Apc-mutant cells act as supercompetitors in intestinal tumour initiation.

A delicate equilibrium of WNT agonists and antagonists in the intestinal stem cell (ISC) niche is critical to maintaining the ISC compartment, as it accommodates the rapid renewal of the gut lining. Disruption of this balance by mutations in the tumour suppressor gene APC, which are found in approximately 80% of all human colon cancers, leads to unrestrained activation of the WNT pathway. It has previously been established that Apc-mutant cells have a competitive advantage over wild-type ISCs.

Chromatin regulators and their impact on DNA repair and G2 checkpoint recovery.

Chromatin plays a pivotal role in regulating the DNA damage response and during DNA double-strand break repair. Upon the generation of DNA breaks, the chromatin structure is altered by post-translational modifications of histones and chromatin remodeling. How the chromatin structure, and the epigenetic information that it carries, is reestablished after the completion of DNA break repair remains unclear though.

Keeping ribosomal DNA intact: a repeating challenge.

More than half of the human genome consists of repetitive sequences, with the ribosomal DNA (rDNA) representing two of the largest repeats. Repetitive rDNA sequences may form a threat to genomic integrity and cellular homeostasis due to the challenging aspects of their transcription, replication, and repair. Predisposition to cancer, premature aging, and neurological impairment in ataxia-telangiectasia and Bloom syndrome, for instance, coincide with increased cellular rDNA repeat instability.

Identification of Two Protein-Signaling States Delineating Transcriptionally Heterogeneous Human Medulloblastoma.

The brain cancer medulloblastoma consists of different transcriptional subgroups. To characterize medulloblastoma at the phosphoprotein-signaling level, we performed high-throughput peptide phosphorylation profiling on a large cohort of SHH (Sonic Hedgehog), group 3, and group 4 medulloblastomas. We identified two major protein-signaling profiles.

Doxorubicin-induced DNA Damage Causes Extensive Ubiquitination of Ribosomal Proteins Associated with a Decrease in Protein Translation.

Protein posttranslational modifications (PTMs) play a central role in the DNA damage response. In particular, protein phosphorylation and ubiquitination have been shown to be essential in the signaling cascade that coordinates break repair with cell cycle progression. Here, we performed whole-cell quantitative proteomics to identify global changes in protein ubiquitination that are induced by DNA double-strand breaks.

Breaks in the 45S rDNA Lead to Recombination-Mediated Loss of Repeats.

rDNA repeats constitute the most heavily transcribed region in the human genome. Tumors frequently display elevated levels of recombination in rDNA, indicating that the repeats are a liability to the genomic integrity of a cell. However, little is known about how cells deal with DNA double-stranded breaks in rDNA.

The core spliceosome as target and effector of non-canonical ATM signalling.

In response to DNA damage, tissue homoeostasis is ensured by protein networks promoting DNA repair, cell cycle arrest or apoptosis. DNA damage response signalling pathways coordinate these processes, partly by propagating gene-expression-modulating signals. DNA damage influences not only the abundance of messenger RNAs, but also their coding information through alternative splicing.

UV-induced G2 checkpoint depends on p38 MAPK and minimal activation of ATR-Chk1 pathway.

In response to UV light, single-stranded DNA intermediates coated with replication protein A (RPA) are generated, which trigger the ATR-Chk1 checkpoint pathway. Recruitment and/or activation of several checkpoint proteins at the damaged sites is important for the subsequent cell cycle arrest. Surprisingly, upon UV irradiation, Rad9 and RPA only minimally accumulate at DNA lesions in G2 phase, suggesting that only a few single-stranded DNA intermediates are generated.

Differential Dynamics of ATR-Mediated Checkpoint Regulators.

The ATR-Chk1 checkpoint pathway is activated by UV-induced DNA lesions and replication stress. Little was known about the spatio and temporal behaviour of the proteins involved, and we, therefore, examined the behaviour of the ATRIP-ATR and Rad9-Rad1-Hus1 putative DNA damage sensor complexes and the downstream effector kinase Chk1. We developed assays for the generation and validation of stable cell lines expressing GFP-fusion proteins.

Mechanisms of ATR-mediated checkpoint signalling.

Cell cycle checkpoints maintain genomic integrity by delaying cell division in the presence of DNA damage or replication problems. A crucial player in this process is the ATR kinase. The rapid localisation of ATR to sites of genotoxic stress and the central role of this kinase in the checkpoint response lead to the suggestion that ATR functions as a sensor of DNA lesions.

Stochastic and reversible assembly of a multiprotein DNA repair complex ensures accurate target site recognition and efficient repair.

To understand how multiprotein complexes assemble and function on chromatin, we combined quantitative analysis of the mammalian nucleotide excision DNA repair (NER) machinery in living cells with computational modeling. We found that individual NER components exchange within tens of seconds between the bound state in repair complexes and the diffusive state in the nucleoplasm, whereas their net accumulation at repair sites evolves over several hours. Based on these in vivo data, we developed a predictive kinetic model for the assembly and function of repair complexes.

Dealing with DNA damage: relationships between checkpoint and repair pathways.

Cell cycle checkpoint activation and DNA repair pathways govern genomic stability after genotoxic stress. Genotoxic insult results in activation of an interwoven network of DNA damage checkpoints and DNA repair pathways. Post-translational modifications on a number of proteins involved in both checkpoint activation and DNA repair play an important role in this cellular response.

Heterochromatin protein 1 is recruited to various types of DNA damage.

Heterochromatin protein 1 (HP1) family members are chromatin-associated proteins involved in transcription, replication, and chromatin organization. We show that HP1 isoforms HP1-alpha, HP1-beta, and HP1-gamma are recruited to ultraviolet (UV)-induced DNA damage and double-strand breaks (DSBs) in human cells. This response to DNA damage requires the chromo shadow domain of HP1 and is independent of H3K9 trimethylation and proteins that detect UV damage and DSBs.

Cell cycle-dependent processing of DNA lesions controls localization of Rad9 to sites of genotoxic stress.

The Rad9/Rad1/Hus1 complex functions to facilitate the ATR-mediated phosphorylation of several substrates that control the checkpoint arrest induced by DNA damage. Here we show that in response to genotoxic stress induced by different types of damaging agents, Rad9 rapidly relocalized to sites of single stranded DNA, as visualized by discrete nuclear foci that co-localize with RPA. UV light-induced Rad9 foci also colocalized with TopBP1 and gamma-H2AX.

ATR and Rad17 collaborate in modulating Rad9 localisation at sites of DNA damage.

The cell cycle checkpoint kinase Chk1 is phosphorylated and activated by ATR in response to DNA damage and is crucial for initiating the DNA damage response. A number of factors act in concert with ATR to facilitate Chk1 phosphorylation, including Rad17-RFC, the Rad9-Rad1-Hus1 complex, TopBP1 and Claspin. Rad17 is required for loading of Rad9-Rad1-Hus1 (9-1-1) onto sites of DNA damage.

From DNA end chemistry to cell-cycle response: the importance of structure, even when it's broken.

In this issue of Molecular Cell, Barlow et al. (2008) show that not all DNA double-strand breaks are processed equally and that the chemical nature of DNA ends guides different paths to DNA repair.

Recruitment of the nucleotide excision repair endonuclease XPG to sites of UV-induced dna damage depends on functional TFIIH.

The structure-specific endonuclease XPG is an indispensable core protein of the nucleotide excision repair (NER) machinery. XPG cleaves the DNA strand at the 3' side of the DNA damage. XPG binding stabilizes the NER preincision complex and is essential for the 5' incision by the ERCC1/XPF endonuclease.