Westmead Institute for Cancer Research, The University of Sydney, Westmead Millennium Institute at Westmead Hospital, Westmead, NSW 2145, Australia.
Int J Biochem Cell Biol. 2012 Sep;44(9):1398-409. doi: 10.1016/j.biocel.2012.05.025. Epub 2012 Jun 5.
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.
MDC1（NFBD1）和 53BP1 是哺乳动物 DNA 损伤反应（DDR）在核斑点中的关键介质。通过定量成像分析，我们发现 MDC1 和 53BP1 的总拷贝数相似（每个斑点 1200 个拷贝），但在正常细胞的复制相关核体和电离辐射（IR）损伤细胞中的 DNA 修复斑点中的动力学有很大差异。大多数 MDC1（80%）非常活跃，处于连续交换状态，只有一小部分（20%）在无论是否受到 IR 处理的情况下在斑点中保持不活跃。相比之下，53BP1 在核体中的活跃部分（35%）较小，不活跃部分（~65%）较大，在 IR 诱导的 DNA 损伤后变得更加活跃（活跃池增加约 20%）。更具体地说，53BP1 的动力学依赖于最小的斑点靶向区域（1231-1709），并由其 N 端（1-1231）和 C 端 tBRCT 结构域（1709-1972）差异调节。此外，MDC1 的敲低或 53BP1-MDC1 相互作用的破坏，使斑点处的 53BP1 分子数量减少了约 60%，但仅适度影响 53BP1 的保留。这种新的体内证据揭示了 MDC1 和 53BP1 在不同类型的核结构中的不同动力学，并表明 MDC1 直接招募并保留了一小部分 53BP1 用于 DNA 修复。
