Clusters of transcription-coupled repair in the human genome

Abstract

A specialized nucleotide excision repair pathway known as transcription-coupled repair (TCR) counteracts the toxic effects of DNA damage in transcriptionally active genes. The clustering of active genes into gene-rich chromosomal domains predicts that the sites of TCR are unevenly distributed through the genome. To elucidate the genomic organization and chromosomal localization of TCR, we isolated DNA fragments encompassing TCR-mediated repair sites from UV-C irradiated xeroderma pigmentosum group C cells, which can only repair the transcribed strand of active genes. This DNA was used as a molecular probe to visualize TCR in normal metaphase spreads by reverse fluorescence in situ hybridization. Whereas DNA repair sites in normal human cells are evenly distributed through the genome, TCR is highly localized at specific chromosomal domains. Particularly, clusters of TCR sites were identified at early-replicating gene-rich bands and telomeric regions of several chromosomes. High gene-density chromosomes such as chromosome 19 and the GC-rich domains of several chromosomes (T bands) are preferential locations of TCR. Our results demonstrate that the intragenomic localization of TCR resembles the uneven distribution of the human transcriptome, CpG islands, and hyperacetylated histones, enforcing the basic link between DNA repair, transcription, and nuclear organization in a complex genome.

Fig 1.

Chromosomal distribution of nucleotide excision repair. Chromosomal distribution of unrepaired (a and c) and repaired (b and d) fractions isolated from wild-type (a and b) and XPC (c and d) cells 24 h after UV irradiation. Both fractions were isolated as described in Materials and Methods; DOP-PCR was labeled with biotin and detected with Alexa 488-conjugated streptavidin as green fluorescence. Chromosomes are counterstained in red color with propidium iodine. Unrepaired and repaired fractions extracted from wild-type cells colored all chromosomes in a similar fashion (a and b). A similar pattern was obtained with the unrepaired fraction in XPC (c). The repaired fraction from XPC cells produced a specific banding pattern indicating clustering of TCR.

Fig 2.

Clusters of sites of TCR at early-replicating chromosomal domains and T bands. (A) Clusters of sites of TCR at early-replicating chromosomal domains. Simultaneous detection of late-replicating DNA with FITC antibodies against BrdUrd incorporated at late S-phase (green) and of the repaired fraction from UV-irradiated XPC cells. The repaired DNA was visualized by FISH after random primed labeling of the probe and immunodetection with tetramethylrhodamine β-isothiocyanate-conjugated antibodies (red). Chromosome 1 (a–c), 4 (d–f), and 19 (g–i) are shown for late-replication banding in green (a, d, and g), repaired DNA in red (b, e, and h), and the merged image (c, f, and i). (B) Clustering of TCR at T bands. Clustering of TCR in red at T bands 11p15, 11q13, 11q23 (a), and 21q22 (b). As in a, green fluorescence indicates late-replicating bands, and the sites of TCR are shown in red fluorescence.