Author Correction:C16orf72/HAPSTR1/TAPR1 functions with BRCA1/Senataxin to modulate replication-associated R-loops and confer resistance to PARP disruption

Correction to: Nature Communications https://doi.org/10.1038/s41467-023-40779-9, published online 17 August 2023

Generation of Knockout Human iPSCs to Investigate Genes Associated with Telomere Length

Telomeres are repetitive sequences located at the ends of human chromosomes. During DNA replication, DNA polymerase is unable to fully replicate telomeric DNA causing a progressive reduction in telomere length with each cell division. Consequently, telomeres shorten with age and when telomere length reaches a critical length the cell becomes dysfunctional or senescent. Increasing numbers of senescent cells results in reduced organ function and telomere length has been associated with age-associated diseases including cardiovascular disease and cancer. Telomere length is highly heritable and genome-wide association studies have identified several loci that associate with telomere length. Interestingly, two of these loci do not contain genes with known roles in telomere maintenance. In this study, CRISPR/Cas9 genome editing was used to knockout telomere length associated genes in human induced pluripotent stem cells (hiPSCs) with the ultimate aim of definitively linking a novel telomere length associated gene to telomere maintenance. CRISPR/Cas9 was used to create hiPSCs with no telomerase activity by knocking out TERT, which encodes the catalytic subunit of telomerase. Telomere length was maintained in control iPSCs, but reduced by approximately 1% per day during extended culture of TERT knockout hiPSCs. Flow cytometric analysis of mutant hiPSCs revealed that they continued to express markers of pluripotency but had increased expression of a differentiation marker. Next, CRISPR/Cas9 was used to generate mutations in the candidate telomere maintenance genes ACYP2 and TSPYL6, however, no difference in telomere length was observed after extended culture of hiPSCs carrying mutations in either gene. In conclusion, CRISPR/Cas9 genome editing was successfully used to generate mutant hiPSCs for TERT, which resulted in telomere shortening and for two candidate telomere maintenance genes, which had no effect on telomere length. Further analysis will be required to determine which gene mediates the association with telomere length at the ACYP2 locus

Photobiomodulation and Antimicrobial Photodynamic Influence of a 650 nm Wavelength on Staphylocoagulase and Viability of Staphylococcus aurous: Antimicrobial Photodynamic Influence of 650 nm Wavelength on Staphylocoagulase Activity

Introduction: Staphylococcus aureus is one of the critical pathological bacteria. This bacterium had developed a variety of genetic mutations that made it resistant to drugs and more harmful to humans. In addition, all attempts to design a specific vaccine against S. aureus have failed. Therefore, this experiment was designed as a trial for vaccine production, by using a photodynamic treatment (PDT) through partial biological inhibition. This study also aimed to evaluate the inhibitory effect of PDT on the total bacterial account (viability) simultaneously with SC assay.Methods: A 650nm wavelength diode laser was used with 100 mW output power and 2 minutes of exposure time. Dye dilutions were 50, 100, 150 and 200 μg/mL. The viability of bacteria after and before laser treatment was calculated using single plate-serial dilution spotting methods. The activity of SC was detected by using human plasma for 4 hours incubation of crude-substrate interaction.Results: The results revealed a significant decrease in enzyme activity and colony-forming units (CFU) after irradiating bacterial suspension with 150 g/mL MB, as well as a decline in CFU. However, irradiation with a laser alone showed a significant increase in SC activity and CFU for the same exposure time.Conclusion: Besides reducing the production of SC activity, PDT significantly inhibited the viability of S. aureus. The application of MB photosensitizer at a concentration of 150 g/mL in combination with a laser wavelength of 650 nm resulted in a complete decrease in the SC activity value as well as the viability of bacteria. Doi:10.34172/jlms.2022.0

Regulation of Rad52-dependent replication fork recovery through serine ADP-ribosylation of PolD3

