The level of origin firing inversely affects the rate of replication fork progression.

DNA damage slows DNA synthesis at replication forks; however, the mechanisms remain unclear. Cdc7 kinase is required for replication origin activation, is a target of the intra-S checkpoint, and is implicated in the response to replication fork stress. Remarkably, we found that replication forks proceed more rapidly in cells lacking Cdc7 function than in wild-type cells. We traced this effect to reduced origin firing, which results in fewer replication forks and a consequent decrease in Rad53 checkpoint signaling. Depletion of Orc1, which acts in origin firing differently than Cdc7, had similar effects as Cdc7 depletion, consistent with decreased origin firing being the source of these defects. In contrast, mec1-100 cells, which initiate excess origins and also are deficient in checkpoint activation, showed slower fork progression, suggesting the number of active forks influences their rate, perhaps as a result of competition for limiting factors.

Functional conservation of beta-hairpin DNA binding domains in the Mcm protein of Methanobacterium thermoautotrophicum and the Mcm5 protein of Saccharomyces cerevisiae.

Mcm proteins are an important family of evolutionarily conserved helicases required for DNA replication in eukaryotes. The eukaryotic Mcm complex consists of six paralogs that form a heterohexameric ring. Because the intact Mcm2-7 hexamer is inactive in vitro, it has been difficult to determine the precise function of the different subunits. The solved atomic structure of an archaeal minichromosome maintenance (MCM) homolog provides insight into the function of eukaryotic Mcm proteins. The N-terminal positively charged central channel in the archaeal molecule consists of beta-hairpin domains essential for DNA binding in vitro. Eukaryotic Mcm proteins also have beta-hairpin domains, but their function is unknown. With the archaeal atomic structure as a guide, yeast molecular genetics was used to query the function of the beta-hairpin domains in vivo. A yeast mcm5 mutant with beta-hairpin mutations displays defects in the G1/S transition of the cell cycle, the initiation phase of DNA replication, and in the binding of the entire Mcm2-7 complex to replication origins. A similar mcm4 mutation is synthetically lethal with the mcm5 mutation. Therefore, in addition to its known regulatory role, Mcm5 protein has a positive role in origin binding, which requires coordination by all six Mcm2-7 subunits in the hexamer.

p21 and p27 induction by silibinin is essential for its cell cycle arrest effect in prostate carcinoma cells.

Recent studies have shown that silibinin induces p21/Cip1 and p27/Kip1 and G1 arrest in different prostate cancer cells irrespective of p53 status; however, biological significance and mechanism of such induction have not been studied. Here, using two different prostate cancer cell lines DU145 and 22Rv1, representing androgen-independent and androgen-dependent stages of malignancy, first we investigated the importance of p21 and p27 induction in silibinin-mediated G1 arrest. Silencing p21 and p27 individually by RNA interference showed marked reversal in G1 arrest; however, their simultaneous ablation showed additional reversal of G1 arrest in 22Rv1 but not DU145 cells. These results suggest that whereas relative importance of these molecules might be cell line specific, their induction by silibinin is essential for its G1 arrest effect. Next, studies were done to examine mechanisms of their induction where cycloheximide-chase experiments showed that silibinin increases p21 and p27 protein half-life. This effect was accompanied by strong reduction in Skp2 level and its binding with p21 and p27 together with strong decrease in phosphorylated Thr(187) p27 without considerable change in proteasomal activity, suggesting a posttranslational mechanism. Skp2 role was further elucidated using Skp2-small interfering RNA-transfected cells, where decreased G1 arrest and attenuated Cip/Kip induction were observed with silibinin treatment. Further, silibinin caused a marked increase in p21 and p27 mRNA levels together with an increase in their promoter activity, also indicating a transcriptional mechanism. Together, our results for the first time identify a central role of p21 and p27 induction and their regulatory mechanism in silibinin-mediated cell cycle arrest.

