SUMMARY We propose that cell cycle-dependent timing of FEN1 nuclease activity is essential for cell cycle progression and the maintenance of genome stability. After DNA replication is complete at the exit point of the S-phase, removal of excess FEN1 may be crucial. Here, we report a mechanism that controls the programmed degradation of FEN1 via a sequential cascade of post-translational modifications. We found that FEN1 phosphorylation stimulated its SUMOylation, which, in turn, stimulated its ubiquitination and ultimately led to its degradation via the proteasome pathway. Mutations or inhibitors that blocked the modification at any step in this pathway suppressed FEN1 degradation. Critically, the presence of SUMOylation- or ubiquitination- defective, non-degradable FEN1 mutant protein caused accumulation of Cyclin B, delays in the G1 and G2/M phases and polyploidy. These findings may represent a newly identified regulatory mechanism used by cells to ensure precise cell cycle progression and to prevent transformation.

DNA polymerase (pol) μ is a DNA-dependent polymerase that incorporates nucleotides during gap-filling synthesis in the non-homologous end-joining pathway of double-strand break repair. Here we report time-lapse X-ray crystallography snapshots of catalytic events during gap-filling DNA synthesis by pol μ. Unique catalytic intermediates and active site conformational changes that underlie catalysis are uncovered, and a transient third (product) metal ion is observed in the product state. The product manganese coordinates phosphate oxygens of the inserted nucleotide and PPi. The product metal is not observed during DNA synthesis in the presence of magnesium. Kinetic analyses indicate that manganese increases the rate constant for deoxynucleoside 5′-triphosphate insertion compared to magnesium. The likely product stabilization role of the manganese product metal in pol μ is discussed. These observations provide insight on structural attributes of this X-family double-strand break repair polymerase that impact its biological function in genome maintenance.

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CBS (cystathionine beta-synthase) domains are found in proteins from all kingdoms of life, and point mutations in these domains are responsible for a variety of hereditary diseases in humans; however, the functions of CBS domains are not well understood. In the present study, we cloned, expressed in Escherichia coli, and characterized a family II PPase (inorganic pyrophosphatase) from Moorella thermoacetica (mtCBS-PPase) that has a pair of tandem 60-amino-acid CBS domains within its N-terminal domain. Because mtCBS-PPase is a dimer and requires transition metal ions (Co2+ or Mn2+) for activity, it resembles common family II PPases, which lack CBS domains. The mtCBS-PPase, however, has lower activity than common family II PPases, is potently inhibited by ADP and AMP, and is activated up to 1.6-fold by ATP. Inhibition by AMP is competitive, whereas inhibition by ADP and activation by ATP are both of mixed types. The nucleotides are effective at nanomolar (ADP) or micromolar concentrations (AMP and ATP) and appear to compete for the same site on the enzyme. The nucleotide-binding affinities are thus 100-10000-fold higher than for other CBS-domain-containing proteins. Interestingly, genes encoding CBS-PPase occur most frequently in bacteria that have a membrane-bound H+-translocating PPase with a comparable PP(i)-hydrolysing activity. Our results suggest that soluble nucleotide-regulated PPases act as amplifiers of metabolism in bacteria by enhancing or suppressing ATP production and biosynthetic reactions at high and low [ATP]/([AMP]+[ADP]) ratios respectively.