Abstract Although Poly(ADP-ribose)-polymerases (PARPs) are key regulators of genome stability, how site-specific ADP-ribosylation regulates DNA repair is unclear. Here, we describe a novel role for PARP1 and PARP2 in regulating Rad52-dependent replication fork repair to maintain cell viability when homologous recombination is dysfunctional, suppress replication-associated DNA damage, and maintain genome stability. Mechanistically, Mre11 and ATM are required for induction of PARP activity in response to replication stress that in turn promotes break-induced replication (BIR) through assembly of Rad52 at stalled/damaged replication forks. Further, by mapping ADP-ribosylation sites induced upon replication stress, we identify that PolD3 is a target for PARP1/PARP2 and that its site-specific ADP-ribosylation is required for BIR activity, replication fork recovery and genome stability. Overall, these data identify a critical role for Mre11-dependent PARP activation and site-specific ADP-ribosylation in regulating BIR to maintain genome integrity during DNA synthesis

C16orf72/HAPSTR1/TAPR1 functions with BRCA1/Senataxin to modulate replication-associated R-loops and confer resistance to PARP disruption

Abstract While the toxicity of PARP inhibitors to cells with defects in homologous recombination (HR) is well established, other synthetic lethal interactions with PARP1/PARP2 disruption are poorly defined. To inform on these mechanisms we conducted a genome-wide screen for genes that are synthetic lethal with PARP1/2 gene disruption and identified C16orf72/HAPSTR1/TAPR1 as a novel modulator of replication-associated R-loops. C16orf72 is critical to facilitate replication fork restart, suppress DNA damage and maintain genome stability in response to replication stress. Importantly, C16orf72 and PARP1/2 function in parallel pathways to suppress DNA:RNA hybrids that accumulate at stalled replication forks. Mechanistically, this is achieved through an interaction of C16orf72 with BRCA1 and the RNA/DNA helicase Senataxin to facilitate their recruitment to RNA:DNA hybrids and confer resistance to PARP inhibitors. Together, this identifies a C16orf72/Senataxin/BRCA1-dependent pathway to suppress replication-associated R-loop accumulation, maintain genome stability and confer resistance to PARP inhibitors

The integrin ligand SVEP1 regulates GPCR-mediated vasoconstriction via integrins alpha 9 beta 1 and alpha 4 beta 1

Background and Purpose: Vascular tone is regulated by the relative contractile state of vascular smooth muscle cells (VSMCs). Several integrins directly modulate VSMC contraction by regulating calcium influx through L-type voltage-gated Ca2+ channels (VGCCs). Genetic variants in ITGA9, which encodes the α9 subunit of integrin α9β1, and SVEP1, a ligand for integrin α9β1, associate with elevated blood pressure; however, neither SVEP1 nor integrin α9β1 has reported roles in vasoregulation. We determined whether SVEP1 and integrin α9β1 can regulate VSMC contraction. Experimental Approach: SVEP1 and integrin binding were confirmed by immunoprecipitation and cell binding assays. Human induced pluripotent stem cell-derived VSMCs were used in in vitro [Ca2+]i studies, and aortas from a Svep1+/− knockout mouse model were used in wire myography to measure vessel contraction. Key Results: We confirmed the ligation of SVEP1 to integrin α9β1 and additionally found SVEP1 to directly bind to integrin α4β1. Inhibition of SVEP1, integrin α4β1 or α9β1 significantly enhanced [Ca2+]i levels in isolated VSMCs to Gαq/11-vasoconstrictors. This response was confirmed in whole vessels where a greater contraction to U46619 was seen in vessels from Svep1+/− mice compared to littermate controls or when integrin α4β1 or α9β1 was inhibited. Inhibition studies suggested that this effect was mediated via VGCCs, PKC and Rho A/Rho kinase dependent mechanisms. Conclusions and Implications: Our studies reveal a novel role for SVEP1 and the integrins α4β1 and α9β1 in reducing VSMC contractility. This could provide an explanation for the genetic associations with blood pressure risk at the SVEP1 and ITGA9 loci