Giuseppe Baldacci

Homologous Recombination Restarts Blocked Replication Forks at the Expense of Genome Rearrangements by Template Exchange

Template switching induced by stalled replication forks has recently been proposed to underlie complex genomic rearrangements. However, the resulting models are not supported by robust physical evidence. Here, we analyzed replication and recombination intermediates in a well-defined fission yeast system that blocks replication forks. We show that, in response to fork arrest, chromosomal rearrangements result from Rad52-dependent nascent strand template exchange occurring during fork restart. This template exchange occurs by both Rad51-dependent and -independent mechanisms. We demonstrate that Rqh1, the BLM homolog, limits Rad51-dependent template exchange without affecting fork restart. In contrast, we report that the Srs2 helicase promotes both fork restart and template exchange. Our data demonstrate that template exchange occurs during recombination-dependent fork restart at the expense of genome rearrangements.

Nearby inverted repeats fuse to generate acentric and dicentric palindromic chromosomes by a replication template exchange mechanism

Gene amplification plays important roles in the progression of cancer and contributes to acquired drug resistance during treatment. Amplification can initiate via dicentric palindromic chromosome production and subsequent breakage-fusion-bridge cycles. Here we show that, in fission yeast, acentric and dicentric palindromic chromosomes form by homologous recombination protein-dependent fusion of nearby inverted repeats, and that these fusions occur frequently when replication forks arrest within the inverted repeats. Genetic and molecular analyses suggest that these acentric and dicentric palindromic chromosomes arise not by previously described mechanisms, but by a replication template exchange mechanism that does not involve a DNA double-strand break. We thus propose an alternative mechanism for the generation of palindromic chromosomes dependent on replication fork arrest at closely spaced inverted repeats.

Peroxiredoxin Tsa1 Is the Key Peroxidase Suppressing Genome Instability and Protecting against Cell Death in Saccharomyces cerevisiae

Peroxiredoxins (Prxs) constitute a family of thiol-specific peroxidases that utilize cysteine (Cys) as the primary site of oxidation during the reduction of peroxides. To gain more insight into the physiological role of the five Prxs in budding yeast Saccharomyces cerevisiae, we performed a comparative study and found that Tsa1 was distinguished from the other Prxs in that by itself it played a key role in maintaining genome stability and in sustaining aerobic viability of rad51 mutants that are deficient in recombinational repair. Tsa2 and Dot5 played minor but distinct roles in suppressing the accumulation of mutations in cooperation with Tsa1. Tsa2 was capable of largely complementing the absence of Tsa1 when expressed under the control of the Tsa1 promoter. The presence of peroxidatic cysteine (Cys47) was essential for Tsa1 activity, while Tsa1C170S lacking the resolving Cys was partially functional. In the absence of Tsa1 activity (tsa1 or tsa1CCS lacking the peroxidatic and resolving Cys) and recombinational repair (rad51), dying cells displayed irregular cell size/shape, abnormal cell cycle progression, and significant increase of phosphatidylserine externalization, an early marker of apoptosis-like cell death. The tsa1CCS rad51– or tsa1 rad51–induced cell death did not depend on the caspase Yca1 and Ste20 kinase, while the absence of the checkpoint protein Rad9 accelerated the cell death processes. These results indicate that the peroxiredoxin Tsa1, in cooperation with appropriate DNA repair and checkpoint mechanisms, acts to protect S. cerevisiae cells against toxic levels of DNA damage that occur during aerobic growth.

The gene for a halophilic glutamate dehydrogenase sequence, transcription analysis and phylogenetic implications

SummaryWe have isolated and sequenced the gene for a putative NADP-dependent glutamate dehydrogenase from the extremely halophilic archaebacterium Halobacterium salinarium. This gene is transcribed as a unique RNA molecule of about 1700 nucleotides. The 5′end of the transcript contains characteristic consensus transcription initiation and promoter sequences observed in halophilic archaebacteria. The encoded polypeptide, with a predicted length of 435 amino acids, shows significant overall homology and conservation of functional domains when compared with different eubacterial and eukaryotic glutamate dehydrogenases. Surprisingly, the archaebacterial protein shares a larger number of identical amino acid residues with homologous polypeptides from higher eukaryotes than with those from unicellular eukaryotes and eubacteria.

p56chk1 protein kinase is required for the DNA replication checkpoint at 37°C in fission yeast

Fission yeast p56chk1 kinase is known to be involved in the DNA damage checkpoint but not to be required for cell cycle arrest following exposure to the DNA replication inhibitor hydroxyurea (HU). For this reason, p56chk1 is considered not to be necessary for the DNA replication checkpoint which acts through the inhibitory phosphorylation of p34cdc2 kinase activity. In a search for Schizosaccharomyces pombe mutants that abolish the S phase cell cycle arrest of a thermosensitive DNA polymerase δ strain at 37°C, we isolated two chk1 alleles. These alleles are proficient for the DNA damage checkpoint, but induce mitotic catastrophe in several S phase thermosensitive mutants. We show that the mitotic catastrophe correlates with a decreased level of tyrosine phosphorylation of p34cdc2. In addition, we found that the deletion of chk1 and the chk1 alleles abolish the cell cycle arrest and induce mitotic catastrophe in cells exposed to HU, if the cells are grown at 37°C. These findings suggest that chk1 is important for the maintenance of the DNA replication checkpoint in S phase thermosensitive mutants and that the p56chk1 kinase must possess a novel function that prevents premature activation of p34cdc2 kinase under conditions of impaired DNA replication at 37°C.

