No abstract

The POLI gene, encoding DNA polymerase a (pola) in Saccharomyces cerevisiae, is transiently transcribed during the cel cycle at the G1/S phase boundary. Here we show that yeast pola is present at every stage of the ceUl cycle, and its level only slightly increases following the peak of POLI transcription. POLI mRNA synthesis driven by a GAL] promoter can be completely abolished without affecting the growth rate of logarithmically growing yeast cultures for several cell divisions, although the amount of the pola polypeptide drops below the physiological level. Moreover, a-factor-arrested cells can enter S phase and divide synchronously even if POLI transcription is abolished. These results indicate that the level of yeast pola is not rate limiting and de novo synthesis of the enzyme is not required for entrance into S phase.Eukaryotic DNA polymerase a (pola), together with the tightly bound DNA primase, plays an essential role in lagging strand synthesis and initiation of DNA replication at an origin (1-3). The gene encoding pola in Saccharomyces cerevisiae (POLI) is transiently transcribed during the cell cycle at the G1/S phase boundary (4) concomitantly with several DNA synthesis genes (for a review, see refs. 5 and 6). A conserved promoter sequence, the Mlu I cell cycle box (MCB), mediates this transcriptional control and is present twice in the POLI gene (7-9). One component of the transcription factor(s) that binds to the MCB is the SWI6 gene product, whose function is relevant for cell cycle-dependent transcription of yeast DNA synthesis genes (10-12). However, SWI6 deletion is not lethal but leads to deregulated constitutive transcription of these genes (10, 11). Moreover, it has been shown that the level of proteins required for Saccharomyces cerevisiae DNA replication (replication factor A) or for entrance into S phase (CDC46 gene product) does not show any large fluctuation during the cell cycle (13,14), although the transcription of the corresponding genes is clearly periodic (14,15). A nearly constant amount of essential replication proteins has been observed also in actively cycling cells from other eukaryotes, including mammalian pola, RF-A, proliferating cell nuclear antigen (PCNA), DNA ligase (13,(16)(17)(18)(19), and Schizosaccharomyces pombe pola, PCNA, and DNA ligase (20)(21)(22). However, the transcription of the corresponding genes in these organisms does not appear to be cell cycle regulated. These findings leave uncertain as to whether the transcriptional activation of DNA replication genes observed in Saccharomyces cerevisiae is required for the onset of DNA replication in S phase.Our goal in the present work was to establish whether the amount of pola in Saccharomyces cerevisiae is rate limitingThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. and periodic transcription of the POLI gene is necessary for entrance into S pha...

The B subunit of the DNA polymerase (pol) ␣-primase complex executes an essential role at the initial stage of DNA replication in Saccharomyces cerevisiae and is phosphorylated in a cell cycle-dependent manner. In this report, we show that the four subunits of the yeast DNA polymerase ␣-primase complex are assembled throughout the cell cycle, and physical association between newly synthesized pol ␣ (p180) and unphosphorylated B subunit (p86) occurs very rapidly. Therefore, B subunit phosphorylation does not appear to modulate p180⅐p86 interaction. Conversely, by depletion experiments and by using a yeast mutant strain, which produces a low and constitutive level of the p180 polypeptide, we found that formation of the p180⅐p86 subcomplex is required for B subunit phosphorylation.

Metastasis is the major cause of mortality in prostate cancer patients. Factors such as genetic makeup and race play critical role in the outcome of therapies. This study was conducted to investigate the relevance of in metastatic prostate cancer disease in Caucasian and African-Americans. We employed race-specific prostate cancer models, clinical specimens, clinical data mining, gene-microarray, transcription-reporter assay, chromatin-immunoprecipitation (ChIP), IHC, transgenic-(tgfl/fl) zebrafish, and mouse metastasis models. BMI1 expression was observed to be elevated in metastatic tumors (lymph nodes, lungs, bones, liver) of Caucasian and African-American prostate cancer patients. The comparative analysis of stage III/IV tumors showed an increased BMI1 expression in African-Americans than Caucasians. TCGA and NIH/GEO clinical data corroborated to our findings. We show that expression (i) positively correlates to metastatic () and (ii) negative correlates to tumor suppressor () levels in tumors. The correlation was prominent in African-American tumors. We show that BMI1 regulates the transcriptional activation of , and We show the effect of pharmacological inhibition of BMI1 on the metastatic genome and invasiveness of tumor cells. Next, we show the anti-metastatic efficacy of BMI1-inhibitor in transgenic zebrafish and mouse metastasis models. Docetaxel as monotherapy has poor outcome on the growth of metastatic tumors. BMI1 inhibitor as an adjuvant improved the taxane therapy in race-based and models. BMI1, a major driver of metastasis, represents a promising therapeutic target for treating advanced prostate cancer in patients (including those belonging to high-risk group).

Gene rearrangement is reported to be associated to the aggressive phenotype and poor prognosis in prostate cancer. We identified a gene fusion between a transcription repressor (BMI1) and transcriptional factor (COMMD3) in human prostate cancer. We show that COMMD3:BMI1 fusion expression is significantly increased in prostate cancer disease in an order: normal tissue < primary < metastatic tumors (Mets). Although elevated TMPRSS-ERG/ETV fusion is reported in prostate cancer, we identified a subtype of Mets exhibiting low TMPRSS:ETV and high COMMD3:BMI1. We delineated the mechanism and function of COMMD3 and COMMD3: BMI1 in prostate cancer. We show that COMMD3 level is elevated in prostate cancer cell models, PDX models (adenocarcinoma, NECaP), and Mets. The analysis of TCGA/NIH/ GEO clinical data showed a positive correlation between increased COMMD3 expression to the disease recurrence and poor survival in prostate cancer. We show that COMMD3 drives proliferation of normal cells and promotes migration/ invasiveness of neoplastic cells. We show that COMMD3:BMI1 and COMMD3 regulate C-MYC transcription and C-MYC downstream pathway. The ChIP analysis showed that COMMD3 protein is recruited at the promoter of C-MYC gene. On the basis of these data, we investigated the relevance of COMMD3:BMI1 and COMMD3 as therapeutic targets using in vitro and xenograft mouse models. We show that siRNAmediated targeting of COMMD3:BMI1 and COMMD3 significantly decreases (i) C-MYC expression in BRD/BET inhibitorresistant cells, (ii) proliferation/invasion in vitro, and (iii) growth of prostate cancer cell tumors in mice. The IHC analysis of tumors confirmed the targeting of COMMD3-regulated molecular pathway under in vivo conditions. We conclude that COMMD3:BMI1 and COMMD3 are potential progression biomarkers and therapeutic targets of metastatic prostate cancer.