Live imaging of cell division in 3D stem-cell organoid cultures.

Examining cell behavior in its correct tissue context is a major challenge in cell biology. The recent development of mammalian stem cell-based organoid cultures offers exciting opportunities to visualize dynamic cellular events in a 3D tissue-like setting. We describe here an approach for live imaging of cell division processes in intestinal organoid cultures derived from human and mouse adult stem cells.

Targeting mutant RAS in patient-derived colorectal cancer organoids by combinatorial drug screening.

Colorectal cancer (CRC) organoids can be derived from almost all CRC patients and therefore capture the genetic diversity of this disease. We assembled a panel of CRC organoids carrying either wild-type or mutant RAS, as well as normal organoids and tumor organoids with a CRISPR-introduced oncogenic mutation. Using this panel, we evaluated RAS pathway inhibitors and drug combinations that are currently in clinical trial for RAS mutant cancers.

Sequential cancer mutations in cultured human intestinal stem cells.

Crypt stem cells represent the cells of origin for intestinal neoplasia. Both mouse and human intestinal stem cells can be cultured in medium containing the stem-cell-niche factors WNT, R-spondin, epidermal growth factor (EGF) and noggin over long time periods as epithelial organoids that remain genetically and phenotypically stable. Here we utilize CRISPR/Cas9 technology for targeted gene modification of four of the most commonly mutated colorectal cancer genes (APC, P53 (also known as TP53), KRAS and SMAD4) in cultured human intestinal stem cells.

DNA damage response and transcription.

A network of DNA damage surveillance systems is triggered by sensing of DNA lesions and the initiation of a signal transduction cascade that activates genome-protection pathways including nucleotide excision repair (NER). NER operates through coordinated assembly of repair factors into pre- and post-incision complexes. Recent work identifies RPA as a key regulator of the transition from dual incision to repair-synthesis in UV-irradiated non-cycling cells, thereby averting the generation of unprocessed repair intermediates.

Replication protein A safeguards genome integrity by controlling NER incision events.

Single-stranded DNA gaps that might arise by futile repair processes can lead to mutagenic events and challenge genome integrity. Nucleotide excision repair (NER) is an evolutionarily conserved repair mechanism, essential for removal of helix-distorting DNA lesions. In the currently prevailing model, NER operates through coordinated assembly of repair factors into pre- and post-incision complexes; however, its regulation in vivo is poorly understood.

UV-induced photolesions elicit ATR-kinase-dependent signaling in non-cycling cells through nucleotide excision repair-dependent and -independent pathways.

Activation of signaling pathways by UV radiation is a key event in the DNA damage response and initiated by different cellular processes. Here we show that non-cycling cells proficient in nucleotide excision repair (NER) initiate a rapid but transient activation of the damage response proteins p53 and H2AX; by contrast, NER-deficient cells display delayed but persistent signaling and inhibition of cell cycle progression upon release from G0 phase. In the absence of repair, UV-induced checkpoint activation coincides with the formation of single-strand DNA breaks by the action of the endonuclease Ape1.

Replication factor C recruits DNA polymerase delta to sites of nucleotide excision repair but is not required for PCNA recruitment.

Nucleotide excision repair (NER) operates through coordinated assembly of repair factors into pre- and postincision complexes. The postincision step of NER includes gap-filling DNA synthesis and ligation. However, the exact composition of this NER-associated DNA synthesis complex in vivo and the dynamic interactions of the factors involved are not well understood.

Three DNA polymerases, recruited by different mechanisms, carry out NER repair synthesis in human cells.

Nucleotide excision repair (NER) is the most versatile DNA repair system that deals with the major UV photoproducts in DNA, as well as many other DNA adducts. The early steps of NER are well understood, whereas the later steps of repair synthesis and ligation are not. In particular, which polymerases are definitely involved in repair synthesis and how they are recruited to the damaged sites has not yet been established.

RNF4 and VHL regulate the proteasomal degradation of SUMO-conjugated Hypoxia-Inducible Factor-2alpha.

Hypoxia-inducible factors (HIFs) are critical transcription factors that mediate cell survival during reduced oxygen conditions (hypoxia). At regular oxygen conditions (normoxia), HIF-1alpha and HIF-2alpha are continuously synthesized in cells and degraded via the ubiquitin-proteasome pathway. During hypoxia, these proteins are stabilized and translocate to the nucleus to activate transcription of target genes that enable cell survival at reduced oxygen levels.

Chinese hamster cell mutant, V-C8, a model for analysis of Brca2 function.

The previously described Chinese hamster cell mutant V-C8 that is defective in Brca2 shows a very complex phenotype, including increased sensitivity towards a wide variety of DNA damaging agents, chromosomal instability, abnormal centrosomes and impaired formation of Rad51 foci in response to DNA damage. Here, we demonstrate that V-C8 cells display biallelic nonsense mutations in Brca2, one in exon 15 and the other in exon 16, both resulting in truncated Brca2 proteins. We generated several independent mitomycin C (MMC)-resistant clones from V-C8 cells that had acquired an additional mutation leading to the restoration of the open reading frame of one of the Brca2 alleles.

The Arabidopsis AtLIG4 gene is required for the repair of DNA damage, but not for the integration of Agrobacterium T-DNA.

The joining of breaks in the chromosomal DNA backbone by ligases in processes of replication, recombination and repair plays a crucial role in the maintenance of genomic stability. Four ATP-dependent ligases, designated DNA ligases I-IV, have been identified in higher eukaryotes, and each one has distinct functions. In mammals and yeast, DNA ligase IV is exclusively involved in the repair of DNA double-strand breaks by non-homologous end joining.