DNA polymerase δ is required for the replication feedback control of cell cycle progression in Schizosaccharomyces pombe

DNA replication and DNA repair are essential cell cycle steps ensuring correct transmission of the genome. The feedback replication control system links mitosis to completion of DNA replication and partially overlaps the radiation checkpoint control. Deletion of the chkl/rad27 gene abolishes the radiation but not the replication feedback control. Thermosensitive mutations in the DNA polymerase λ, cdc18 or cdc20 genes lead cells to arrest in the S phase of the cell cycle. We show that strains carrying any of these mutations enter lethal mitosis in the absence of the radiation checkpoint chk1/rad27. We interpret these data as an indication that an assembled replisome is essential for replication dependent control of mitosis and we propose that the arrest of the cell cycle in the thermosensitive mutants is due to the chk1+/rad27+ pathway, which monitors directly DNA for signs of damage.

Putative origins of replication in the mitochondrial genome of yeast

The mitochondrial genomes of cytoplasmic spontaneous ‘petite’ mut~ts of ~~cc~~ro~~c~s c~rev~s~ue arise by a mechanism in which a segment of a mitochondrial genome unit of a wild-type cell is excised and tandemly amplified (reviewed [I]; fig.1). Excised segments may originate from different regions of the wild-type genome unit, may have different sizes, and are highly conserved in sequence [2]. It has been suggested that, in the case of spontaneous ‘petite’ mutants, such segments contain an origin of replication, implying that the wild-type genome unit contains several origins of replication [3 1. In agreement with this suggestion, our recent work [4] has shown that 3 ‘petite’ mutants (which we call supersuppress~ve) have mitochondrial genomes characterized by the following features: (i) They are selectively transmitted to the progeny in crosses with wild-type cells; (ii) They are made up of very short repeat units (400-900 basepairs); (iii) They share a sequence of 80 nueleotides, in spite of the fact that they derive from two different regions of the parental wild-type genome. These results have been interpreted as indicating that the common sequence corresponds to an origin of replication; if this is so, the multiple copies of the origin of replication in supersuppressive genomes explain why these can compete out those of wildtype cells and become the only ones present in the progeny. Here we have shown that the common 80 nucleotide sequence exhibited by the 3 supersuppressive ‘pet&es in [4] also exists in the genome of

A role for DNA polymerase θ in the timing of DNA replication

Although DNA polymerase θ (Pol θ) is known to carry out translesion synthesis and has been implicated in DNA repair, its physiological function under normal growth conditions remains unclear. Here we present evidence that Pol θ plays a role in determining the timing of replication in human cells. We find that Pol θ binds to chromatin during early G1, interacts with the Orc2 and Orc4 components of the Origin recognition complex and that the association of Mcm proteins with chromatin is enhanced in G1 when Pol θ is downregulated. Pol θ-depleted cells exhibit a normal density of activated origins in S phase, but early-to-late and late-to-early shifts are observed at a number of replication domains. Pol θ overexpression, on the other hand, causes delayed replication. Our results therefore suggest that Pol θ functions during the earliest steps of DNA replication and influences the timing of replication initiation.

Supersuppressive “petite” mutants of yeast

SummaryA class of suppressive “petite” mutants of S. cerevisiae, called here supersuppressive, is characterized by a) the fact that their unmodified mitochondrial genomes are the only ones found in the progeny of crosses with wild-type cells; b) very short repeat units (400–900 base pairs) in their mitochondrial genomes. The repeat units of the three supersuppressive “petites” investigated here share a common 83 nucleotide sequence, which seems to correspond to an initiation site of DNA replication; the multiple copies of this site in the mitochondrial genomes of supersuppressive “petites” might explain why these genomes can compete out those of wild-type cells.

A new putative gene in the mitochondrial genome of Saccharomyces cerevisiae

The 2200-bp ori2-ori7 region of the mitochondrial (mt) genome of Saccharomyces cerevisiae has been sequenced on the genome of a petite, b7, excised at those ori sequences from wild-type strain B. The region contains an open reading frame, ORF5, which is transcribed into a 900-nucleotide (nt) RNA in both the parental wild-type strain and its derived petite, b7. This RNA uses as a template the strand used by most mt transcripts. Its start point is located 337 nt upstream of ORF5; and a messenger termination site has been found 900 nt downstream of the initiation site. These data suggest that ORF5 is a new mitochondrial gene. The G + C content of ORF5 is only 15.7%; 90% of the G + C base pairs of ORF5 are comprised in a palindromic G + C cluster similar to that present in the varl gene. The coding capacity of ORF5 is 46 amino acids (aa), mainly represented by methionine, phenylalanine, arginine, valine, asparagine, isoleucine and tyrosine. The aa composition and the codon usage of ORF5 are reminiscent of those of varl and of other intergenic ORFs.