<i>De novo</i> synthesis of budding yeast DNA polymerase alpha and <i>POL1</i> transcription at the G₁/S boundary are not required for entrance into S phase

The POL1 gene, encoding DNA polymerase α(pol α) in Saccharomyces cerevisiae, is transiently transcribed during the cell cycle at the G1/S phase boundary. Here we show that yeast pol α is present at every stage of the cell cycle, and its level only slightly increases following the peak of POL1 transcription. POL1 mRNA synthesis driven by a GAL1 promoter can be completely abolished without affecting the growth rate of logarithmically growing yeast cultures for several cell divisions, although the amount of the pol α polypeptide drops below the physiological level. Moreover, α-factor-arrested cells can enter S phase and divide synchronously even if POL1 transcription is abolished. These results indicate that the level of yeast pol α is not rate limiting and de novo synthesis of the enzyme is not required for entrance into S phase

Targeting human apurinic/apyrimidinic endonuclease 1 (APE1) in phosphatase and tensin homolog (PTEN) deficient melanoma cells for personalized therapy

Phosphatase and tensin homolog (PTEN) loss is associated with genomic instability. APE1 is a key player in DNA base excision repair (BER) and an emerging drug target in cancer. We have developed small molecule inhibitors against APE1 repair nuclease activity. In the current study we explored a synthetic lethal relationship between PTEN and APE1 in melanoma. Clinicopathological significance of PTEN mRNA and APE1 mRNA expression was investigated in 191 human melanomas. Preclinically, PTEN-deficient BRAF-mutated (UACC62, HT144, and SKMel28), PTEN-proficient BRAF-wildtype (MeWo), and doxycycline-inducible PTEN-knockout BRAF-wildtype MeWo melanoma cells were DNA repair expression profiled and investigated for synthetic lethality using a panel of four prototypical APE1 inhibitors. In human tumours, low PTEN mRNA and high APE1 mRNA was significantly associated with reduced relapse free and overall survival. Pre-clinically, compared to PTEN-proficient cells, PTEN-deficient cells displayed impaired expression of genes involved in DNA double strand break (DSB) repair. Synthetic lethality in PTEN-deficient cells was evidenced by increased sensitivity, accumulation of DSBs and induction of apoptosis following treatment with APE1 inhibitors. We conclude that PTEN deficiency is not only a promising biomarker in melanoma, but can also be targeted by a synthetic lethality strategy using inhibitors of BER, such as those targeting APE1

Amyloid β peptides are differentially vulnerable to preanalytical surface exposure, an effect incompletely mitigated by the use of ratios

INTRODUCTION: We tested the hypothesis that the amyloid β (Aβ) peptide ratios are more stable than Aβ42 alone when biofluids are exposed to two preanalytical conditions known to modify measurable Aβ concentration. METHODS: Human cerebrospinal fluid (CSF) and culture media (CM) from human cortical neurons were exposed to a series of volumes and polypropylene surfaces. Aβ42, Aβ40, and Aβ38 peptide concentrations were measured using a multiplexed electrochemiluminescence immunoassay. Data were analyzed using mixed models in R. RESULTS: Decrease of measurable Aβ peptide concentrations was exaggerated in longer peptides, affecting the Aβ42:Aβ40 and Aβ42:Aβ38 ratios. However, the effect size of surface treatment was reduced in Aβ peptide ratios versus Aβ42 alone. For Aβ42:Aβ40, the effect was reduced by approximately 50% (volume) and 75% (transfer) as compared to Aβ42 alone. DISCUSSION: Use of Aβ ratios, in conjunction with concentrations, may mitigate confounding factors and assist the clinical diagnostic process for Alzheimer's disease

Kinesins relocalize the chromosomal passenger complex to the midzone for spindle disassembly.

Mitotic spindle disassembly after chromosome separation is as important as spindle assembly, yet the molecular mechanisms for spindle disassembly are unclear. In this study, we investigated how the chromosomal passenger complex (CPC), which contains the Aurora B kinase Ipl1, swiftly concentrates at the spindle midzone in late anaphase, and we researched the role of this dramatic relocalization during spindle disassembly. We showed that the kinesins Kip1 and Kip3 are essential for CPC relocalization. In cells lacking Kip1 and Kip3, spindle disassembly is severely delayed until after contraction of the cytokinetic ring. Purified Kip1 and Kip3 interact directly with the CPC and recruit it to microtubules in vitro, and single-molecule experiments showed that the CPC diffuses dynamically on microtubules but that diffusion stops when the CPC encounters a Kip1 molecule. We propose that Kip1 and Kip3 trap the CPC at the spindle midzone in late anaphase to ensure timely spindle disassembly

Dna2 processes behind the fork long ssDNA flaps generated by Pif1 and replication-dependent strand displacement

Dna2 is a DNA helicase-endonuclease mediating DSB resection and Okazaki fragment processing. Dna2 ablation is lethal and rescued by inactivation of Pif1, a helicase assisting Okazaki fragment maturation, Pol32, a DNA polymerase \u3b4 subunit, and Rad9, a DNA damage response (DDR) factor. Dna2 counteracts fork reversal and promotes fork restart. Here we show that Dna2 depletion generates lethal DNA structures activating the DDR. While PIF1 deletion rescues the lethality of Dna2 depletion, RAD9 ablation relieves the first cell cycle arrest causing genotoxicity after few cell divisions. Slow fork speed attenuates DDR in Dna2 deprived cells. Electron microscopy shows that Dna2-ablated cells accumulate long ssDNA flaps behind the forks through Pif1 and fork speed. We suggest that Dna2 offsets the strand displacement activity mediated by the lagging strand polymerase and Pif1, processing long ssDNA flaps to prevent DDR activation. We propose that this Dna2 function has been hijacked by Break Induced Replication in DSB processing

Cerebrospinal fluid in the differential diagnosis of Alzheimer's disease: clinical utility of an extended panel of biomarkers in a specialist cognitive clinic.

