Direct Quantitative Monitoring of Homology-Directed DNA Repair of Damaged Telomeres

Abstract

Homology-directed DNA repair (HDR) is an evolutionary conserved mechanism that is required for genome integrity and organismal fitness across species. While a myriad of different factors and mechanisms are able to execute HDR, all forms necessitate common steps of DNA damage recognition, homology search and capture, and assembly of a DNA polymerase complex to conduct templated DNA synthesis. The central question of what determines HDR mechanism utilization in mammalian cells has been limited by an inability to directly monitor the DNA damage response and products of repair as they arise from a defined genomic lesion. In this chapter, we describe several methodologies to delineate major steps of HDR during alternative lengthening of telomeres in human cells. This includes procedures to visualize interchromosomal telomere homology searches in real time and quantitatively detect HDR synthesis of nascent telomeres emanating from synchronous activation of telomere DNA double-strand breaks. We highlight the critical details of these methods and their applicability to monitoring HDR at telomeres in a broad variety of mammalian cell types.

The FANCM-BLM-TOP3A-RMI complex suppresses alternative lengthening of telomeres (ALT)

“…BrdU pulldown was performed as described previously, with some adaptations 67 . Cells were harvested after pulsing with 100 μM BrdU (Sigma) for 5 h. Genomic DNA (gDNA) was extracted and resuspended in 100 μl of EB buffer (Qiagen).…”

Nuclear body phase separation drives telomere clustering in ALT cancer cells

“…Such an increase in PML localization to telomeres was not seen when the SIM mutant was recruited, agreeing with the hypothesis that SUMO–SIM interactions drive APB condensation. We then looked at proteins involved in the DNA damage response and repair pathways: 53BP1, PCNA, and POLD3, which localize to APBs induced by DNA damage ( Cho et al , 2014 ; Dilley et al , 2016 ; Verma et al , 2018 , 2019 ). None of these factors were recruited after dimerization-induced condensation ( Figure 4, D–F ; Figure 4–Supplemental Figure S1), indicating that they are recruited to the APB condensates via additional signals emanating from damaged DNA.…”

RAD52 and SLX4 act nonepistatically to ensure telomere stability during alternative telomere lengthening

“…1E-K;Supplemental Fig. S1A,B;Cho et al 2014;Dilley et al 2016;Verma et al 2018). Spontaneous mitotic telomere synthesis was present at higher levels in several ALT cell lines compared with telomerase lines (Supplemental Fig.…”

“…BrdU pull-down of 2 µg of gDNA was performed as described previously with two major differences (Dilley et al 2016;Verma et al 2018). First, sonication was omitted in order to preserve C-circle structure.…”