DNA replication is central to cell proliferation. Studies in the past six decades since the proposal of a semiconservative mode of DNA replication have confirmed the high degree of conservation of the basic machinery of DNA replication from prokaryotes to eukaryotes. However, the need for replication of a substantially longer segment of DNA in coordination with various internal and external signals in eukaryotic cells has led to more complex and versatile regulatory strategies. The replication program in higher eukaryotes is under a dynamic and plastic regulation within a single cell, or within the cell population, or during development. We review here various regulatory mechanisms that control the replication program in eukaryotes and discuss future directions in this dynamic field.

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DNA replication is controlled by the stepwise assembly of a pre-replicative complex and the replication apparatus. Cdt1 is a novel component of the pre-replicative complex and plays a role in loading the minichromosome maintenance (MCM) 2-7 complex onto chromatin. Cdt1 activity is inhibited by geminin, which is essential for the G 2 /M transition in metazoan cells. To understand the molecular basis of the Cdt1-geminin regulatory mechanism in mammalian cells, we cloned and expressed the mouse Cdt1 homologue cDNA in bacterial cells and purified mouse Cdt1 to near homogeneity. We found by yeast two-hybrid analysis that mouse Cdt1 associates with geminin, MCM6, and origin recognition complex 2. MCM6 interacts with the Cdt1 carboxyl-terminal region (amino acids 407-477), which is conserved among eukaryotes, whereas geminin associates with the Cdt1 central region (amino acids 177-380), which is conserved only in metazoans. In addition, we found that Cdt1 can bind DNA in a sequence-, strand-, and conformation-independent manner. The Cdt1 DNA binding domain overlaps with the geminin binding domain, and the binding of Cdt1 to DNA is inhibited by geminin. Taken together, we have defined structural domains and novel biochemical properties for mouse Cdt1 that suggest that Cdt1 behaves as an intrinsic DNA binding factor in the pre-replicative complex.Chromosomal DNA replication is subject to strict cell cycle control, which ensures that cells enter S phase once and only once per cell cycle. A considerable body of evidence from both genetic analyses of yeast mutants and biochemical studies using Xenopus egg extracts has shown that the initiation of replication requires the stepwise assembly of protein complexes on chromatin to form a pre-replicative complex (pre-RC) 1 (1-8).The pre-RC includes the origin recognition complex (ORC), the minichromosome maintenance protein complex (MCM), and the Cdc6 and Cdt1 proteins. After the activation of S phasepromoting kinases, CDKs, and the Dbf4-dependent kinase, DNA helicase unwinds the two DNA strands, and replication protein A stabilizes single-stranded DNA, thereby allowing an initiation complex to be formed by the loading of DNA polymerases onto the pre-RC. Because most of the components of the pre-RC identified in Saccharomyces cerevisiae and Xenopus have been found in other eukaryotes including humans, it is believed that the mechanisms controlling the initiation of replication are conserved in all eukaryotes. However, the DNA helicase that is associated with the replication fork has not yet been identified, even in S. cerevisiae (1,6,8,9). The best candidate for the replicative DNA helicase is the MCM2-7 complex. The MCM2-7 complex was first identified as a set of genes required for minichromosome maintenance in S. cerevisiae, and it was subsequently identified as a critical component of the replication licensing system in Xenopus egg extracts (3, 9, 10). MCM2-7 proteins are loaded onto chromatin at late telophase and gradually released as replication forks proceed, and concomitan...

