The in vivo dynamic interplay of MDC1 and 53BP1 at DNA damage-induced nuclear foci

Abstract

MDC1 (NFBD1) and 53BP1 are critical mediators of the mammalian DNA damage response (DDR) at nuclear foci. Here we show by quantitative imaging assays that MDC1 and 53BP1 are similar in total copy number (∼1200 copies per focus), but differ substantially in dynamics at both replication-associated nuclear bodies in normal cells and DNA repair foci in ionizing radiation (IR)-damaged cells. The majority of MDC1 (∼80%) is extremely mobile and under continuous exchange, with only a small fraction (∼20%) remaining immobile at foci irrespective of IR treatment. By contrast, 53BP1 has a smaller mobile fraction (∼35%) and a larger immobile fraction (∼65%) at nuclear bodies, and becomes more dynamic (∼20% increase in mobile pool) upon IR-induced DNA damage. More specifically, the dynamics of 53BP1 is dependent on a minimal foci-targeting region (1231–1709), and differentially regulated by its N-terminus (1–1231) and C-terminal tBRCT domain (1709–1972). Furthermore, MDC1 knockdown, or disruption of 53BP1–MDC1 interaction, reduced the number of 53BP1 molecules at foci by ∼60%, but only modestly affected 53BP1 retention. This novel in vivo evidence reveals distinct dynamics of MDC1 and 53BP1 at different types of nuclear structures, and shows that MDC1 directly recruits and retains a subset of 53BP1 for DNA repair.