Biology subjects

Tyler, J. K.

Publications and source records attributed to Tyler, J. K..

5 recordsLinked to original sources

bioRxiv2025

Overexpression of Ssd1 and calorie restriction extend yeast replicative lifespan by preventing deleterious age-dependent iron uptake

Overexpression of the mRNA binding protein Ssd1 extends the yeast replicative lifespan. Using microfluidics to trap and image single cells throughout their lifespans, we find that lifespan extension by Ssd1 overexpression is accompanied by formation of cytoplasmic Ssd1 foci. The age-dependent Ssd1 foci are condensates that appear dynamically in a cell cycle-dependent manner and their failure to resolve during mitosis coincided with the end of lifespan. Ssd1 overexpression was epistatic with calorie restriction (CR) for lifespan extension and yeast overexpressing Ssd1 or undergoing CR were resistant to iron supplementation-induced lifespan shortening while their lifespans were reduced by iron chelation. The nuclear translocation of the Aft1 transcriptional regulator of the iron regulon occurred during aging in a manner that predicted remaining lifespan, but was prevented by CR. Accordingly, age-dependent induction of the Fit2 and Arn1 high-affinity iron transporters within the iron regulon was reduced by CR and Ssd1 overexpression. Consistent with age-dependent activation of the iron regulon, intracellular iron accumulated during aging but was prevented by CR and Ssd1 overexpression. Moreover, lifespan extension by Ssd1 overexpression or CR was epistatic to inactivation of the iron regulon. These studies reveal that CR and Ssd1 overexpression extend the yeast replicative lifespan by blocking deleterious age-dependent iron uptake, identifying novel therapeutic targets for lifespan extension and providing insight into how CR may extend the lifespan and healthspan in humans.

cell biology↗

bioRxiv2024

Senataxin and DNA-PKcs Redundantly Promote Non-Homologous End Joining Repair of DNA Double Strand Breaks During V(D)J Recombination

Non-homologous end joining (NHEJ) is required for repairing DNA double strand breaks (DSBs) generated by the RAG endonuclease during lymphocyte antigen receptor gene assembly by V(D)J recombination. The Ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) kinases regulate functionally redundant pathways required for NHEJ. Here we report that loss of the senataxin helicase leads to a significant defect in RAG DSB repair upon inactivation of DNA-PKcs. The NHEJ function of senataxin is redundant with the RECQL5 helicase and the HLTF translocase and is epistatic with ATM. Co-inactivation of ATM, RECQL5 and HLTF results in an NHEJ defect similar to that from the combined deficiency of DNA-PKcs and senataxin or losing senataxin, RECQL5 and HLTF. These data suggest that ATM and DNA-PKcs regulate the functions of senataxin and RECQL5/HLTF, respectively to provide redundant support for NHEJ.

cell biology↗

bioRxiv2023

Transcriptional inhibition after irradiation occurs preferentially at highly expressed genes in a manner dependent on cell cycle progression

In response to DNA double strand damage, ongoing transcription is inhibited to facilitate accurate DNA repair while transcriptional recovery occurs after DNA repair is complete. However, the mechanisms at play and identity of the transcripts being regulated in this manner are unclear. In contrast to the situation following UV damage, we found that transcriptional recovery after ionizing radiation (IR) occurs in a manner independent of the HIRA histone chaperone. Sequencing of the nascent transcripts identified a programmed transcriptional response, where certain transcripts and pathways are rapidly downregulated after IR, while other transcripts and pathways are upregulated. Specifically, most of the loss of nascent transcripts occurring after IR is due to inhibition of transcriptional initiation of the highly transcribed histone genes and the rDNA. To identify factors responsible for transcriptional inhibition after IR in an unbiased manner, we performed a whole genome gRNA library CRISPR / Cas9 screen. Many of the top hits in our screen were factors required for protein neddylation. However, at short times after inhibition of neddylation, transcriptional inhibition still occurred after IR, even though neddylation was effectively inhibited. Persistent inhibition of neddylation blocked transcriptional inhibition after IR, and it also leads to cell cycle arrest. Indeed, we uncovered that many inhibitors and conditions that lead to cell cycle arrest in G1 or G2 phase also prevent transcriptional inhibition after IR. As such, it appears that transcriptional inhibition after IR occurs preferentially at highly expressed genes in cycling cells.

molecular biology↗

bioRxiv2022

A novel Single Alpha-Helix-DNA-binding domain in CAF-1 promotes gene silencing and DNA damage survival through tetrasome-length DNA selectivity and spacer function

The histone chaperone chromatin assembly factor 1 (CAF-1) deposits nascent histone H3/H4 dimers onto newly replicated DNA forming the central core of the nucleosome known as the tetrasome. How CAF-1 ensures there is sufficient space for the assembly of tetrasomes remains unknown. Structural and biophysical characterization of the lysine/glutamic acid/arginine-rich (KER) region of CAF-1 revealed a 128 [A] single alpha helix (SAH) motif with unprecedented DNA binding properties. Distinct KER sequence features and length of the SAH drive the selectivity of CAF-1 for tetrasome-length DNA and facilitate function in budding yeast. In vivo, the KER cooperates with the DNA-binding winged helix domain in CAF-1 to overcome DNA damage sensitivity and maintain silencing of gene expression. We propose that the KER SAH links functional domains within CAF-1 with structural precision, acting as a DNA binding spacer element during chromatin assembly.

biochemistry↗

bioRxiv2021

DNA-PK Promotes DNA End Resection at DNA Double Strand Breaks in G0 cells

DNA double-strand break (DSB) repair by homologous recombination is confined to the S and G2 phases of the cell cycle partly due to 53BP1 antagonizing DNA end resection in G1 phase and non-cycling quiescent (G0) cells where DSBs are predominately repaired by non-homologous end joining (NHEJ). Unexpectedly, we uncovered extensive MRE11- and CtIP-dependent DNA end resection at DSBs in G0 mammalian cells. A whole genome CRISPR/Cas9 screen revealed the DNA-dependent kinase (DNA-PK) complex as a key factor in promoting DNA end resection in G0 cells. In agreement, depletion of FBXL12, which promotes ubiquitylation and removal of the KU70/KU80 subunits of DNA-PK from DSBs, promotes even more extensive resection in G0 cells. In contrast, a requirement for DNA-PK in promoting DNA end resection in proliferating cells at the G1 or G2 phase of the cell cycle was not observed. Our findings establish that DNA-PK uniquely promotes DNA end resection in G0, but not in G1 or G2 phase cells, and has important implications for DNA DSB repair in quiescent cells.

molecular biology↗