PubMed Health⌕ Search

Biomedical subjects

Marco Muzi-Falconi

Publications and source records attributed to Marco Muzi-Falconi.

11 recordsLinked to original sources
PubMed2006

Yeast Rev1 is cell cycle regulated, phosphorylated in response to DNA damage and its binding to chromosomes is dependent upon MEC1.

Translesion DNA synthesis (TLS) is one of the mechanisms involved in lesion bypass during DNA replication. Three TLS polymerases (Pol) are present in the yeast Saccharomyces cerevisiae: Pol zeta, Pol eta and the product of the REV1 gene. Rev1 is considered a deoxycytidyl transferase because it almost exclusively inserts a C residue in front of the lesion. Even though REV1 is required for most of the UV-induced and spontaneous mutagenesis events, the role of Rev1 is poorly understood since its polymerase activity is often dispensable. Rev1 interacts with several TLS polymerases in mammalian cells and may act as a platform in the switching mechanism required to substitute a replicative polymerase with a TLS polymerase at the sites of DNA lesions. Here we show that yeast Rev1 is a phosphoprotein, and the level of this modification is cell cycle regulated under normal growing conditions. Rev1 is unphosphorylated in G1, starts to be modified while cells are passing S phase and it becomes hyper-phosphorylated in mitosis. Rev1 is also hyper-phosphorylated in response to a variety of DNA damaging agents, including treatment with a radiomimetic drug mostly causing double-strand breaks (DSB). By using the chromosome spreading technique we found the Rev1 is bound to chromosomes throughout the cell cycle, and its binding does not significantly increase in response to genotoxic stress. Therefore, Rev1 phosphorylation does not appear to modulate its binding to chromosomes, suggesting that such modification may influence other aspects of the TLS process. Rev1 binding under damaged and undamaged conditions, is at least partially dependent on MEC1, a gene playing a pivotal role in the DNA damage checkpoint cascade. This genetic dependency may suggest a role for MEC1 in spontaneous mutagenesis events, which require a functional REV1 gene.

Chromosomes, Fungal↗
PubMed2006

Alk1 and Alk2 are two new cell cycle-regulated haspin-like proteins in budding yeast.

Haspin is a protein kinase identified in mouse and human cells, and genes coding for haspin-like proteins are present in virtually all eukaryotic genomes sequenced so far. Two haspin homologues, called Alk1 and Alk2, are present in the yeast Saccharomyces cerevisiae. Both Alk1 and Alk2 exhibit a weak auto-kinase activity in vitro, are phosphoproteins in vivo and are hyperphosphorylated in response to DNA damage. The amount and modification of the two proteins is greatly regulated during the cell cycle. In fact, Alk1 and Alk2 levels peak in mitosis and late-S/G2, respectively, and phosphorylation of both proteins is maximal in mitosis. Control of protein stability plays a major role in Alk2 regulation. The half-life of Alk2 is particularly short in G1; mutagenesis and genetic analysis indicate that its degradation is controlled by the APC pathway. Overexpression of ALK2, but not of ALK1, causes a mitotic arrest, which is correlated to the kinase activity of the protein. This finding, together with its cell cycle regulation, suggests a role for Alk2 in the control of mitosis.

Amino Acid Sequence↗
PubMed2005

The 9-1-1 checkpoint clamp physically interacts with polzeta and is partially required for spontaneous polzeta-dependent mutagenesis in Saccharomyces cerevisiae.

The use of translesion synthesis (TLS) polymerases to bypass DNA lesions during replication constitutes an important mechanism to restart blocked/stalled DNA replication forks. Because TLS polymerases generally have low fidelity on undamaged DNA, the cell must regulate the interaction of TLS polymerases with damaged versus undamaged DNA to maintain genome integrity. The Saccharomyces cerevisiae checkpoint proteins Ddc1, Rad17, and Mec3 form a clamp-like structure (the 9-1-1 clamp) that has physical similarity to the homotrimeric sliding clamp proliferating cell nuclear antigen, which interacts with and promotes the processivity of the replicative DNA polymerases. In this work, we demonstrate both an in vivo and in vitro physical interaction between the Mec3 and Ddc1 subunits of the 9-1-1 clamp and the Rev7 subunit of the Polzeta TLS polymerase. In addition, we demonstrate that loss of Mec3, Ddc1, or Rad17 results in a decrease in Polzeta-dependent spontaneous mutagenesis. These results suggest that, in addition to its checkpoint signaling role, the 9-1-1 clamp may physically regulate Polzeta-dependent mutagenesis by controlling the access of Polzeta to damaged DNA.

Base Sequence↗
PubMed2005

The DNA damage checkpoint response requires histone H2B ubiquitination by Rad6-Bre1 and H3 methylation by Dot1.

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.

Binding Sites↗
PubMed2004

DNA decay and limited Rad53 activation after liquid holding of UV-treated nucleotide excision repair deficient S. cerevisiae cells.