Silibinin upregulates the expression of cyclin-dependent kinase inhibitors and causes cell cycle arrest and apoptosis in human colon carcinoma HT-29 cells.

Silymarin, a defined mixture of natural flavonoid, has recently been shown to have potent cancer chemopreventive efficacy against colon carcinogenesis in rat model; however, the mechanism of such efficacy is not elucidated. Here, using pure active agent in silymarin, namely silibinin, we show its antiproliferative and apoptotic effects, and associated molecular alterations in human colon carcinoma HT-29 cells. Silibinin treatment of cells at 50-100 microg/ml doses resulted in a moderate to very strong growth inhibition in a dose- and a time-dependent manner, which was largely due to a G0/G1 arrest in cell cycle progression; higher dose and longer treatment time also caused a G2/M arrest. In mechanistic studies related its effect on cell cycle progression, silibinin treatment resulted in an upregulation of Kip1/p27 and Cip1/p21 protein as well as mRNA levels, and decreased CDK2, CDK4, cyclin E and cyclin D1 protein levels together with an inhibition in CDK2 and CDK4 kinase activities. In other studies, we observed that G2/M arrest by silibinin was associated with a decrease in cdc25C, cdc2/p34 and cyclin B1 protein levels, as well as cdc2/p34 kinase activity. In the studies assessing biological fate of silibinin-treated cells, silibinin-induced cell cycle arrest and growth inhibition were not associated with cellular differentiation, but caused apoptotic death. The quantitative apoptosis analysis showed up to 15% apoptotic cell death after 48 h of silibinin treatment. Interestingly, silibinin-induced apoptosis in HT-29 cells was independent of caspases activation, as all caspases inhibitor did not reverse silibinin-induced apoptosis. This observation was further confirmed by the findings showing a lack in caspases activity increase and caspases and PARP cleavage as well as a lack in cytochrome c release in cytosol following silibinin treatment of HT-29 cells. Additional studies conducted in mice showed that silibinin doses found effective in HT-29 cells are achievable in plasma, which increases the significance of the present findings and their possible translation in in vivo anticancer efficacy of silibinin against colon cancer. Together, these results identify molecular mechanisms of silibinin efficacy as a cell cycle regulator and apoptosis inducer in human colon carcinoma HT-29 cells, and justify further studies to investigate potential usefulness of this nontoxic agent in colon cancer prevention and intervention.

The mcm5-bob1 bypass of Cdc7p/Dbf4p in DNA replication depends on both Cdk1-independent and Cdk1-dependent steps in Saccharomyces cerevisiae.

The roles in DNA replication of two distinct protein kinases, Cdc7p/Dbf4p and Cdk1p/Clb (B-type cyclin), were studied. This was accomplished through a genetic and molecular analysis of the mechanism by which the mcm5-bob1 mutation bypasses the function of the Cdc7p/Dbf4p kinase. Genetic experiments revealed that loss of either Clb5p or Clb2p cyclins suppresses the mcm5-bob1 mutation and prevents bypass. These two cyclins have distinct roles in bypass and presumably in DNA replication as overexpression of one could not complement the loss of the other. Furthermore, the ectopic expression of CLB2 in G1 phase cannot substitute for CLB5 function in bypass of Cdc7p/Dbf4p by mcm5-bob1. Molecular experiments revealed that the mcm5-bob1 mutation allows for constitutive loading of Cdc45p at early origins in arrested G1 phase cells when both kinases are inactive. A model is proposed in which the Mcm5-bob1 protein assumes a unique molecular conformation without prior action by either kinase. This conformation allows for stable binding of Cdc45p to the origin. However, DNA replication still cannot occur without the combined action of Cdk1p/Clb5p and Cdk1p/Clb2p. Thus Cdc7p and Cdk1p kinases catalyze the initiation of DNA replication at several distinct steps, of which only a subset is bypassed by the mcm5-bob1 mutation.