DNA double-strand breaks (DSBs) can arise at unpredictable locations after DNA damage or in a programmed manner during meiosis. DNA damage checkpoint response to accidental DSBs during mitosis requires the Rad53 effector kinase. On the other hand, the meiosis-specific Mek1 kinase, together with Red1 and Hop1, mediates the recombination checkpoint in response to programmed Spo11-dependent meiotic DSBs that are required for meiotic recombination to take place. In Saccharomyces cerevisiae, the Sae2 protein and the Mre11- Rad50-Xrs2 complex are necessary to remove the covalently attached Spo11 protein from the DNA ends of meiotic DSBs, which are then resected by so far unknown nucleases. As several aspects of the control of the response to DSBs during meiosis are still obscure, I focused my research as Ph. D. student on two different aspects of this control: 1) the possible role of Rad53 and inter-relationships with Mek1 in the response to accidental and programmed DSBs during meiosis and 2) the mechanisms responsible for processing Spo11-induced meiotic DSBs. 1. We have provided evidence that exogenous DSBs lead to Rad53 phosphorylation during meiosis, whereas programmed meiotic DSBs do not. However, the latter can trigger phosphorylation of a protein fusion between Rad53 and the Mec1-interacting protein Ddc2, suggesting that the inability of Rad53 to transduce the meiosis-specific DSB signals might be due to its failure to access the meiotic recombination sites. Rad53 phosphorylation/activation is elicited when unrepaired meiosis-specific DSBs escape the recombination checkpoint. This activation requires homologous chromosome segregation and delays the second meiotic division. Altogether, these data indicate that Rad53 phosphorylation prevents sister chromatid segregation in the presence of unrepaired programmed meiotic DSBs, thus providing a salvage mechanism ensuring genetic integrity in the gametes even in the absence of the recombination checkpoint. 2. By using site directed mutagenesis and gene replacements, we have demonstrated that phosphorylation of Sae2 Ser-267 by cyclin-dependent kinase 1 (Cdk1) is required to initiate meiotic DSB resection by allowing Spo11 removal from DSB ends. This finding suggests that Cdk1 activity is required for the processing of Spo11-induced DSBs, thus providing a mechanism for coordinating DSB resection with progression through meiotic prophase. Furthermore, we used different genetic and biochemical tools to demonstrate that the helicase Sgs1 and the nucleases Exo1 and Dna2 participate in lengthening the 5’-3’ resection tracts during meiosis by controlling a step subsequent to Spo11 removal. Our findings suggest that, once Spo11 has catalyzed meiosis-specific DSB formation, it is removed from the DSB ends by endonucleolytic cleavage that necessitates CDK-mediated Sae2 phosphorylation and the nuclease activity of MRX. This cleavage is in turn required for resection of the break by either Exo1 or Dna2-Sgs1 activities, thus allowing completion of meiosis-specific DSB processing.

(2011). Maintenance of genome integrity in gametes: coping with accidental and programmed DNA double-strand breaks during meiosis. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2011).

Maintenance of genome integrity in gametes: coping with accidental and programmed DNA double-strand breaks during meiosis

MANFRINI, NICOLA
2011

Abstract

DNA double-strand breaks (DSBs) can arise at unpredictable locations after DNA damage or in a programmed manner during meiosis. DNA damage checkpoint response to accidental DSBs during mitosis requires the Rad53 effector kinase. On the other hand, the meiosis-specific Mek1 kinase, together with Red1 and Hop1, mediates the recombination checkpoint in response to programmed Spo11-dependent meiotic DSBs that are required for meiotic recombination to take place. In Saccharomyces cerevisiae, the Sae2 protein and the Mre11- Rad50-Xrs2 complex are necessary to remove the covalently attached Spo11 protein from the DNA ends of meiotic DSBs, which are then resected by so far unknown nucleases. As several aspects of the control of the response to DSBs during meiosis are still obscure, I focused my research as Ph. D. student on two different aspects of this control: 1) the possible role of Rad53 and inter-relationships with Mek1 in the response to accidental and programmed DSBs during meiosis and 2) the mechanisms responsible for processing Spo11-induced meiotic DSBs. 1. We have provided evidence that exogenous DSBs lead to Rad53 phosphorylation during meiosis, whereas programmed meiotic DSBs do not. However, the latter can trigger phosphorylation of a protein fusion between Rad53 and the Mec1-interacting protein Ddc2, suggesting that the inability of Rad53 to transduce the meiosis-specific DSB signals might be due to its failure to access the meiotic recombination sites. Rad53 phosphorylation/activation is elicited when unrepaired meiosis-specific DSBs escape the recombination checkpoint. This activation requires homologous chromosome segregation and delays the second meiotic division. Altogether, these data indicate that Rad53 phosphorylation prevents sister chromatid segregation in the presence of unrepaired programmed meiotic DSBs, thus providing a salvage mechanism ensuring genetic integrity in the gametes even in the absence of the recombination checkpoint. 2. By using site directed mutagenesis and gene replacements, we have demonstrated that phosphorylation of Sae2 Ser-267 by cyclin-dependent kinase 1 (Cdk1) is required to initiate meiotic DSB resection by allowing Spo11 removal from DSB ends. This finding suggests that Cdk1 activity is required for the processing of Spo11-induced DSBs, thus providing a mechanism for coordinating DSB resection with progression through meiotic prophase. Furthermore, we used different genetic and biochemical tools to demonstrate that the helicase Sgs1 and the nucleases Exo1 and Dna2 participate in lengthening the 5’-3’ resection tracts during meiosis by controlling a step subsequent to Spo11 removal. Our findings suggest that, once Spo11 has catalyzed meiosis-specific DSB formation, it is removed from the DSB ends by endonucleolytic cleavage that necessitates CDK-mediated Sae2 phosphorylation and the nuclease activity of MRX. This cleavage is in turn required for resection of the break by either Exo1 or Dna2-Sgs1 activities, thus allowing completion of meiosis-specific DSB processing.
LUCCHINI, GIOVANNA
Longhese, Maria Pia
DSBs, meiosis, yeast, S. cerevisiae, recombination, checkpoint, CDK, Mek1, Rad53
BIO/18 - GENETICA
English
Scuola di dottorato di Scienze
BIOLOGIA - 48R
23
2009/2010
(2011). Maintenance of genome integrity in gametes: coping with accidental and programmed DNA double-strand breaks during meiosis. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2011).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/18999
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