Genome integrity is constantly challenged by various processes including DNA damage, structured DNA, transcription, and DNA-protein crosslinks. During DNA replication, active replication forks that encounter these obstacles can result in their stalling and collapse. Accurate DNA replication requires the ability of forks to navigate these threats, which is aided by DNA repair proteins. Histone acetylation participates in this process through an ability to signal and recruit proteins to regions of replicating DNA. For example, the histone acetyltransferase PCAF promotes the recruitment of the DNA repair factors MRE11 and EXO1 to stalled forks by acetylating histone H4 at lysine 8 (H4K8ac). These highly dynamic processes can be detected and analyzed using a modified proximity ligation assay (PLA) method, known as SIRF (in situ protein interactions with nascent DNA replication forks). This single-cell assay combines PLA with EdU-coupled Click-iT chemistry reactions and fluorescence microscopy to detect these interactions at sites of replicating DNA. Here we provide a detailed protocol utilizing SIRF that detects the HAT PCAF and histone acetylation at replication forks. This technique provides a robust methodology to determine protein recruitment and modifications at the replication fork with single-cell resolution.
Keywords: DNA replication; Genome integrity; Histone acetylation; PCAF; PLA; Replication stress; SIRF; Stalled replication forks.
© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.