analysis-extracellular-vesicle-mediated-vascular-calcification-using

Analysis of Extracellular Vesicle-Mediated Vascular Calcification Using <em>In Vitro</em> and <em>In Vivo</em> Models

Cardiovascular disease is the leading cause of death in the world, and vascular calcification is the most significant predictor of cardiovascular events; ...

Florida International University

DNA damage and base excision repair (BER) are actively involved in the modulation of DNA methylation and demethylation. However, the underlying molecular mechanisms remain unclear. In this study, we seek to understand the mechanisms by exploring the effects of oxidative DNA damage on the DNA methylation pattern of the tumor suppressor breast cancer 1 (BRCA1) gene in the human embryonic kidney (HEK) HEK293H cells. We found that oxidative DNA damage simultaneously induced DNA demethylation and generation of new methylation sites at the CpGs located at the promoter and transcribed regions of the gene ranging from -189 to +27 in human cells. We demonstrated that DNA damage-induced demethylation was mediated by nucleotide misincorporation by DNA polymerase β (pol β). Surprisingly, we found that the generation of new DNA methylation sites was mediated by coordination between pol β and the  DNA methyltransferase, DNA methyltransferase 3b (DNMT3b), through the interaction between the two enzymes in the promoter and encoding regions of the BRCA1 gene. Our study provides the first evidence that oxidative DNA damage can cause dynamic changes in DNA methylation in the BRCA1 gene through the crosstalk between BER and  DNA methylation.

Oxidative DNA Damage Modulates DNA Methylation Pattern in Human Breast Cancer 1 (BRCA1) Gene via the Crosstalk between DNA Polymerase β and a  DNA Methyltransferase.

Oxidative stress can damage organs, tissues, and cells through reactive oxygen species (ROS) by oxidizing DNA, proteins, and lipids, thereby resulting in diseases. However, the underlying molecular mechanisms remain to be elucidated. In this study, employing scanning ion conductance microscopy (SICM), we explored the early responses of human embryonic kidney (HEK293H) cells to oxidative DNA damage induced by potassium chromate (KCrO). We found that the short term (1-2 h) exposure to a low concentration (10 M) of KCrO damaged the lipid membrane of HEK293H cells, resulting in structural defects and depolarization of the cell membrane and reducing cellular secretion activity shortly after the treatment. We further demonstrated that the KCrO treatment decreased the expression of the cytoskeleton protein, -actin, by inducing oxidative DNA damage in the exon 4 of the -actin gene. These results suggest that KCrO caused oxidative DNA damage in cytoskeleton genes such as -actin and reduced their expression, thereby disrupting the organization of the cytoskeleton beneath the cell membrane and inducing cell membrane damages. Our study provides direct evidence that oxidative DNA damage disrupted human cell membrane integrity by deregulating cytoskeleton gene expression.

Scanning Ion Conductance Microscopy Study Reveals the Disruption of the Integrity of the Human Cell Membrane Structure by Oxidative DNA Damage.

Flap endonuclease 1 (FEN1) is an essential enzyme that removes RNA primers and base lesions during DNA lagging strand maturation and long-patch base excision repair (BER). It plays a crucial role in maintaining genome stability and integrity. FEN1 is also implicated in RNA processing and biogenesis. A recent study from our group has shown that FEN1 is involved in trinucleotide repeat deletion by processing the RNA strand in R-loops through BER, further suggesting that the enzyme can modulate genome stability by facilitating the resolution of R-loops. However, it remains unknown how FEN1 can process RNA to resolve an R-loop. In this study, we examined the FEN1 cleavage activity on the RNA:DNA hybrid intermediates generated during DNA lagging strand processing and BER in R-loops. We found that both human and yeast FEN1 efficiently cleaved an RNA flap in the intermediates using its endonuclease activity. We further demonstrated that FEN1 was recruited to R-loops in normal human fibroblasts and senataxin-deficient (AOA2) fibroblasts, and its R-loop recruitment was significantly increased by oxidative DNA damage. We showed that FEN1 specifically employed its endonucleolytic cleavage activity to remove the RNA strand in an R-loop during BER. We found that FEN1 coordinated its DNA and RNA endonucleolytic cleavage activity with the 3'-5' exonuclease of APE1 to resolve the R-loop. Our results further suggest that FEN1 employed its unique tracking mechanism to endonucleolytically cleave the RNA strand in an R-loop by coordinating with other BER enzymes and cofactors during BER. Our study provides the first evidence that FEN1 endonucleolytic cleavage can result in the resolution of R-loops via the BER pathway, thereby maintaining genome integrity.

Article	Year
Oxidative DNA Damage Modulates DNA Methylation Pattern in Human Breast Cancer 1 (BRCA1) Gene via the Crosstalk between DNA Polymerase β and a DNA Methyltransferase. Cells\| PubMed ID: 31963223	2020
Scanning Ion Conductance Microscopy Study Reveals the Disruption of the Integrity of the Human Cell Membrane Structure by Oxidative DNA Damage. ACS applied bio materials\| PubMed ID: 34430802	2021
Flap Endonuclease 1 Endonucleolytically Processes RNA to Resolve R-Loops through DNA Base Excision Repair. Genes\| PubMed ID: 36672839	2022

Mohammad Shaver

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