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To maintain genetic stability, the entire mammalian genome must replicate only once per cell cycle. This is largely achieved by strictly regulating the stepwise formation of the pre-replication complex (pre-RC), followed by the activation of individual origins of DNA replication by Cdc7/Dbf4 kinase. However, the mechanism how Cdc7 itself is regulated in the context of cell cycle progression is poorly understood. Here we report that Cdc7 is phosphorylated by a Cdk1-dependent manner during prometaphase on multiple sites, resulting in its dissociation from origins. In contrast, Dbf4 is not removed from origins in prometaphase, nor is it degraded as cells exit mitosis. Our data thus demonstrates that constitutive phosphorylation of Cdc7 at Cdk1 recognition sites, but not the regulation of Dbf4, prevents the initiation of DNA replication in normally cycling cells and under conditions that promote re-replication in G2/M. As cells exit mitosis, PP1a associates with and dephosphorylates Cdc7. Together, our data support a model where Cdc7 (de)phosphorylation is the molecular switch for the activation and inactivation of DNA replication in mitosis, directly connecting Cdc7 and PP1a/Cdk1 to the regulation of once-per-cell cycle DNA replication in mammalian cells.

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Uracil DNA glycosylases are an important class of enzymes that hydrolyze the N-glycosidic bond between the uracil base and the deoxyribose sugar to initiate uracil excision repair. Uracil may arise in DNA either because of its direct incorporation (against A in the template) or because of cytosine deamination. Mycobacteria with G, C rich genomes are inherently at high risk of cytosine deamination. Uracil DNA glycosylase activity is thus important for the survival of mycobacteria. A limitation in evaluating the druggability of this enzyme, however, is the absence of a rapid assay to evaluate catalytic activity that can be scaled for medium to high-throughput screening of inhibitors. Here we report a fluorescence-based method to assay uracil DNA glycosylase activity. A hairpin DNA oligomer with a fluorophore at its 5′ end and a quencher at its 3′ ends was designed incorporating five consecutive U:A base pairs immediately after the first base pair (5′ C:G 3’) at the top of the hairpin stem. Enzyme assays performed using this fluorescent substrate were seen to be highly sensitive thus enabling investigation of the real time kinetics of uracil excision. Here we present data that demonstrate the feasibility of using this assay to screen for inhibitors of Mycobacterium tuberculosis uracil DNA glycosylase. We note that this assay is suitable for high-throughput screening of compound libraries for uracil DNA glycosylase inhibitors.

Objective To assess the feasibility and accuracy of preoperative wire localization performed one day prior to surgery and the relationship between the time interval following wire placement with migration distance within the time-window examined. Methods Two trials were performed with next-day mammography to assess migration. Trial 1 used a standard hooked wire (50 patients, 61 wires). Trial 2 employed a looped wire (50 patients, 59 wires). A third trial was subsequently performed (16 patients, 18 wires) using the looped wire without repeat mammograms. Complications were recorded. Comparative statistical analyses were performed between patients in Trial 1 and Trial 2. Results In Trials 1 and 2, no wires required readjustment on the day of surgery. Mean and maximum migration were less with the looped wire (range: 0–7 mm) compared to the hooked wire (range: 0–18 mm), allowing for the elimination of next-day mammograms in Trial 3. A Mann–Whitney U test showed no significant difference between the migration distances for the first two trials (P = 0.11). A Chi-square test showed no significant difference in the direction of the migration between the two trials (P = 0.15). There was no correlation between the time interval of localization and needle migration in the first two trials (r = -0.16, P = 0.22 and -0.12, P = 0.36). Specimen radiographs demonstrated the lesion/biopsy marker clip in all cases in all three trials. No infections or bleeding occurred. Two patients developed an allergic reaction to adhesive. Conclusion Wire localization performed on the day before surgery is feasible, inexpensive, did not compromise accuracy, and successfully unlinked the radiologic and surgical procedures.

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