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The DNA replication checkpoint response stabilizes stalled replication forks


In response to DNA damage and blocks to replication, eukaryotes activate the checkpoint pathways that prevent genomic instability and cancer by coordinating cell cycle progression with DNA repair1,2,3,4,5. In budding yeast, the checkpoint response requires the Mec1-dependent activation of the Rad53 protein kinase3,4,6. Active Rad53 slows DNA synthesis when DNA is damaged7 and prevents firing of late origins of replication8,9. Further, rad53 mutants are unable to recover from a replication block10. Mec1 and Rad53 also modulate the phosphorylation state of different DNA replication and repair enzymes6,11,12,13. Little is known of the mechanisms by which checkpoint pathways interact with the replication apparatus when DNA is damaged or replication blocked. We used the two-dimensional gel technique14 to examine replication intermediates in response to hydroxyurea-induced replication blocks. Here we show that hydroxyurea-treated rad53 mutants accumulate unusual DNA structures at replication forks. The persistence of these abnormal molecules during recovery from the hydroxyurea block correlates with the inability to dephosphorylate Rad53. Further, Rad53 is required to properly maintain stable replication forks during the block. We propose that Rad53 prevents collapse of the fork when replication pauses.

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Figure 1: rad53-K227A mutant cells accumulate abnormal DNA structures at ARS305 in response to hydroxyurea treatment.
Figure 2: Analysis of replication intermediates in chromosome III regions adjacent to the ARS305 origin.
Figure 3: Y-shaped molecules in rad53-K227A mutant cells persist after hydroxyurea removal.
Figure 4: Proper maintenance of hydroxyurea-arrested replication forks requires Rad53 activity.


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We thank S. Alberti, L. Fabiani, A. Gambetta, S. Pintus and J. Theis for technical advice and support. We also thank A. Carr, J. Diffley, J. Haber, R. Rothstein, J. Sogo and all the members of our laboratory for helpful discussions. This work was supported by Associazione Italiana per la Ricerca sul Cancro and partially by grants from Telethon–Italy, Cofinanziamento MURST–Università di Milano, MURST (5%) Biomolecole per la Salute Umana, and CNR Target Project on Biotechnology, by a EU TMR contract, and by a NIH grant to C.S.N.

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Correspondence to Marco Foiani.

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Addentum to Figure 1

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Protein extracts prepared from cells taken at the indicated time points were analysed by western blotting using anti-Rad53 antibodies. The DNA content of wt and rad53 cells was analysed at the indicated time points by FACS analysis.

Addentum to Figure 4

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Protein extracts prepared from cells taken at the indicated time points were analysed by western blotting using anti-DNA polymerase alpha B subunit antibodies. The appearence of the hyperphosphorylated form of the B subunit indicates that the checkpoint has been inactivated (for further details see ref.6). The DNA content of wt and rad53 cells was analysed at the indicated time points by FACS analysis.

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Lopes, M., Cotta-Ramusino, C., Pellicioli, A. et al. The DNA replication checkpoint response stabilizes stalled replication forks. Nature 412, 557–561 (2001).

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