Biology subjects

Myler, L. R.

Publications and source records attributed to Myler, L. R..

4 recordsLinked to original sources

bioRxiv2024

Tetrameric TRF2 forms t-loop to protect telomeres from ATM signaling and cNHEJ

Telomeres often occur in the t-loop structure, formed through the base-pairing of the 3 telomeric overhang is base-paired with more internal telomeric DNA. T-loops are proposed to avert ATM kinase signaling and classical non-homologous end joining (cNHEJ) by sequestering the telomere end. The shelterin subunit TRF2 is required for t-loop formation but how it generates t-loops is not known and the t-loop model for telomere protection remains untested. We show that TRF2 binds telomeric DNA as a tetramer using two dimerization domains and that tetramerization is critical for telomere protection. Furthermore, TRF1, the TRF2 paralog that binds DNA as a dimer and does not form t-loops, gained the ability to form t-loops in vivo upon its tetramerization through the addition of a second dimerization domain. We propose that TRF2 tetramers loop telomeric DNA, resulting in torsional stress and opening of the helix, which is a requirement for t-loop formation. Importantly, t-loops formed by tetrameric TRF1 were sufficient to avert ATM signaling and blocked most cNHEJ at telomeres, demonstrating that t-loops per se protect telomeres from these threats.

cell biology↗

bioRxiv2023

DNA-PK and the TRF2 iDDR inhibit MRN-initiated resection at leading-end telomeres

Telomeres replicated by leading-strand synthesis lack the 3 overhang required for telomere protection. Surprisingly, resection of these blunt telomere is initiated by the telomere-specific 5 exonuclease Apollo rather than the Mre11-Rad50-Nbs1 (MRN) complex, the nuclease that acts at DNA breaks. Without Apollo, leading-end telomeres undergo fusion, which, as demonstrated here, are mediated by alternative End Joining. Here, we show that DNA-PK and TRF2 coordinate the repression of MRN at blunt telomeres. DNA-PK represses an MRN-dependent long range resection at blunt telomeres, while the endonuclease activity of MRN/CtIP, which could cleave DNA-PK off of blunt telomere ends, is inhibited in vitro and in vivo by the iDDR of TRF2. AlphaFold-Multimer predicts a conserved association of the iDDR with Rad50 potentially interfering with CtIP binding and MRN endonuclease activation. We propose that repression of MRN-mediated resection is a conserved aspect of telomere maintenance and represents an ancient feature of DNA-PK and the iDDR.

molecular biology↗

bioRxiv2019

RPA phosphorylation regulates DNA resection

Genetic recombination in all kingdoms of life initiates when helicases and nucleases process (resect) the free DNA ends to expose single-stranded (ss) DNA overhangs. Resection termination in bacteria is programmed by a DNA sequence but the mechanisms limiting resection in eukaryotes have remained elusive. Using single-molecule imaging of reconstituted human DNA repair factors, we identify a general mechanism that limits DNA resection. BLM helicase together with EXO1 and DNA2 nucleases catalyze kilobase-length DNA resection on nucleosome-coated DNA. The resulting ssDNA is rapidly bound by RPA, which is in turn phosphorylated as part of the DNA damage response (DDR). Remarkably, phosphorylated RPA (pRPA) inhibits DNA resection via regulation of BLM helicase. pRPA suppresses BLM initiation at DNA ends and promotes the intrinsic helicase strand-switching activity. These findings establish that pRPA is a critical regulator of DNA repair enzymes and provides a feedback loop between the DDR and DNA resection termination.

biophysics↗

bioRxiv2017

Assembly and translocation of a CRISPR-Cas primed acquisition complex

Bacteria and archaea destroy foreign nucleic acids by mounting an RNA-based CRISPR-Cas adaptive immune response1-3. In type I CRISPR-Cas systems, the most frequently found type of CRISPR in bacteria and archaea3,4, foreign DNAs that trigger efficient immunity can also provoke primed acquisition of protospacers into the CRISPR locus5-12. Both interference and primed acquisition require Cascade (CRISPR-associated complex for antiviral defense) and the Cas3 helicase/nuclease. Primed acquisition also requires the Cas1-Cas2 integrase; however, the biophysical mechanisms of how interference and primed acquisition are coordinated have remained elusive. Here, we present single-molecule characterization of the type I-E Thermobifida fusca (Tfu) primed acquisition complex (PAC). TfuCascade rapidly samples non-specific DNA for its target via facilitated one-dimensional (1D) diffusion. An evolutionary-conserved positive patch on the Cse1 subunit increases the target recognition efficiency by promoting this 1D diffusion. Cas3 loads at target-bound Cascade and the Cascade/Cas3 complex initiates processive translocation via a looped DNA intermediate. Moving Cascade/Cas3 complexes stall and release the DNA loop at protein roadblocks. Cas1-Cas2 samples DNA transiently via 3D collisions, but stably associates with target-bound Cascade. Cas1-Cas2 also remains associated with translocating Cascade/Cas3, forming the PAC. By directly imaging all key subcomplexes involved in target recognition, interference, and primed acquisition, this work provides a molecular basis for the coordinated steps in CRISPR-based adaptive immunity.

biophysics↗