Endogenous formaldehyde is produced by numerous biochemical pathways fundamental to life, and it can crosslink both DNA and proteins. However, the consequences of its accumulation are unclear. Here we show that endogenous formaldehyde is removed by the enzyme alcohol dehydrogenase 5 (ADH5/GSNOR), and Adh5(-/-) mice therefore accumulate formaldehyde adducts in DNA. The repair of this damage is mediated by FANCD2, a DNA crosslink repair protein. Adh5(-/-)Fancd2(-/-) mice reveal an essential requirement for these protection mechanisms in hematopoietic stem cells (HSCs), leading to their depletion and precipitating bone marrow failure. More widespread formaldehyde-induced DNA damage also causes karyomegaly and dysfunction of hepatocytes and nephrons. Bone marrow transplantation not only rescued hematopoiesis but, surprisingly, also preserved nephron function. Nevertheless, all of these animals eventually developed fatal malignancies. Formaldehyde is therefore an important source of endogenous DNA damage that is counteracted in mammals by a conserved protection mechanism.

Co-transcriptional RNA-DNA hybrids (R loops) cause genome instability. To prevent harmful R loop accumulation, cells have evolved specific eukaryotic factors, one being the BRCA2 double-strand break repair protein. As BRCA2 also protects stalled replication forks and is the FANCD1 member of the Fanconi Anemia (FA) pathway, we investigated the FA role in R loop-dependent genome instability. Using human and murine cells defective in FANCD2 or FANCA and primary bone marrow cells from FANCD2 deficient mice, we show that the FA pathway removes R loops, and that many DNA breaks accumulated in FA cells are R loop-dependent. Importantly, FANCD2 foci in untreated and MMC-treated cells are largely R loop dependent, suggesting that the FA functions at R loop-containing sites. We conclude that co-transcriptional R loops and R loop-mediated DNA damage greatly contribute to genome instability and that one major function of the FA pathway is to protect cells from R loops. R loops are co-transcriptional RNA-DNA hybrids that can have a physiological role in transcription and replication, but also may be a major threat to genome stability. To avoid the deleterious effects of R loops, specific factors prevent their formation or facilitate their removal. The double-strand break repair factor BRCA2 is among those that prevent R-loop accumulation. As BRCA2 also protects stalled replication forks and is the FANCD1 member of the Fanconi Anemia (FA) pathway, we studied the role of this pathway in preventing R loop accumulation and R loop-dependent genome instability. Using human and murine cells defective in FANCD2 or FANCA and primary bone marrow cells derived from FANCD2 deficient mice, we show that the FA pathway removes R loops and that many DNA breaks accumulated in FA cells are R loop-dependent. Importantly, FANCD2 foci accumulation is largely R loop-dependent, suggesting that the FA functions at R loop-containing sites. The FA pathway is primarily known as a DNA interstrand crosslinks (ICLs) repair pathway. Our findings reveal a novel function of the FA pathway in preventing R loop-mediated DNA damage, providing new clues to understand the relevance of R-loops as a natural source of genome instability and the way they are processed.

Righting Repair Pathways: The genetic disease Fanconi anemia (FA) results from mutations in a series of genes involved in a DNA repair pathway that helps process the damage caused by erroneous chemical cross-links between the two strands of the DNA double helix. The double-stranded breaks in DNA that arise from such cross-links can be repaired in an error-free manner or through an error-prone repair pathway. Pace et al. (p. 219 , published online 10 June) show that the FA pathway can drive repair through the error-free pathway. The FA FANCC gene shows a genetic interaction with a component of the error-prone repair pathway, Ku70, inhibiting its action and thereby promoting the error-free pathway.

Ketan Patel and colleagues report a new mouse model of Fanconi anemia. The authors show that mice deficient in Btbd12, the mouse ortholog of SLX4 (a new Fanconi anemia gene), phenocopy the human disease. The evolutionarily conserved SLX4 protein, a key regulator of nucleases, is critical for DNA damage response. SLX4 nuclease complexes mediate repair during replication and can also resolve Holliday junctions formed during homologous recombination. Here we describe the phenotype of the Btbd12 knockout mouse, the mouse ortholog of SLX4, which recapitulates many key features of the human genetic illness Fanconi anemia. Btbd12-deficient animals are born at sub-Mendelian ratios, have greatly reduced fertility, are developmentally compromised and are prone to blood cytopenias. Btbd12−/− cells prematurely senesce, spontaneously accumulate damaged chromosomes and are particularly sensitive to DNA crosslinking agents. Genetic complementation reveals a crucial requirement for Btbd12 (also known as Slx4) to interact with the structure-specific endonuclease Xpf-Ercc1 to promote crosslink repair. The Btbd12 knockout mouse therefore establishes a disease model for Fanconi anemia and genetically links a regulator of nuclease incision complexes to the Fanconi anemia DNA crosslink repair pathway.