretic analysis of replication intermediates revealed a strict orientation dependence of the replication block, evident only when the homopurine sequence was in the lagging-strand template. Fork accumulation was further increased in the presence of the G4-DNA stabilizer pyridostatin, implicating G4-DNA in the process. This stalling was followed by an accumulation of reversed forks and further analysis revealed roles for SMARCAL1 and RAD51 in this process. Biochemical studies showed that SMARCAL1, with assistance from RPA, efficiently remodels a fork carrying the G4-forming AAGGG repeat in the ssDNA gap, but not a substrate carrying the repeat in triplex conformation. We conclude that stalling results from G4-DNA formation by the repeat, which then triggers SMARCAL1-mediated fork reversal at the repeat. Given the mirror symmetry of the sequence, the nascent strands at the reversed forks likely adopt a triplex conformation, leading to a thorough accumulation of branched structures.

molecular biology↗

bioRxiv2024

Anti-gene oligonucleotides targeting Friedreichs ataxia expanded GAA:TTC repeats increase Frataxin expression

Friedreichs ataxia (FRDA) is a progressive, autosomal recessive ataxia caused, in the majority of cases, by homozygous expansion of GAA*TTC triplet-repeats in the first intron of the frataxin (FXN) gene. GAA*TTC repeat expansion results in the formation of non-B DNA intramolecular triplex structure (H-DNA) as well as changes in the epigenetic landscape at FXN loci and heterochromatin formation. Expansion of intronic GAA*TTC repeats is associated with reduced levels of FXN mRNA and protein resulting in disease development. Previously, we reported that DNA-binding anti-gene oligonucleotides (AGOs) targeting the GAA*TTC repeat expansion abolished H-DNA formation. Here, we demonstrate that targeting repeat-expanded chromosomal DNA using single-strand locked nucleic acid (LNA)-DNA mixmer AGOs increases FXN mRNA and protein expression in patient-derived cells. We examined numerous LNA-DNA AGOs and found that the design, length and their LNA composition have a high impact on the effectiveness of the treatment. Collectively, our results demonstrate the unique capability of specifically designed ONs targeting the GAA*TTC DNA repeats to upregulate FXN gene expression.

molecular biology↗

bioRxiv2022

Large-scale expansions of Friedreich's ataxia GAA·TTC repeats in human cells are prevented by LNA-DNA oligonucleotides and PNA oligomers

The human disease Friedreichs ataxia (FRDA) is caused by expansions of GAA*TTC repeats in the first intron intron of the frataxin (FXN) gene, and both intergenerational and somatic expansions are crucial for disease development. We and others have shown earlier that expanded GAA*TTC repeats can form an intramolecular triplex structure (H-DNA). Here we studied the effects of locked nucleic acid (LNA)-DNA mixmer oligonucleotides and peptide nucleic acid (PNA) oligomers on the expansion of GAA*TTC repeats in cultured human cells. Our experimental system employes a mammalian/yeast shuttle plasmid containing a selectable cassette to detect repeat expansions. Using our in-house in vitro triplex-specific DNA cleavage assay, we first confirmed H-DNA formation by the (GAA)100*(TTC)100 repeat in the selectable cassette and demonstrated that the designed LNA-DNA oligonucleotides as well as PNA oligomers are able to disrupt this structure. We then found that both LNA-DNA mixmers and PNA oligomers prevent repeat expansions in human cells. In the accompanying paper, we show that expansions of GAA*TTC repeats in this experimental system occur during replication fork stalling, regression and restart at the repetitive run. We hypothesize, therefore, that triplex DNA formation by the GAA*TTC repeats is a key to their instability, while LNA-DNA oligonucleotides and PNA oligomers counteract repeat expansions by disrupting the triplex at the fork or preventing triplex formation upon fork reversal.

genetics↗

bioRxiv2021

Metabolomic Signature of Amino Acids in Plasma of Patients with non-segmental Vitiligo

IntroductionVitiligo is an asymptomatic disorder that results from the loss of pigments (melanin), causing skin or mucosal depigmentation and impairs beauty. ObjectiveDue to the complexity of the pathogenesis of this disease and various theories including self-safety theory, oxidative stress, neurological theory and internal defects of melanocytes behind it, and finally, the vast role of amino acids in body metabolism and various activities of the body, amino acids targeted based metabolomics was set up to follow any fluctuation inside this disease. MethodologyThe study of amino acid profiles in plasma of people with non-segmental vitiligo using a liquid chromatography equipped with fluorescent detector was performed to find remarkable biomarkers for the diagnosis and evaluation of disease severity of patients with vitiligo. Twenty-two amino acids derivatized with o-phthalaldehyde (OPA) and fluorylmethyloxycarbonyl chloride (FMOC), were precisely determined. Next, the concentrations of these twenty-two amino acids and their corresponding molar ratios were calculated in 37 patients (including 18 females and 19 males) and corresponding 34 healthy individuals (18 females and 16 males). Using R programing, the data were completely analyzed between the two groups of patients and healthy to find suitable and reliable biomarkers. ResultsInterestingly, comparing the two groups, in the patient group, tyrosine, cysteine, the ratio of tyrosine to lysine and the ratio of cysteine to ornithine were increased while, arginine, lysine, ornithine and glycine ratios to cysteine have been decreased. These amino acids were selected for identification of patients with accuracy of detection of approximately 0.95 using the assessment of logistic regression. ConclusionThese results indicate a disruption of the production of melanin, increased immune activity and oxidative stress, which are also involved in the effects of vitiligo. Therefore, these amino acids can be used as biomarker for the evaluation of risk, prevention of complications in individuals at risk and monitoring of treatment process.

biochemistry↗