The cellular response to DNA lesions entails the recruitment of several checkpoint and repair factors to damaged DNA, and chromatin modifications may play a role in this process. Here we show that in Saccharomyces cerevisiae epigenetic modification of histones is required for checkpoint activity in response to a variety of genotoxic stresses. We demonstrate that ubiquitination of histone H2B on lysine 123 by the Rad6-Bre1 complex, is necessary for activation of Rad53 kinase and cell cycle arrest. We found a similar requirement for Dot1-dependent methylation of histone H3. Loss of H3-Lys 79 methylation does not affect Mec1 activation, whereas it renders cells checkpoint-defective by preventing phosphorylation of Rad9. Such results suggest that histone modifications may have a role in checkpoint function by modulating the interactions of Rad9 with chromatin and active Mec1 kinase.

Pregnancy is increasingly undertaken in patients with chronic kidney disease (CKD) and, conversely, CKD is increasingly diagnosed in pregnancy: up to 3 % of pregnancies are estimated to be complicated by CKD. The heterogeneity of CKD (accounting for stage, hypertension and proteinuria) and the rarity of several kidney diseases make risk assessment difficult and therapeutic strategies are often based upon scattered experiences and small series. In this setting, the aim of this position statement of the Kidney and Pregnancy Study Group of the Italian Society of Nephrology is to review the literature, and discuss the experience in the clinical management of CKD in pregnancy. CKD is associated with an increased risk for adverse pregnancy-related outcomes since its early stage, also in the absence of hypertension and proteinuria, thus supporting the need for a multidisciplinary follow-up in all CKD patients. CKD stage, hypertension and proteinuria are interrelated, but they are also independent risk factors for adverse pregnancy-related outcomes. Among the different kidney diseases, patients with glomerulonephritis and immunologic diseases are at higher risk of developing or increasing proteinuria and hypertension, a picture often difficult to differentiate from preeclampsia. The risk is higher in active immunologic diseases, and in those cases that are detected or flare up during pregnancy. Referral to tertiary care centres for multidisciplinary follow-up and tailored approaches are warranted. The risk of maternal death is, almost exclusively, reported in systemic lupus erythematosus and vasculitis, which share with diabetic nephropathy an increased risk for perinatal death of the babies. Conversely, patients with kidney malformation, autosomal-dominant polycystic kidney disease, stone disease, and previous upper urinary tract infections are at higher risk for urinary tract infections, in turn associated with prematurity. No risk for malformations other than those related to familiar urinary tract malformations is reported in CKD patients, with the possible exception of diabetic nephropathy. Risks of worsening of the renal function are differently reported, but are higher in advanced CKD. Strict follow-up is needed, also to identify the best balance between maternal and foetal risks. The need for further multicentre studies is underlined.

Following genotoxic insults, eukaryotic cells trigger a signal transduction cascade known as the DNA damage checkpoint response, which involves the loading onto DNA of an apical kinase and several downstream factors. Chromatin modifications play an important role in recruiting checkpoint proteins. In budding yeast, methylated H3-K79 is bound by the checkpoint factor Rad9. Loss of Dot1 prevents H3-K79 methylation, leading to a checkpoint defect in the G 1 phase of the cell cycle and to a reduction of checkpoint activation in mitosis, suggesting that another pathway contributes to Rad9 recruitment in M phase. We found that the replication factor Dpb11 is the keystone of this second pathway. dot1⌬ dpb11-1 mutant cells are sensitive to UV or Zeocin treatment and cannot activate Rad53 if irradiated in M phase. Our data suggest that Dpb11 is held in proximity to damaged DNA through an interaction with the phosphorylated 9-1-1 complex, leading to Mec1-dependent phosphorylation of Rad9. Dpb11 is also phosphorylated after DNA damage, and this modification is lost in a nonphosphorylatable ddc1-T602A mutant. Finally, we show that, in vivo, Dpb11 cooperates with Dot1 in promoting Rad9 phosphorylation but also contributes to the full activation of Mec1 kinase.The cellular response to DNA damage is based on signal transduction mechanisms that are essential for the maintenance of genome integrity. The molecules involved and the organization of the pathway are generally conserved in all eukaryotes (2,29,30,42). A major output of this response is a controlled delay in cell cycle progression that regulates the G 1 -S transition (G 1 checkpoint) or the G 2 -M transition (G 2 /M checkpoint; in budding yeast, this response does not regulate the passage from G 2 to M but prevents the anaphase-to-metaphase transition). This is achieved by regulating Cdk kinase or anaphase-promoting complex activities. The current model predicts that genotoxin treatments activate the DNA damage checkpoint response through the recruitment of the ATM and ATR phosphoinositide 3-kinase-related kinases to damaged chromatin (42, 51). The molecular details of the DNA damage signaling pathway in fission and budding yeasts have been mostly worked out by following the phosphorylation of critical kinase substrates in appropriately mutated genetic backgrounds (5, 25). In budding yeast, the prevalent apical kinase is represented by Mec1, which is associated with a Ddc2 subunit. Processing of DNA lesions by repair mechanisms generates single-stranded DNA (ssDNA) filaments that are rapidly coated by replication protein A (RPA). This structure seems to be responsible for the recruitment of 38,51). The first biochemical event in the signal transduction cascade seems to be the direct phosphorylation of Ddc2 (33, 37). A heterotrimeric complex (9-1-1) composed of Rad17, Mec3, and Ddc1 is loaded onto damaged DNA by a replication factor C-like complex and is itself phosphorylated by Mec1 on the Ddc1 subunit (25,28,34). Another Mec1 target is checkpoint factor Rad9, the orth...

The mechanisms used by checkpoints to identify DNA lesions are poorly understood and may involve the function of repair proteins. Looking for mutants specifically defective in activating the checkpoint following UV lesions, but proficient in the response to methyl methane sulfonate and double-strand breaks, we isolated cdu1-1, which is allelic to RAD14, the homolog of human XPA, involved in lesion recognition during nucleotide excision repair (NER). Rad14 was also isolated as a partner of the Ddc1 checkpoint protein in a two-hybrid screening, and physical interaction was proven by co-immunoprecipitation. We show that lesion recognition is not sufficient for checkpoint activation, but processing, carried out by repair factors, is required for recruiting checkpoint proteins to damaged DNA. Mutations affecting the core NER machinery abolish G1 and G2 checkpoint responses to UV, preventing activation of the Mec1 kinase and its binding to chromosomes. Conversely, elimination of transcriptioncoupled or global genome repair alone does not affect checkpoints, suggesting a possible interpretation for the heterogeneity in cancer susceptibility observed in different NER syndrome patients.

Template switching (TS) mediates damage-bypass via a recombination-related mechanism involving PCNA polyubiquitylation and Polymerase δ-dependent DNA synthesis. Using two-dimensional gel electrophoresis and electron microscopy, here we characterize TS intermediates arising in Saccharomyces cerevisiae at a defined chromosome locus, identifying five major families of intermediates. Single-stranded DNA gaps in the range of 150-200 nucleotides, and not DNA ends, initiate TS by strand invasion. This causes re-annealing of the parental strands and exposure of the non-damaged newly synthesized chromatid as template for replication by the other blocked nascent strand. Structures resembling double Holliday Junctions, postulated to be central double-strand break repair intermediates, but so far only visualized in meiosis, mediate late stages of TS, before being processed to hemicatenanes. Our results reveal the DNA transitions accounting for recombination-mediated DNA damage tolerance in mitotic cells and for replication under conditions of genotoxic stress.