Minichromosome maintenance (MCM) proteins play an essential role in eukaryotic DNA replication and bind to chromatin before the initiation of DNA replication. We reported that MCM protein complexes consisting of MCM2, -4, -6, and -7 bind strongly to a histone-Sepharose column (Ishimi, Y., Ichinose, S., Omori, A., Sato, K., and Kimura, H. (1996) J. Biol. Chem. 271, 24115-24122). Here, we have analyzed this interaction at the molecular level. We found that among six mouse MCM proteins, only MCM2 binds to histone; amino acid residues 63-153 are responsible for this binding. The region required for nuclear localization of MCM2 was mapped near this histone-binding domain. Far-Western blotting analysis of truncated forms of H3 histone indicated that amino acid residues 26 -67 of H3 histone are required for binding to MCM2. We have also shown that mouse MCM2 can inhibit the DNA helicase activity of the human MCM4, -6, and -7 protein complex. These results suggest that MCM2 plays a different role in the initiation of DNA replication than the other MCM proteins. Minichromosome maintenance (MCM)1 proteins play an essential role in eukaryotic DNA replication. Six MCM proteins (MCM2-7) have been identified from yeast to mammals, and each of them plays a distinct role in DNA replication (reviewed in Refs. 1-3). Several lines of evidence suggest that MCM proteins are required for the initiation of DNA replication (4 -6). Consistent with this idea, MCM proteins associate with the chromatin before the onset of DNA replication and detach from the chromatin during DNA replication (7-9). Biochemical analyses have indicated that these six MCM proteins interact. In extracts from Schizosaccharomyces pombe (10), mouse cells (11) and mitotic human cells (12), a complex of approximately 600 kDa, containing all six MCM proteins, have been identified. Based on the molecular mass of each MCM protein, this complex is probably a hexamer containing a single molecule of the six MCM proteins. However, sub-complexes containing MCM2, -4, -6, and -7 or MCM3 and -5 have also been isolated from human cell extracts (13-17), Xenopus egg extracts (18), and mouse cell extracts (19). Our group recently found that the DNA helicase activity was associated with the human MCM4, -6, and -7 protein complex (20). The 600-kDa complex containing the six MCM proteins is detected in soluble cell extracts; however, the structures of the chromatin-bound MCM protein heterocomplexes (21) remain to be elucidated.Origin recognition complex (ORC) and CDC6 protein are both required for the initiation of DNA replication and are necessary for the binding of MCM proteins to chromatin in Xenopus egg extracts (22, 23). CDC6-dependent loading of MCM proteins onto chromatin (24, 25) and origins (26, 27) has also been observed in Saccharomyces cerevisiae. The genetic interaction between MCM proteins and ORC in S. cerevisiae supports these findings (28, 29). These results suggest that MCM proteins form a complex with ORC and CDC6 at the replication origin. In S. cerevisiae the numb...

Background Studies of non-cutaneous and cutaneous malignancies support the hypothesis that poor risk-perception status contributes to health disparity. Objective We evaluated skin cancer risk perceptions across race and other demographic markers using the Health Information National Trends Survey (HINTS) and compared them to discover differences in perception that may contribute to the disparities in skin cancer diagnosis and treatment. Methods Respondents with no prior history of skin cancer were randomly selected to answer questions assessing perceived risk and knowledge of preventive strategies of skin cancer. Logistic regression was performed to identify associations between perceptions of skin cancer and demographic variables including self-described race, age, sex, education, income, and health insurance status. Results Blacks, the elderly, and people with less education perceived themselves as at lower risk of developing skin cancer. They, along with Hispanics, were also more likely to believe that one cannot lower their skin cancer risk and that there are so many different recommendations on how to prevent skin cancer that it makes it difficult to know which ones to follow. Lower education also correlated with greater reluctance to have a skin exam. Limitations HINTS is a cross-sectional instrument, thus it only provides a snapshot of skin cancer perceptions. Conclusion Uncertainty and altered perceptions are more common in the skin cancer risk perceptions of ethnic minorities, the elderly, and those with less education. These are the same groups that are subject to disparities in skin cancer outcomes. Educational programs directed at these demographic groups may help to reduce the skin cancer-related health disparities.

Hyperuricemia may contribute to endothelial dysfunction in CKD. We evaluated whether lowering serum uric acid levels with allopurinol improves endothelial dysfunction in 80 participants ≥18 years of age with stage 3 CKD and asymptomatic hyperuricemia (≥7 mg/dl in men and ≥6 mg/dl in women) randomized in a double-blinded manner to receive placebo or allopurinol for 12 weeks. Randomization was stratified according to presence or absence of diabetes mellitus. We measured vascular endothelial function by brachial artery flow-mediated dilation. No significant differences existed between groups at baseline; 61% of the participants had diabetes mellitus in both groups. The placebo and the allopurinol groups had baseline serum uric acid levels (SDs) of 8.7 (1.6) mg/dl and 8.3 (1.4) mg/dl, respectively, and baseline flow-mediated dilation values (SDs) of 6.0% (5.0%) and 4.8% (5.0%), respectively. Compared with placebo, allopurinol lowered serum uric acid significantly but did not improve endothelial function. In participants without diabetes mellitus, allopurinol associated with a trend toward improved flow-mediated dilation (+1.4% [3.9%] versus -0.7% [4.1%] with placebo), but this was not statistically significant (=0.26). Furthermore, we did not detect significant differences between groups in BP or serum levels of markers of inflammation and oxidative stress. In conclusion, allopurinol effectively and safely lowered serum uric acid levels in adults with stage 3 CKD and asymptomatic hyperuricemia but did not improve endothelial function in this sample of patients.

