The overall goal of this project is to help elucidate the mechanism by which cells distinguish newly-replicated DNA, and its associated proteins, in S and G2 phases of the cell cycle and prevent that DNA from reinitiating replication in the same cycle. A primary hypothesis is that alterations in activity of an MCM4, 6, 7 helicase complex, necessary for initiation or reinitiation, are mediated by CDK-dependent phosphorylation. Intracellular levels of the MCM helicase proteins are high in replicating cells vs. quiescent cells and are particularly high in lung carcinoma cells vs. adjacent non-cancerous cells. We shall pursue our findings that cyclin AJCDK2 and sequence-specific single-stranded DNA-binding protein, Puralpha, are colocalized with once-replicated DNA in S and G2, that Puralpha modulates activity of CDK2 and that Puralpha colocalizes with MCM7 on chromatin. We shall ascertain using chromatin immunoprecipitation (ChIP) and re-ChIP with successive antibodies the location and timing of assembly and dissociation of MCM4, 6 and 7 helicase components and the helicase inhibitor, MCM2, upstream of the c-Myc gene in the cell cycle of NCI-H146 SCLC cells and normal controls. We shall identify amino acid sites of phosphorylation of MCM proteins in nuclei and by purified cyclin/CDK combinations, generated in the baculovirus expression vector system, and determine the effects of such phosphorylation on kinetics of MCM4, 6, 7 helicase activity. We shall determine whether Puralpha modulates the MCM4, 6, 7 helicase, either by association with cyclin A/CDK2 or by DNA unwinding. We shall determine whether the timing of formation and phosphorylation of the MCM4, 6,7 complex on chromatin is altered in SCLC cells transfected to express dominant-negative RB. Results will allow identification of links between control of initiation of replication and imposition of checkpoint controls that are critical in preventing progression to cancer.
| Wortman, Margaret J; Hanson, Laura K; Martinez-Sobrido, Luis et al. (2010) Regulation of PURA gene transcription by three promoters generating distinctly spliced 5-prime leaders: a novel means of fine control over tissue specificity and viral signals. BMC Mol Biol 11:81 |
| Liu, Xiaomei; Gomez-Pinillos, Alejandro; Liu, Xiaojun et al. (2010) Induction of bicalutamide sensitivity in prostate cancer cells by an epigenetic Puralpha-mediated decrease in androgen receptor levels. Prostate 70:179-89 |
| Johnson, Edward M (2010) Structural evaluation of new human polyomaviruses provides clues to pathobiology. Trends Microbiol 18:215-23 |
| Kinoshita, Yayoi; Johnson, Edward M (2004) Site-specific loading of an MCM protein complex in a DNA replication initiation zone upstream of the c-MYC gene in the HeLa cell cycle. J Biol Chem 279:35879-89 |
