Abstract
The cellular response to DNA damage can be marked by the appearance of immunofluorescent foci of DNA Damage Response (DDR) proteins. These serve as surrogates for DNA damage and are frequently interpreted as denoting specific lesions. For example, double-strand breaks (DSBs) are potent inducers of the DDR, whose best-known factor is the phosphorylated histone variant H2AX (γ-H2AX). The association with DSBs is so well established that the reverse interpretation that γ-H2AX invariably implies DSBs is routine. However, this conclusion is inferential and has been challenged. The resolution of this question has been hampered by the lack of methods for distinguishing the location of DDR proteins relative to DSBs caused by sequence indifferent agents. Here, we describe an approach for marking the location of DDR factors in relation to DSBs on DNA fibers, illustrated by γ-H2AX. We synthesized a two-arm "Y" conjugate containing biotin and trimethylpsoralen (TMP) coupled to a secondary antibody. After exposure to ionizing radiation, which introduces multiple lesions, including DSBs, cells were fixed, permeabilized, and incubated with a primary antibody against γ-H2AX followed by binding of the conjugated secondary antibody to the primary antibody. Exposure to long-wave UV light covalently linked psoralen to the DNA. DNA fibers were spread, and the immunofluorescence of the biotin tag indicated the location of the target protein. This technique denotes the location of γ-H2AX along DNA fibers following ionizing irradiation of the cells and the relationship between DSBs and γ-H2AX. Our results demonstrate that, after radiation, most γ-H2AX signals detected on DNA fibers are located at internal sites rather than at ends.