Measuring PARP1 mobility at DNA damage sites by Segmented Fluorescence Correlation Spectroscopy (FCS).

Abstract

Segmented Fluorescence Correlation Spectroscopy (FCS) improves the accuracy of FCS measurements in cells by analyzing data in short temporal segments. We have recently demonstrated the possibility of performing segmented FCS using a commercial confocal laser scanning microscope, enabling the measurement of molecular diffusion in different subcellular regions. In this study, we apply segmented FCS to investigate the dynamics of poly(ADP-ribose) polymerase 1 (PARP1), a protein playing a central role in DNA damage response. We perform fast line scanning across the nucleoplasm of live cells to measure the recruitment kinetics of PARP1 at DNA damage sites following laser microirradiation. The segmentation of FCS data allows us to distinguish between the damaged and the undamaged region, and to measure the mobility of PARP1 in the two regions. We find a reduced mobility of PARP1 at DNA damage sites, described as the appearance of a binding fraction, whereas the diffusion of PARP1 is unaltered outside the DNA damage region. Additionally, we investigate the effect of photobleaching on the measured dynamics.