Phosphorylation of the DNA Polymerase -Primase B Subunit Is Dependent on Its Association with the p180 Polypeptide

The B subunit of the DNA polymerase (pol) alpha-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 alpha-primase complex are assembled throughout the cell cycle, and physical association between newly synthesized pol alpha (p180) and unphosphorylated B subunit (p86) occurs very rapidly. Therefore, B subunit phosphorylation does not appear to modulate p180.p86 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 p180.p86 subcomplex is required for B subunit phosphorylation

Affinity labeling of the active center and ribonucleoside triphosphate binding site of yeast DNA primase.

Abstract A highly selective affinity labeling procedure has been applied to map the active center of DNA primase from the yeast Saccharomyces cerevisiae. Enzyme molecules that have been modified by covalent attachment of benzaldehyde derivatives of adenine nucleotides are autocatalytically labeled by incubation with a radioactive ribonucleoside triphosphate. The affinity labeling of primase requires a template DNA, is not affected by DNase and RNase treatments, but is sensitive to proteinase K. Both the p58 and p48 subunits of yeast DNA primase appear to participate in the formation of the catalytic site of the enzyme, although UV-photocross-linking with [alpha-32P]ATP locates the ribonucleoside triphosphate binding site exclusively on the p48 polypeptide. The fixation of the radioactive product has been carried out also after the enzymatic reaction. Under this condition the RNA primers synthesized by the DNA polymerase-primase complex under uncoupled DNA synthesis conditions are linked to both DNA primase and DNA polymerase. When DNA synthesis is allowed to proceed first, the labeled RNA chains are fixed exclusively to the DNA polymerase polypeptide. These results, in accord with previous data, have been used to propose a model illustrating the interactions and the putative roles of the polypeptides of the DNA polymerase-primase complex

<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

Exploring the gap between needs and practice in facilitating breastfeeding within the neonatal intensive care setting : An Italian survey on organizational factors

Introduction: The system-level factors of the neonatal intensive care unit work environment contribute to breastfeeding promotion in the preterm population. The aim of this study was to investigate the operative policies related to breastfeeding support in a sample of Italian Neonatal Intensive Care Units. Materials and Methods: A multicenter cross-sectional survey was conducted, including a sample of 17 head nurses. The items of the questionnaire investigated the following areas: breastfeeding policies, staff education, family centered care, and breastfeeding promotion and support both in the neonatal intensive care units and after discharge. Results: Written breastfeeding policies were available for staff in all the neonatal intensive care units, most commonly addressing procedures related to skin-to-skin contact, human milk expression, and preterm infant breastfeeding. Most of the neonatal intensive care units correctly advised the mothers to initiate milk expression within 6 h from delivery and to pump milk at least 6 times/days. Breastfeeding training for the nursing staff was planned in the majority of the neonatal intensive care units although according to different schedules. With regard to the family centered care implementation, time restrictions were present in seven neonatal intensive care units, mostly occurring during the night shift, and the morning hours concomitantly with medical rounds. Moreover, in the majority of the investigated neonatal intensive care units, the parents were asked to leave the ward when their infant underwent a major invasive procedure or during the nurse/physician shift change report. With regard to breastfeeding promotion and support, eight neonatal care units had a multidisciplinary team with several health care professionals and 10 provided information about community-based support services. Most of the units assessed breastfeeding after discharge. Conclusion: Based on the present findings, enrolled Neonatal Intensive Care Units appear to provide breastfeeding-supportive environments with special regard to breastfeeding policies, milk expression practices, interprofessional collaboration, and continuity of care. Health care professionals should exert efforts to ensure continuous and updated breastfeeding staff education and promote parent-infant closeness and family centered care

Human exonuclease 1 role in response to UV irradiation

DNA damage checkpoints are surveillance mechanisms that monitor the integrity of the genome. Nucleotide excision repair (NER) is a DNA repair mechanism that cells use to remove UV-induced DNA lesions. Previous publication from our laboratory demonstrated that recognition and processing of UV-induced damage by NER is required for proper activation of checkpoint through interactions between NER proteins and checkpoint factors in yeast and human primary fibroblasts. From a two hybrid screening in yeast exonuclease 1 (Exo1) was identified as a 9-1-1 complex interactor. Exo1 is a 5\u2019-3\u2019 exonuclease and 5'-flap-endonuclease with many different roles in DNA metabolism such as meiotic and mitotic recombination, mismatch repair and telomere processing. Characterization of an exo1 yeast deleted strain has shown that this protein is involved in the early steps of UV-induced DNA damage checkpoint. In human cells EXO1 is present as two isoforms named hEXO1a and hEXO1b genetarated by alternative splicing. We are analyzing the role of EXO1 in checkpoint activation in response to UV-C damage in human cells: using siRNA against both a and b isoform of hEXO1 in G1 cells we were able to observe a defect in Chk1 and p53 phosphorylation induced by UV-C irradiation

