Visualization of Replisome Encounters with an Antigen Tagged Blocking Lesion

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Cellular responses to DNA double-strand breaks (DSBs) are well-studied, with established methods for identifying break sites and recruited factors.
• However, technologies for analyzing interactions with repair proteins during the processing of DNA adducts, like interstrand crosslinks (ICLs), are lacking.
• ICLs, formed by common cancer chemotherapeutics, can lead to breaks upon encountering replication forks, but their interactions with replication proteins remain unmonceived.

Purpose of the Study:

• To develop a novel methodology for monitoring cellular responses to DNA interstrand crosslinks (ICLs) during replication fork encounters.
• To visualize ICLs within living cells and identify proteins closely associated with these DNA adducts.

Main Methods:

• A steroid antigen was conjugated to psoralen to create photoactivation-dependent ICLs in cell nuclei.
• Immunofluorescence was used to visualize the ICLs via the antigen tag.
• Proximity Ligation Assay (PLA) was employed, utilizing the antigen tag to detect proteins in close proximity to the ICLs.

Main Results:

• The study successfully visualized ICLs using immunofluorescence against the antigen tag.
• The Proximity Ligation Assay (PLA) effectively distinguished proteins associated with tagged ICLs from those that were not.
• Key replisome proteins retained and lost upon encountering ICLs were identified.

Conclusions:

• A novel strategy was established for tracking cellular responses to DNA interstrand crosslinks (ICLs).
• This approach allows for the characterization of protein interactions with ICLs and other immunologically detectable DNA adducts.
• The methodology offers a powerful tool for studying DNA repair mechanisms and the effects of genotoxic agents.