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The current concept regarding cell cycle regulation of DNA replication is that Cdt1, together with origin recognition complex and CDC6 proteins, constitutes the machinery that loads the minichromosome maintenance complex, a candidate replicative helicase, onto chromatin during the G 1 phase. The actions of origin recognition complex and CDC6 are suppressed through phosphorylation by cyclin-dependent kinases (Cdks) after S phase to prohibit rereplication. It has been suggested in metazoan cells that the function of Cdt1 is blocked through binding to an inhibitor protein, geminin. However, the functional relationship between the Cdt1-geminin system and Cdks remains to be clarified. In this report, we demonstrate that human Cdt1 is phosphorylated by cyclin A-dependent kinases dependent on its cyclin-binding motif. Cdk phosphorylation resulted in the binding of Cdt1 to the F-box protein Skp2 and subsequent degradation. In contrast, in vitro DNA binding activity of Cdt1 was inhibited by the phosphorylation. However, geminin binding to Cdt1 was not affected by the phosphorylation. Finally we provide evidence that inactivation of Cdk1 results in Cdt1 dephosphorylation and rebinding to chromatin in murine FT210 cells synchronized around the G 2 /M phase. Taken together, these findings suggest that Cdt1 function is also negatively regulated by the Cdk phosphorylation independent of geminin binding.

We describe a Drosophila gene, orbit, that encodes a conserved 165-kD microtubule-associated protein (MAP) with GTP binding motifs. Hypomorphic mutations in orbit lead to a maternal effect resulting in branched and bent mitotic spindles in the syncytial embryo. In the larval central nervous system, such mutants have an elevated mitotic index with some mitotic cells showing an increase in ploidy. Amorphic alleles show late lethality and greater frequencies of hyperploid mitotic cells. The presence of cells in the hypomorphic mutant in which the chromosomes can be arranged, either in a circular metaphase or an anaphase-like configuration on monopolar spindles, suggests that polyploidy arises through spindle and chromosome segregation defects rather than defects in cytokinesis. A role for the Orbit protein in regulating microtubule behavior in mitosis is suggested by its association with microtubules throughout the spindle at all mitotic stages, by its copurification with microtubules from embryonic extracts, and by the finding that the Orbit protein directly binds to MAP-free microtubules in a GTP-dependent manner.

A recombinant plasmid for expression of rat DNA polymerase beta was constructed in a plasmid/phage chimeric vector, pUC118, by an oligonucleotide-directed mutagenesis technique. The insert contained a 1005 bp coding sequence for the whole rat DNA polymerase beta. The recombinant plasmid was designed to use the regulatory sequence of Escherichia coli lac operon and the initiation ATG codon for beta-galactosidase as those for DNA polymerase beta. The recombinant clone, JMp beta 5, obtained by transfection of E. coli JM109 with the plasmid, produced high levels of DNA polymerase activity and a 40-kDa polypeptide that were not detected in JM109 cell extract. Inducing this recombinant E. coli with isopropyl beta-thiogalactopyranoside (IPTG) yielded amounts of 40-kDa polypeptide as high as 19.3% of total protein. Another recombinant clone, JMp beta 2-1, which was constructed by an oligonucleotide-directed mutagenesis to use the second ATG codon for the initiation codon, thus deleting the first 17 amino acid residues from the amino terminus, produced neither high DNA polymerase activity nor the 40-kDa polypeptide. The evidence suggests that this amino-terminal structure is important for stability of this enzyme in E. coli. The DNA polymerase was purified to homogeneity from the IPTG-induced JMp beta 5 cells by fewer steps than the procedure for purification of DNA polymerase beta from animal cells. The properties of this enzyme in activity, chromatographic behavior, size, antigenicity, and also lack of associated nuclease activity were indistinguishable from those of DNA polymerase beta purified from rat cells, indicating the identity of the overproduced DNA polymerase in the JMp beta 5 and the rat DNA polymerase beta.