Structural and Dynamical Signatures of Local DNA Damage in Live Cells
The dynamic organization of chromatin inside the cell nucleus plays a key role in gene regulation, genome replication as well as maintaining genome integrity. While the static folded state of the genome has been extensively studied, dynamical signatures of processes such as transcription or DNA repair remain an open question.
Here, we investigate the interphase chromatin dynamics in human cells in response to local DNA damage, specifically, DNA double strand breaks (DSBs). Using simultaneous two-color spinning disk confocal microscopy, we monitor the DSB dynamics and the compaction of the surrounding chromatin, visualized by fluorescently labeled 53BP1 and histone H2B, respectively. Our study reveals a surprising difference between the mobility of DSBs located in the nuclear interior vs. periphery (less than 1 micron from the nuclear envelope), with the interior DSBs being almost twice as mobile as the periphery DSBs.
Remarkably, we find that the DSB sites possess a robust structural signature in a form of a unique chromatin compaction profile. Moreover, our data show that the DSB motion is subdiffusive, ATP-dependent and exhibiting unique dynamical signatures, different from those of undamaged chromatin. Our findings reveal that the DSB mobility follows a universal relationship defined solely by the physical parameters describing the DSBs and their local environment, such as the DSB focus size (represented by the local accumulation of 53BP1), DSB density and the local chromatin compaction.
This suggests that the DSB-related repair processes are robust and likely deterministic, as the observed dynamical signatures (DSB mobility) can be explained solely by their structural features (DSB focus size, local chromatin compaction). Such knowledge might help in detection of local DNA damage in live cells as well as aid our biophysical understanding of genome integrity in health and disease.
J. Eaton and A. Zidovska, Biophys. J., 118: 2168-2180 (2020)
Mechanical stress affects dynamics and rheology of the human genome
Material properties of the genome are critical for proper cellular function – they directly affect timescales and length scales of DNA transactions such as transcription, replication and DNA repair, which in turn impact all cellular processes via the central dogma of molecular biology. Hence, elucidating the genome's rheology in vivo may help reveal physical principles underlying the genome's organization and function. Here, we present a novel noninvasive approach to study the genome's rheology and its response to mechanical stress in form of nuclear injection in live human cells.
Specifically, we use Displacement Correlation Spectroscopy to map nucleus-wide genomic motions pre/post injection, during which we deposit rheological probes inside the cell nucleus. While the genomic motions inform on the bulk rheology of the genome pre/post injection, the probe's motion informs on the local rheology of its surroundings. Our results reveal that mechanical stress of injection leads to local as well as nucleus-wide changes in the genome's compaction, dynamics and rheology. We find that the genome pre-injection exhibits subdiffusive motions, which are coherent over several micrometers. In contrast, genomic motions post-injection become faster and uncorrelated, moreover, the genome becomes less compact and more viscous across the entire nucleus. In addition, we use the injected particles as rheological probes and find the genome to condense locally around them, mounting a local elastic response. Taken together, our results show that mechanical stress alters both dynamics and material properties of the genome. These changes are consistent with those observed upon DNA damage, suggesting that the genome experiences similar effects during the injection process.
C.M. Caragine, N. Kanellakopoulos and A. Zidovska, Soft Matter, 18: 107-116 (2022)