The ATM repair pathway inhibits RNA polymerase I transcription in response to chromosome breaks

Experimental Immunology, NCI.
Nature (Impact Factor: 41.46). 07/2007; 447(7145):730-4. DOI: 10.1038/nature05842
Source: PubMed


DNA lesions interfere with DNA and RNA polymerase activity. Cyclobutane pyrimidine dimers and photoproducts generated by ultraviolet irradiation cause stalling of RNA polymerase II, activation of transcription-coupled repair enzymes, and inhibition of RNA synthesis. During the S phase of the cell cycle, collision of replication forks with damaged DNA blocks ongoing DNA replication while also triggering a biochemical signal that suppresses the firing of distant origins of replication. Whether the transcription machinery is affected by the presence of DNA double-strand breaks remains a long-standing question. Here we monitor RNA polymerase I (Pol I) activity in mouse cells exposed to genotoxic stress and show that induction of DNA breaks leads to a transient repression in Pol I transcription. Surprisingly, we find Pol I inhibition is not itself the direct result of DNA damage but is mediated by ATM kinase activity and the repair factor proteins NBS1 (also known as NLRP2) and MDC1. Using live-cell imaging, laser micro-irradiation, and photobleaching technology we demonstrate that DNA lesions interfere with Pol I initiation complex assembly and lead to a premature displacement of elongating holoenzymes from ribosomal DNA. Our data reveal a novel ATM/NBS1/MDC1-dependent pathway that shuts down ribosomal gene transcription in response to chromosome breaks.

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Available from: Michael J Kruhlak, May 20, 2015
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    • "Consistent with this hypothesis, generation of several DSBs distal to the promoter of a reporter gene in mammalian cells leads to ATM-dependent transcriptional repression of this reporter gene (Shanbhag et al., 2010), possibly through phosphorylation of the transcriptional elongation factor ENL (Ui et al., 2015). Similarly, RNA polymerase I-mediated transcription of rDNA is inhibited in an ATM-dependent manner in the vicinity of DSBs (Kruhlak et al., 2007). Furthermore, the steady state RNA levels of genes proximal to a single DSB have been observed to decrease by microarray analysis also in S. cerevisiae cells (Lee et al., 2000). "
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    ABSTRACT: eLife digest DNA is constantly under assault from harmful chemicals; some of which are produced inside the cell, while others come from outside of the cell. Breaks that form across both strands in a DNA double helix are considered the most dangerous type of DNA damage, and can cause a cell to die or become cancerous if they are not repaired accurately. ‘Homologous recombination’ is one of the main mechanisms used by cells to repair DNA double-strand breaks. This mechanism requires enzymes to eat away at the end of one of the DNA strands on each side of the double-strand break. This process is called ‘resection’ and it exposes single strands of DNA. These single-stranded DNA ‘tails’ are then free to interact with an intact copy of the same DNA sequence from elsewhere in the cell's nucleus, which is used as a guide when repairing the damage. The proteins involved in homologous recombination have to work around other processes that go on inside the nucleus, such as the transcription of DNA in genes into RNA molecules. Previous research has reported that forming a double-strand break in the DNA reduces the levels of transcription for the genes that surround the break, but it was not clear how this occurred. In mammalian cells, inhibiting the transcription of genes around a double-strand DNA break depends on a signaling pathway that is activated whenever DNA damage is detected. Manfrini et al. now show that this is not the case for budding yeast (Saccharomyces cerevisiae). Instead, the experiments indicate that it is the resection of the DNA around a double-strand break to form single-stranded tails that inhibits transcription in budding yeast. One of the next challenges will be to see if the resection process makes any contribution to changes in the transcription of genes that surround a double-strand break in mammals as well. DOI:
    Full-text · Article · Jul 2015 · eLife Sciences
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    • "By combining these results with our recent work on the genomic architecture of NORs, we can now present a model for how integrity of rDNA arrays is maintained against an onslaught of DNA damage (Fig. 7). While the use of γ-irradiation and laser microirradiation to induce DSBs has yielded conflicting results (Kruhlak et al. 2007; Moore et al. 2011; Larsen et al. 2014), here we unequivocally demonstrated that DSBs within rDNA are sufficient to induce ATM-dependent inhibition of Pol I transcription. The introduction of the CRISPR/Cas9 allowed us to demonstrate that DSBs in both transcribed and nontranscribed regions of the rDNA repeat induce this response. "
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    ABSTRACT: DNA double-strand breaks (DSBs) are repaired by two main pathways: nonhomologous end-joining and homologous recombination (HR). Repair pathway choice is thought to be determined by cell cycle timing and chromatin context. Nucleoli, prominent nuclear subdomains and sites of ribosome biogenesis, form around nucleolar organizer regions (NORs) that contain rDNA arrays located on human acrocentric chromosome p-arms. Actively transcribed rDNA repeats are positioned within the interior of the nucleolus, whereas sequences proximal and distal to NORs are packaged as heterochromatin located at the nucleolar periphery. NORs provide an opportunity to investigate the DSB response at highly transcribed, repetitive, and essential loci. Targeted introduction of DSBs into rDNA, but not abutting sequences, results in ATM-dependent inhibition of their transcription by RNA polymerase I. This is coupled with movement of rDNA from the nucleolar interior to anchoring points at the periphery. Reorganization renders rDNA accessible to repair factors normally excluded from nucleoli. Importantly, DSBs within rDNA recruit the HR machinery throughout the cell cycle. Additionally, unscheduled DNA synthesis, consistent with HR at damaged NORs, can be observed in G1 cells. These results suggest that HR can be templated in cis and suggest a role for chromosomal context in the maintenance of NOR genomic stability. © 2015 van Sluis and McStay; Published by Cold Spring Harbor Laboratory Press.
    Full-text · Article · Jun 2015 · Genes & Development
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    • "Interestingly, Treacle, like MDC1, contains several phoshorylated SDT domains that mediate NBS1 binding, suggesting a conserved mechanism of NBS1 retention in the nucleus and nucleolus. It is notable that while MDC1 is not required for NBS1 retention to nucleoli, MDC1 deficient cells failed to silence rDNA transcription after IR treatment [57]. This may reflect a difference between the techniques used, as IR could induce damage directly in nucleolar DNA, or could indicate that MRE11 complex localization per se is not sufficient for rDNA silencing. "
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    ABSTRACT: Newly identified interactions with the FHA and BRCT domains of NBS1 influence subcellular localization of the MRE11 complex.•The MRE11 complex promotes TOPBP1 recruitment and ATR activation during replication stress.•The MRE11 complex is a barrier to oncogene-induced tumorigenesis.
    Full-text · Article · Oct 2014 · Experimental Cell Research
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