The cellular response to chromosome breakage

Abstract

SummaryDNA double-strand breaks (DSBs) are among the most deleterious types of damage that can occur in the genome of eukaryotic cells because failure to repair them can lead to loss of genetic information and chromosome rearrangements. DSBs can arise by failures in DNA replication and by exposure to environmental factors, such as ionizing radiations and radiomimetic chemicals. Moreover, they might arise when telomeres undergo extensive erosion, leading to the activation of the DNA damage response pathways and the onset of apoptosis and/or senescence. Importantly, DSBs can also form in a programmed manner during development. For example, meiotic recombination and rearrangement of the immunoglobulin genes in lymphocytes require the generation of site-or region-specific DSBs through the action of specific endonucleases. Efficient DSB repair is crucial in safeguarding genome integrity, whose maintenance in the face of DSBs involves branched signalling networks that switch on DNA damage checkpoints, activate DNA repair, induce chromatin reorganization and modulate numerous cellular processes. Not surprisingly, defects in these networks result in a variety of diseases ranging from severe genetic disorders to cancer predisposition and accelerated ageing.

Budding Yeast Sae2 is an In Vivo Target of the Mec1 and Tel1 Checkpoint Kinases During Meiosis

“…ApaI digestion of the integrative plasmids pML469.14, pML473.15, pML474.35 and pML488.15 described in Baroni et al, 2004 44 was used to direct the integration of these plasmids to the SAE2 promoter region of a SK1-derivative sae2∆ strain, giving rise to heterozygous diploid strains carrying, respectively, single copies of the SAE2, sae2 [1][2][3][4][5] , sae2 6-9 and sae2 2,5,6,8,9 alleles at the SAE2 chromosomal locus. To generate SAE2, sae2 [1][2][3][4][5] , sae2 6-9 and sae2 2,5,6,8,9 homozygous diploid strains, the above heterozygous strains were allowed to sporulate, followed by tetrad dissection and self-diploidization of spores with the desired SAE2 alleles.…”

“…To generate SAE2, sae2 [1][2][3][4][5] , sae2 6-9 and sae2 2,5,6,8,9 homozygous diploid strains, the above heterozygous strains were allowed to sporulate, followed by tetrad dissection and self-diploidization of spores with the desired SAE2 alleles. PCR one-step tagging was then used to obtain strains YLL1772/7D, YLL1773/14D, YLL1791/3D and YLL1774/5B, carrying one copy of the HA-tagged SAE2, sae2 [1][2][3][4][5] , sae2 6-9 and sae2 2,5,6,8,9 alleles, respectively. The accuracy of all gene replacements and integrations was verified by Southern blot analysis or PCR.…”

“…All these residues were numbered progressively from 1 to 9 starting from the most N-terminal one. 44 The derivative sae2 [1][2][3][4][5] (alanine substitutions of S72, S73, T75, S76, T90), sae2 [6][7][8][9] (alanine substitutions of S249, S278, T279, S289) and sae2 2,5,6,8,9 (alanine substitutions of S73, T90, S249, T279 and S289) alleles were fused in frame to a three HA epitope coding sequence before the stop codon in order to follow phosphorylation of the corresponding gene products by western blot analysis.…”

“…1 Key players in these pathways belong to an evolutionarily conserved protein kinase family including mammalian ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and RAD3-related (ATR), as well as their S. cerevisiae orthologs Tel1 and Mec1. 2,3 Other checkpoint factors, among which the S. cerevisiae PCNA-like complex Mec3/Ddc1/Rad17 and the RFC-like complex Rad24/Rfc2-5, as well as their mammalian orthologs, participate in the DNA damage sensing step and are thought to modulate Mec1/ATR activation.…”

“…5 It has therefore been proposed that ATM/Tel1 and the MRN/MRX complex are the first proteins localizing at DSBs to initiate signaling and attempt repair, whereas 5'-3' exonucleolytic degradation of DSB termini generates 3'-ended ssDNA that is detected by ATR/Mec1 and is prerequisite for homologous recombination to occur. 1 The DNA damage sensing functions are linked with the downstream effectors by Mec1/ATR-or Tel1/ATM-dependent phosphorylation of adaptor proteins represented by the archetypal S. cerevisiae protein Rad9. In particular, Mec1/ATR-or Tel1/ATM-dependent phosphorylation of Rad9 triggers its interaction with Rad53 and consequent release of active Rad53 kinase.…”