Measuring PARP1 mobility at DNA damage sites by segmented fluorescence correlation spectroscopy

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 that plays a central role in the 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 after laser micro-irradiation. The segmentation of FCS data allows us to distinguish between the damaged and undamaged regions and to measure the mobility of PARP1 in the two regions. We find 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.