The DNA damage checkpoint is a surveillance mechanism activated by DNA lesions and devoted to the maintenance of genome stability. It is considered as a signal transduction cascade, involving a sensing step, the activation of a set of protein kinases and the transmission and amplification of the damage signal through several phosphorylation events. In budding yeast many players of this pathway have been identified. Recent work showed that G1 and G2 checkpoint activation in response to UV irradiation requires prior recognition and processing of UV lesions by nucleotide excision repair (NER) factors that likely recruit checkpoint proteins near the damage. However, another report suggested that NER was not required for checkpoint function. Since the functional relationship between repair mechanisms and checkpoint activation is a very important issue in the field, we analyzed, under different experimental conditions, whether lesion processing by NER is required for checkpoint activation. We found that DNA damage checkpoint can be triggered in an NER-independent manner only if cells are subjected to liquid holding after UV treatment. This incubation causes a time-dependent breakage of DNA strands in NER-deficient cells and leads to partial activation of the checkpoint kinase. The analysis of the genetic requirements for this alternative activation pathway suggest that it requires Mec1 and the Rad17 complex and that the observed DNA breaks are likely to be due to spontaneous decay of damaged DNA.

Cell Cycle Proteins↗
PubMed2004

Physical and functional interactions between nucleotide excision repair and DNA damage checkpoint.

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 transcription-coupled 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.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Correlation between checkpoint activation and in vivo assembly of the yeast checkpoint complex Rad17-Mec3-Ddc1.

Rad17-Mec3-Ddc1 forms a proliferating cell nuclear antigen-like complex that is required for the DNA damage response in Saccharomyces cerevisiae and acts at an early step of the signal transduction cascade activated by DNA lesions. We used the mec3-dn allele, which causes a dominant negative checkpoint defect in G1 but not in G2, to test the stability of the complex in vivo and to correlate its assembly and disassembly with the mechanisms controlling checkpoint activation. Under physiological conditions, the mutant complex is formed both in G1 and G2, although the mutant phenotype is detectable only in G1, suggesting that is not the presence of the mutant complex per se to cause a checkpoint defect. Our data indicate that the Rad17-Mec3-Ddc1 complex is very stable, and it takes several hours to replace Mec3 with Mec3-dn within a wild type complex. On the other hand, the mutant complex is rapidly assembled when starting from a condition where the complex is not pre-assembled, indicating that the critical factor for the substitution is the disassembly step rather than complex formation. Moreover, the kinetics of mutant complex assembly, starting from conditions in which the wild type form is present, parallels the kinetics of checkpoint inactivation, suggesting that the complex acts in a stoichiometric way, rather than catalytically.

Cell Cycle↗
PubMed2003

The DNA polymerase alpha-primase complex: multiple functions and interactions.

DNA polymerase alpha (pol alpha) holds a special position among the growing family of eukaryotic DNA polymerases. In fact, pol alpha 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 alpha-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.

Animals↗
PubMed2002

A dominant-negative MEC3 mutant uncovers new functions for the Rad17 complex and Tel1.

The Rad17-Mec3-Ddc1 complex is essential for the cellular response to genotoxic agents and is thought to be important for sensing DNA lesions. Deletion of any of the RAD17, MEC3 or DDC1 genes abolishes the G(1) and G(2) and impairs the intra-S DNA-damage checkpoints. We characterize a dominant-negative mec3-dn mutation that has an unexpected phenotype. It inactivates the G(1) checkpoint while it leaves the G(2) response functional, thus revealing a difference in the requirements of the DNA-damage response in different phases of the cell cycle. In an attempt to identify the molecular defect imparted by the mutation, we dissected step-by-step the signaling cascade, which is triggered by DNA lesions and requires the activity of Mec1 and Rad53 kinases. The analysis of the phosphorylation state of checkpoint factors and critical protein interactions showed that, in mec3-dn cells, the signal transduction cascade is triggered normally, and the central kinase Mec1 can be activated. In G(1) cells expressing the mutation, the signaling cannot proceed any further along the pathway, indicating that the Rad17 complex acts after the activation of Mec1, possibly recruiting targets for the kinase. We also show that the function of the G(2) checkpoint in mutant cells is maintained by an uncharacterized activity of Tel1, the yeast homologue of ATM. This work thus reports a previously undiscovered role for Tel1 in checkpoint control.

Cell Cycle↗
PubMedDate not supplied

Mechanisms controlling the integrity of replicating chromosomes in budding yeast.

Cells are continually challenged by genomic insults that originate from chemical and physical agents diffused in the environment, but also normal cellular metabolism produces genotoxic effects. Moreover, DNA replication and recombination generate intermediates potentially dangerous for genome stability. Growing evidence show that many genetic disorders are characterized by high levels of chromosome alterations due to genomic instability, which is also a hallmark of cancer cells. Recent work shed some light on the molecular events that maintain the integrity of chromosomes during unperturbed S phase and in the face of odds.

Cell Cycle↗