DNA replication and repair kinetics of Alu, LINE-1 and satellite III genomic repetitive elements.

BACKGROUND: Preservation of genome integrity by complete, error-free DNA duplication prior to cell division and by correct DNA damage repair is paramount for the development and maintenance of an organism. This holds true not only for protein-encoding genes, but also it applies to repetitive DNA elements, which make up more than half of the human genome. Here, we focused on the replication and repair kinetics of interspersed and tandem repetitive DNA elements. RESULTS: We integrated genomic population level data with a single cell immunofluorescence in situ hybridization approach to simultaneously label replication/repair and repetitive DNA elements. We found that: (1) the euchromatic Alu element was replicated during early S-phase; (2) LINE-1, which is associated with AT-rich genomic regions, was replicated throughout S-phase, with the majority being replicated according to their particular histone marks; (3) satellite III, which constitutes pericentromeric heterochromatin, was replicated exclusively during the mid-to-late S-phase. As for the DNA double-strand break repair process, we observed that Alu elements followed the global genome repair kinetics, while LINE-1 elements repaired at a slower rate. Finally, satellite III repeats were repaired at later time points. CONCLUSIONS: We conclude that the histone modifications in the specific repeat element predominantly determine its replication and repair timing. Thus, Alu elements, which are characterized by euchromatic chromatin features, are repaired and replicated the earliest, followed by LINE-1 elements, including more variegated eu/heterochromatic features and, lastly, satellite tandem repeats, which are homogeneously characterized by heterochromatic features and extend over megabase-long genomic regions. Altogether, this work reemphasizes the need for complementary approaches to achieve an integrated and comprehensive investigation of genomic processes.

Identification of the elementary structural units of the DNA damage response.

Histone H2AX phosphorylation is an early signalling event triggered by DNA double-strand breaks (DSBs). To elucidate the elementary units of phospho-H2AX-labelled chromatin, we integrate super-resolution microscopy of phospho-H2AX during DNA repair in human cells with genome-wide sequencing analyses. Here we identify phospho-H2AX chromatin domains in the nanometre range with median length of ∼75 kb. Correlation analysis with over 60 genomic features shows a time-dependent euchromatin-to-heterochromatin repair trend. After X-ray or CRISPR-Cas9-mediated DSBs, phospho-H2AX-labelled heterochromatin exhibits DNA decondensation while retaining heterochromatic histone marks, indicating that chromatin structural and molecular determinants are uncoupled during repair. The phospho-H2AX nano-domains arrange into higher-order clustered structures of discontinuously phosphorylated chromatin, flanked by CTCF. CTCF knockdown impairs spreading of the phosphorylation throughout the 3D-looped nano-domains. Co-staining of phospho-H2AX with phospho-Ku70 and TUNEL reveals that clusters rather than nano-foci represent single DSBs. Hence, each chromatin loop is a nano-focus, whose clusters correspond to previously known phospho-H2AX foci.