Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

The overall goal of this protocol is to describe how to use laser microirradiation to induce different types of DNA damage, including simple strand breaks, and complex damage, to study DNA damage signaling and repair factor assembly in vivo. In the 1960s and 70s, we focused a laser beam through a microscope that you can see behind me, and was able to damage a small region of a chromosome. The cell divided, survived, and we couldn't understand why.

Well, 40 years later, we are now using this method to selectively study DNA repair, and that's what you're going to see today. This method can help answer key questions in the DNA repair field, such as how DNA damage is recognized and processed and how damage signal propagates in the cell. Laser microirradiation allows for real time, high resolution, single cell analysis of macromolecular dynamics in response to DNA damage confounded to a several micrometer region within the cell nucleus.