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

The DNA Polymerase _-Primase Complex: Multiple Functions and Interactions

DNA polymerase _ (pol _) holds a special position among the growing family of eukaryotic DNA polymerases. In fact, pol _ is associated with DNA primase to form a four subunit complex and, as a consequence, is the only enzyme able to start DNA synthesis de novo. Because of this peculiarity the major role of the DNA polymerase _-primase complex (pol-prim) is in the initiation of DNA replication at chromosomal origins and in the discontinuous synthesis of Okazaki fragments on the lagging strand of the replication fork. However, pol-prim seems to play additional roles in other complex cellular processes, such as the response to DNA damage, telomere maintenance, and the epigenetic control of higher order chromatin assembly

Preventing replication stress to maintain genome stability: Resolving conflicts between replication and transcription

DNA and RNA polymerases clash along the genome as they compete for the same DNA template. Cells have evolved specialized strategies to prevent and resolve replication and transcription interference. Here, we review the topology and architecture at sites of replication fork clashes with transcription bubbles as well as the regulatory circuits that control replication fork passage across transcribed genes. In the case of RNA polymerase II-transcribed genes, cotranscriptional processes such as mRNA maturation, splicing, and export influence the integrity of replication forks and transcribed loci. Fork passage likely contributes to reset the epigenetic landscape, influencing gene expression and transcriptional memory. When any of these processes are not properly coordinated, aberrant outcomes such as fork reversal and R-loop formation arise and trigger unscheduled recombinogenic events and genome rearrangements. The evolutionary implications of such conflicts on genome dynamics and their potential impact on oncogenic stress are discussed. © 2012 Elsevier Inc..Peer Reviewe

PP2A controls genome integrity by integrating nutrient-sensing and metabolic pathways with the DNA damage response

21 p.-7 fig. Ferrari, Elisa et al.Mec1ATR mediates the DNA damage response (DDR), integrating chromosomal signals and mechanical stimuli. We show that the PP2A phosphatases, ceramide-activated enzymes, couple cell metabolism with the DDR. Using genomic screens, metabolic analysis, and genetic and pharmacological studies, we found that PP2A attenuates the DDR and that three metabolic circuits influence the DDR by modulating PP2A activity. Irc21, a putative cytochrome b5 reductase that promotes the condensation reaction generating dihydroceramides (DHCs), and Ppm1, a PP2A methyltransferase, counteract the DDR by activating PP2A; conversely, the nutrient-sensing TORC1-Tap42 axis sustains DDR activation by inhibiting PP2A. Loss-of-function mutations in IRC21, PPM1, and PP2A and hyperactive tap42 alleles rescue mec1 mutants. Ceramides synergize with rapamycin, a TORC1 inhibitor, in counteracting the DDR. Hence, PP2A integrates nutrient-sensing and metabolic pathways to attenuate the Mec1ATR response. Our observations imply that metabolic changes affect genome integrity and may help with exploiting therapeutic options and repositioning known drugs.The work was supported by grants from the Associazione Italiana per la Ricerca sul Cancro (IG 16770 to M.F. and IG 15210 to M.P.L.), the European Union and Telethon-Italy (GGP12171 to M.F.), and Progetti di Ricerca di Interesse Nazionale (PRIN) 2015 (to M.F. and M.P.L).Peer reviewe

Srs2 DNA helicase is involved in checkpoint response and its regulation requires a functional Mec1-dependent pathway and Cdk1 activity

In Saccharomyces cerevisiae the rate of DNA replication is slowed down in response to DNA damage as a result of checkpoint activation, which is mediated by the Mec1 and Rad53 protein kinases. We found that the Srs2 DNA helicase, which is involved in DNA repair and recombination, is phosphorylated in response to intra-S DNA damage in a checkpoint-dependent manner. DNA damage-induced Srs2 phosphorylation also requires the activity of the cyclin-dependent kinase Cdk1, suggesting that the checkpoint pathway might modulate Cdk1 activity in response to DNA damage. Moreover, srs2 mutants fail to activate Rad53 properly and to slow down DNA replication in response to intra-S DNA damage. The residual Rad53 activity observed in srs2 cells depends upon the checkpoint proteins Rad17 and Rad24. Moreover, DNA damage-induced lethality in rad17 mutants depends partially upon Srs2, suggesting that a functional Srs2 helicase causes accumulation of lethal events in a checkpoint-defective context. Altogether, our data implicate Srs2 in the Mec1 and Rad53 pathway and connect the checkpoint response to DNA repair and recombination

Papillary Meningioma in the dog: A clinicopathological case series study.

Papillary meningioma (PM) is one of the most aggressive variants of meningioma in humans and clas- sified as grade III by WHO system. To date, the biological behavior of PM is still not clear in dogs. This study investigated the correlation between histopathological findings of 16 canine PMs and follow up data. Moreover, the expression of doublecortin, E-cadherin, and N-cadherin was investigated by immu- nohistochemistry. The supratentorial compartment resulted the most common involved. Despite the low grade of histological malignancy, 87.5% of dogs that underwent surgery experienced tumor recurrence. Intratumoral necrosis was observed in a strict correlation with malignancy histological parameter and tumor recurrence. The post-surgery mean survival time was much lower than thus observed in the most common histological subtypes. This data were also confirmed in dogs that received a conservative treat- ment alone. Tumors with a severe clinical behavior showed a high N-cadherin expression versus a low or absent E-cadherin expression