one step high throughput assay for quantitative detection of β galactosidase activity in intact gram negative bacteria yeast and mammalian cells

n/

Haspin regulates Ras localization to promote Cdc24-driven mitotic depolarization

Cell polarization is of paramount importance for proliferation, differentiation, development, and it is altered during carcinogenesis. Polarization is a reversible process controlled by positive and negative feedback loops. How polarized factors are redistributed is not fully understood and is the focus of this work. In Saccharomyces cerevisiae, mutants defective in haspin kinase exhibit stably polarized landmarks and are sensitive to mitotic delays. Here, we report a new critical role for haspin in polarisome dispersion; failure to redistribute polarity factors, in turn, leads to nuclear segregation defects and cell lethality. We identified a mitotic role for GTP-Ras in regulating the local activation of the Cdc42 GTPase, resulting in its dispersal from the bud tip to a homogeneous distribution over the plasma membrane. GTP-Ras2 physically interacts with Cdc24 regulateing its mitotic distribution. Haspin is shown to promote a mitotic shift from a bud tip-favored to a homogenous PM fusion of Ras-containing vesicles. In absence of haspin, active Ras is not redistributed from the bud tip; Cdc24 remains hyperpolarized promoting the activity of Cdc42 at the bud tip, and the polarisome fails to disperse leading to erroneously positioned mitotic spindle, defective nuclear segregation, and cell death after mitotic delays. These findings describe new functions for key factors that modulate cell polarization and mitotic events, critical processes involved in development and tumorigenesis

TLS Polymerases are involved in processing of EXO1-dependent lesions after UV-induced damage

UV light mainly damages DNA by generating CPDs and 6-4PP photoproducts, which are responsible for the pathological effects of sunlight. In a healthy organism, such DNA helix distorting lesions are removed by Nucleotide Excision Repair (NER), a multistep process. Mutations in NER genes cause the onset of severe pathologies. The principal symptom common to all diseases is the strong sensitivity to UV. A high predisposition to tumors development arises in xeroderma pigmentosum (XP) patients, while neurological dysfunctions have been observed in both XP and Cockayne syndrome patients. Upon DNA damage sensing, checkpoints are activated allowing a block or delay of cell cycle progression to ensure repair of the DNA lesions. Intriguingly, while in normal cells UV irradiation activates DNA damage checkpoints in all phases of the cell cycle NER yeast mutant strains and human fibroblasts derived from XP patients fail activate the checkpoint in G1 and G2. Recently, we demonstrated that the checkpoint response to UV light in cells that are not actively replicating their genome requires prior processing of the UV lesions. This involves NER factors but also the Exo1 nuclease. In particular, acting on NER intermediates, Exo1 generates structures containing long tracts of ssDNA in response to UV irradiation. This role of Exo1 is only observed at a subset of problematic lesions that cannot properly repaired by canonic NER. It is these Exo1-induced structures that provide the signal for checkpoint activation both in yeast and human non-replicating cells. The essential role of Exo1 in UV-induced checkpoint activation in vivo has been recently supported by in vitro reconstitution of the activation pathway. What are the problematic lesions that require EXO1 activity is still unknown. We hypothesized that Closely Opposing UV Lesions (COLs) on the two DNA strands could exist and may be a likely candidate. This scenario would require TLS polymerases bypass during repair synthesis step. Therefore, we are investigating Y-family polymerase recruitment at EXO1-positive local UV damage sites (LUDs). We found that Pol h is recruited at both EXO1-positive and EXO1-negative LUDs, while Pol \u3b9 and\uf020Pol \u3ba always co-localize with the nuclease\uf02e Using the CRISPR-Cas9 system, we generated EXO1 knock out cell lines that demonstrated a requirement for EXO1 in Pol \u3b9 and\uf020Pol \u3ba recruitment, consistently with our working model\uf02e Finally, when we silenced TLS polymerases we observed a hyper-activation of UV-induced DNA damage checkpoint, suggesting that EXO1 continues to process UV damaged DNA enlarging the gap and eventually producing DSBs. TLS polymerases, thus are crucial to prevent dangerous situations in non-replicating UV irradiated cells