BACKGROUND: Cerebrospinal fluid (CSF) biomarkers are increasingly being used to support a diagnosis of Alzheimer's disease (AD). Their clinical utility for differentiating AD from non-AD neurodegenerative dementias, such as dementia with Lewy bodies (DLB) or frontotemporal dementia (FTD), is less well established. We aimed to determine the diagnostic utility of an extended panel of CSF biomarkers to differentiate AD from a range of other neurodegenerative dementias. METHODS: We used immunoassays to measure conventional CSF markers of amyloid and tau pathology (amyloid beta (Aβ)1-42, total tau (T-tau), and phosphorylated tau (P-tau)) as well as amyloid processing (AβX-38, AβX-40, AβX-42, soluble amyloid precursor protein (sAPP)α, and sAPPβ), large fibre axonal degeneration (neurofilament light chain (NFL)), and neuroinflammation (YKL-40) in 245 patients with a variety of dementias and 30 controls. Patients fulfilled consensus criteria for AD (n = 156), DLB (n = 20), behavioural variant frontotemporal dementia (bvFTD; n = 45), progressive non-fluent aphasia (PNFA; n = 17), and semantic dementia (SD; n = 7); approximately 10% were pathology/genetically confirmed (n = 26). Global tests based on generalised least squares regression were used to determine differences between groups. Non-parametric receiver operating characteristic (ROC) curves and area under the curve (AUC) analyses were used to quantify how well each biomarker discriminated AD from each of the other diagnostic groups (or combinations of groups). CSF cut-points for the major biomarkers found to have diagnostic utility were validated using an independent cohort which included causes of AD (n = 104), DLB (n = 5), bvFTD (n = 12), PNFA (n = 3), SD (n = 9), and controls (n = 10). RESULTS: There were significant global differences in Aβ1-42, T-tau, T-tau/Aβ1-42 ratio, P-tau-181, NFL, AβX-42, AβX-42/X-40 ratio, APPα, and APPβ between groups. At a fixed sensitivity of 85%, AβX-42/X-40 could differentiate AD from controls, bvFTD, and SD with specificities of 93%, 85%, and 100%, respectively; for T-tau/Aβ1-42 these specificities were 83%, 70%, and 86%. AβX-42/X-40 had similar or higher specificity than Aβ1-42. No biomarker or ratio could differentiate AD from DLB or PNFA with specificity > 50%. Similar sensitivities and specificities were found in the independent validation cohort for differentiating AD and other dementias and in a pathology/genetically confirmed sub-cohort. CONCLUSIONS: CSF AβX-42/X-40 and T-tau/Aβ1-42 ratios have utility in distinguishing AD from controls, bvFTD, and SD. None of the biomarkers tested had good specificity at distinguishing AD from DLB or PNFA

An inducible chaperone adapts proteasome assembly to stress

The proteasome is essential for the selective degradation of most cellular proteins. To survive overwhelming demands on the proteasome arising during environmental stresses, cells increase proteasome abundance. Proteasome assembly is known to be complex. How stressed cells overcome this vital challenge is unknown. In an unbiased suppressor screen aimed at rescuing the defects of a yeast Rpt6 thermosensitive proteasome mutant, we identified a protein, hereafter named Adc17, as it functions as an ATPase dedicated chaperone. Adc17 interacts with the amino terminus of Rpt6 to assist formation of the Rpt6-Rpt3 ATPase pair, an early step in proteasome assembly. Adc17 is important for cell fitness, and its absence aggravates proteasome defects. The abundance of Adc17 increases upon proteasome stresses, and its function is crucial to maintain homeostatic proteasome levels. Thus, cells have mechanisms to adjust proteasome assembly when demands increase, and Adc17 is a critical effector of this process

Cell cycle-dependent phosphorylation and dephosphorylation of the yeast DNA polymerase alpha-primase B subunit

The yeast DNA polymerase α-primase B subunit functions in initiation of DNA replication. This protein is present in two forms, of 86 and 91 kDa, and the p91 polypeptide results from cell cycle-regulated phosphorylation of p86. The B subunit present in G1 arises by dephosphorylation of p91 while cells are exiting from mitosis, becomes phosphorylated in early S phase, and is competent and sufficient to initiate DNA replication. The B subunit transiently synthesized as a consequence of periodic transcription of the POL12 gene is phosphorylated no earlier than G2. Phosphorylation of the B subunit does not require execution of the CDC7-dependent step and ongoing DNA synthesis. We suggest that posttranslational modifications of the B subunit might modulate the role of DNA polymerase α-primase in DNA replication