Two Aldehyde Clearance Systems Are Essential to Prevent Lethal Formaldehyde Accumulation in Mice and Humans.

Reactive aldehydes arise as by-products of metabolism and are normally cleared by multiple families of enzymes. We find that mice lacking two aldehyde detoxifying enzymes, mitochondrial ALDH2 and cytoplasmic ADH5, have greatly shortened lifespans and develop leukemia. Hematopoiesis is disrupted profoundly, with a reduction of hematopoietic stem cells and common lymphoid progenitors causing a severely depleted acquired immune system. We show that formaldehyde is a common substrate of ALDH2 and ADH5 and establish methods to quantify elevated blood formaldehyde and formaldehyde-DNA adducts in tissues. Bone-marrow-derived progenitors actively engage DNA repair but also imprint a formaldehyde-driven mutation signature similar to aging-associated human cancer mutation signatures. Furthermore, we identify analogous genetic defects in children causing a previously uncharacterized inherited bone marrow failure and pre-leukemic syndrome. Endogenous formaldehyde clearance alone is therefore critical for hematopoiesis and in limiting mutagenesis in somatic tissues.

Mouse SLX4 is a tumor suppressor that stimulates the activity of the nuclease XPF-ERCC1 in DNA crosslink repair.

SLX4 binds to three nucleases (XPF-ERCC1, MUS81-EME1, and SLX1), and its deficiency leads to genomic instability, sensitivity to DNA crosslinking agents, and Fanconi anemia. However, it is not understood how SLX4 and its associated nucleases act in DNA crosslink repair. Here, we uncover consequences of mouse Slx4 deficiency and reveal its function in DNA crosslink repair. Slx4-deficient mice develop epithelial cancers and have a contracted hematopoietic stem cell pool. The N-terminal domain of SLX4 (mini-SLX4) that only binds to XPF-ERCC1 is sufficient to confer resistance to DNA crosslinking agents. Recombinant mini-SLX4 enhances XPF-ERCC1 nuclease activity up to 100-fold, directing specificity toward DNA forks. Mini-SLX4-XPF-ERCC1 also vigorously stimulates dual incisions around a DNA crosslink embedded in a synthetic replication fork, an essential step in the repair of this lesion. These observations define vertebrate SLX4 as a tumor suppressor, which activates XPF-ERCC1 nuclease specificity in DNA crosslink repair.

The structure of Escherichia coli ExoIX--implications for DNA binding and catalysis in flap endonucleases.

Escherichia coli Exonuclease IX (ExoIX), encoded by the xni gene, was the first identified member of a novel subfamily of ubiquitous flap endonucleases (FENs), which possess only one of the two catalytic metal-binding sites characteristic of other FENs. We have solved the first structure of one of these enzymes, that of ExoIX itself, at high resolution in DNA-bound and DNA-free forms. In the enzyme-DNA cocrystal, the single catalytic site binds two magnesium ions. The structures also reveal a binding site in the C-terminal domain where a potassium ion is directly coordinated by five main chain carbonyl groups, and we show this site is essential for DNA binding. This site resembles structurally and functionally the potassium sites in the human FEN1 and exonuclease 1 enzymes. Fluorescence anisotropy measurements and the crystal structures of the ExoIX:DNA complexes show that this potassium ion interacts directly with a phosphate diester in the substrate DNA.

Active site substitutions delineate distinct classes of eubacterial flap endonuclease.

FENs (flap endonucleases) play essential roles in DNA replication, pivotally in the resolution of Okazaki fragments. In eubacteria, DNA PolI (polymerase I) contains a flap processing domain, the N-terminal 5'-->3' exonuclease. We present evidence of paralogous FEN-encoding genes present in many eubacteria. Two distinct classes of these independent FEN-encoding genes exist with four groups of eubacteria, being identified based on the number and type of FEN gene encoded. The respective proteins possess distinct motifs hallmarking their differentiation. Crucially, based on primary sequence and predicted secondary structural motifs, we reveal key differences at their active sites. These results are supported by biochemical characterization of two family members--ExoIX (exonuclease IX) from Escherichia coli and SaFEN (Staphylococcus aureus FEN). These proteins displayed marked differences in their ability to process a range of branched and linear DNA structures. On bifurcated substrates, SaFEN exhibited similar substrate specificity to previously characterized FENs. In quantitative exonuclease assays, SaFEN maintained a comparable activity with that reported for PolI. However, ExoIX showed no observable enzymatic activity. A threaded model is presented for SaFEN, demonstrating the probable interaction of this newly identified class of FEN with divalent metal ions and a branched DNA substrate. The results from the present study provide an intriguing model for the cellular role of these FEN sub-classes and illustrate the evolutionary importance of processing aberrant DNA, which has led to their maintenance alongside DNA PolI in many eubacteria.

Molecular interactions of Escherichia coli ExoIX and identification of its associated 3'-5' exonuclease activity.

The flap endonucleases (FENs) participate in a wide range of processes involving the structure-specific cleavage of branched nucleic acids. They are also able to hydrolyse DNA and RNA substrates from the 5'-end, liberating mono-, di- and polynucleotides terminating with a 5' phosphate. Exonuclease IX is a paralogue of the small fragment of Escherichia coli DNA polymerase I, a FEN with which it shares 66% similarity. Here we show that both glutathione-S-transferase-tagged and native recombinant ExoIX are able to interact with the E. coli single-stranded DNA binding protein, SSB. Immobilized ExoIX was able to recover SSB from E. coli lysates both in the presence and absence of DNA. In vitro cross-linking studies carried out in the absence of DNA showed that the SSB tetramer appears to bind up to two molecules of ExoIX. Furthermore, we found that a 3'-5' exodeoxyribonuclease activity previously associated with ExoIX can be separated from it by extensive liquid chromatography. The associated 3'-5' exodeoxyribonuclease activity was excised from a 2D gel and identified as exonuclease III using matrix-assisted laser-desorption ionization mass spectrometry.

"Ring-fencing" BRCA1 tumor suppressor activity.

BRCA1 is a crucial human breast and ovarian cancer tumor suppressor gene. The article by Drost et al. in this issue of Cancer Cell together with a recent paper in Science now provide a clearer picture of how this large and complex protein suppresses tumorigenesis.