Background High‐resistance inspiratory muscle strength training (IMST) is a novel, time‐efficient physical training modality. Methods and Results We performed a double‐blind, randomized, sham‐controlled trial to investigate whether 6 weeks of IMST (30 breaths/day, 6 days/week) improves blood pressure, endothelial function, and arterial stiffness in midlife/older adults (aged 50–79 years) with systolic blood pressure ≥120 mm Hg, while also investigating potential mechanisms and long‐lasting effects. Thirty‐six participants completed high‐resistance IMST (75% maximal inspiratory pressure, n=18) or low‐resistance sham training (15% maximal inspiratory pressure, n=18). IMST was safe, well tolerated, and had excellent adherence (≈95% of training sessions completed). Casual systolic blood pressure decreased from 135±2 mm Hg to 126±3 mm Hg ( P <0.01) with IMST, which was ≈75% sustained 6 weeks after IMST ( P <0.01), whereas IMST modestly decreased casual diastolic blood pressure (79±2 mm Hg to 77±2 mm Hg, P =0.03); blood pressure was unaffected by sham training (all P >0.05). Twenty‐four hour systolic blood pressure was lower after IMST versus sham training ( P =0.01). Brachial artery flow‐mediated dilation improved ≈45% with IMST ( P <0.01) but was unchanged with sham training ( P =0.73). Human umbilical vein endothelial cells cultured with subject serum sampled after versus before IMST exhibited increased NO bioavailability, greater endothelial NO synthase activation, and lower reactive oxygen species bioactivity ( P <0.05). IMST decreased C‐reactive protein ( P =0.05) and altered select circulating metabolites (targeted plasma metabolomics) associated with cardiovascular function. Neither IMST nor sham training influenced arterial stiffness ( P >0.05). Conclusions High‐resistance IMST is a safe, highly adherable lifestyle intervention for improving blood pressure and endothelial function in midlife/older adults with above‐normal initial systolic blood pressure. Registration URL: https://www.clinicaltrials.gov ; Unique identifier: NCT03266510.

The association of overweight/obesity with disease progression in patients with autosomal dominant polycystic kidney disease (ADPKD) remains untested. We hypothesized that overweight/obesity associates with faster progression in early-stage ADPKD. Overall, 441 nondiabetic participants with ADPKD and an eGFR>60 ml/min per 1.73 m who participated in the Halt Progression of Polycystic Kidney Disease Study A were categorized on the basis of body mass index (BMI; calculated using nonkidney and nonliver weight) as normal weight (18.5-24.9 kg/m; reference; =192), overweight (25.0-29.9 kg/m; =168), or obese (≥30 kg/m; =81). We evaluated the longitudinal (5-year) association of overweight/obesity with change in total kidney volume (TKV) by magnetic resonance imaging using linear regression and multinomial logistic regression models. Among participants, mean±SD age was 37±8 years, annual percent change in TKV was 7.4%±5.1%, and BMI was 26.3±4.9 kg/m The annual percent change in TKV increased with increasing BMI category (normal weight: 6.1%±4.7%, overweight: 7.9%±4.8%, obese: 9.4%±6.2%; <0.001). In the fully adjusted model, higher BMI associated with greater annual percent change in TKV (=0.79; 95% confidence interval [95% CI], 0.18 to 1.39, per 5-unit increase in BMI). Overweight and obesity associated with increased odds of annual percent change in TKV ≥7% compared with <5% (overweight: odds ratio, 2.02; 95% CI, 1.15 to 3.56; obese: odds ratio, 3.76; 95% CI, 1.81 to 7.80). Obesity also independently associated with greater eGFR decline (slope) versus normal weight (fully adjusted =-0.08; 95% CI, -0.15 to -0.02). In conclusion, overweight and, particularly, obesity are strongly and independently associated with rate of progression in early-stage ADPKD.