12/9/2023 0 Comments Dot blot apparaturElectron microscopy methods for studying in vivo DNA replication intermediates. Telomere damage induces internal loops that generate telomeric circles. Measuring UV photoproduct repair in isolated telomeres and bulk genomic DNA. Analysis of mammalian telomere position effect. Mammalian polymerase theta promotes alternative NHEJ and suppresses recombination. Break-induced telomere synthesis underlies alternative telomere maintenance. ![]() Human telomeres replicate using chromosome-specific, rather than universal, replication programs. elegans telomeres contain G-strand and C-strand overhangs that are bound by distinct proteins. Telomere loops and homologous recombination-dependent telomeric circles in a Kluyveromyces lactis telomere mutant strain. Telomeric DNA in ALT cells is characterized by free telomeric circles and heterogeneous t-loops. J., Quinney, N., Willcox, S., Subramanian, D. Twenty years of t-loops: a case study for the importance of collaboration in molecular biology. Mammalian telomeres end in a large duplex loop. Structure and variability of human chromosome ends. A highly conserved repetitive DNA sequence, (TTAGGG) n, present at the telomeres of human chromosomes. thermophila cross hybridizes with human telomeres. Mammalian telomeres resemble fragile sites and require TRF1 for efficient replication. ![]() Semi-conservative DNA replication through telomeres requires Taz1. Protection of telomeres through independent control of ATM and ATR by TRF2 and POT1. DNA damage foci at dysfunctional telomeres. Heterogeneity in telomere length of human chromosomes. The procedure described here can be adapted to the enrichment of other repetitive elements, based on the use of restriction enzymes that do not cut into the repeat of interest. The latter can be combined with specific labeling for single-molecule analysis of replicating DNA or for long-read sequencing analysis of telomeric repeats. A smaller-scale version of the protocol that involves one round of digestion and purification requires 200 µg of DNA and enriches telomeres ~50-fold in 4 d is also provided. No special skills are required for the enrichment procedure, while some assistance is needed in harvesting a large number of plates in a timely fashion at the beginning of the procedure. The purified material is suitable for single-molecule analysis of telomere structure, visualizing telomere replication and recombination intermediates by electron microscopy or performing molecular combing at telomeric repeats. Around 2 mg of genomic DNA is required, and the procedure lasts 5–6 d and yields preparations enriched >800-fold in telomeres. ![]() The procedure consists of two successive rounds of digestion with frequently cutting restriction enzymes followed by size fractionation. Here we provide a detailed protocol for the enrichment of telomeric repeats from mouse and human cells.
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