Mrc1 is a replication fork component whose phosphorylation in response to DNA replication stress activates Rad53
- 1 Verna and Marrs MacLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030, USA
- 2 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA
- 3 Howard Hughes Medical Institute, Baylor College of Medicine, Houston, Texas 77030, USA
Abstract
When DNA replication is stalled, a signal transduction pathway is activated that promotes the stability of stalled forks and resumption of DNA synthesis. In budding yeast, this pathway includes the kinases Mec1 and Rad53. Here we report that the Mediator protein Mrc1, which is required for normal DNA replication and for activation of Rad53, is present at replication forks. Mrc1 initially binds early-replicating sequences and moves along chromatin with the replication fork. Blocking initiation of DNA replication blocks Mrc1 loading onto origins, providing an explanation for why so many mutants in DNA replication show checkpoint defects. In the presence of replication blocks, we find that Mec1 is recruited to regions of stalled replication, where it encounters and presumably phosphorylates Mrc1. Mutation of the canonical Mec1 phosphorylation sites on Mrc1 prevents Mrc1 phosphorylation and blocks Rad53 activation, but does not alter Mrc1's role in DNA replication. Our results suggest a model whereby in response to DNA replication interference, the Mec1 kinase is recruited to sites of replication blocks and phosphorylates a component of the DNA replication complex, Mrc1, thereby setting up a solid-state Rad53 activation platform to initiate the checkpoint response.
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Footnotes
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Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/gad.1098303.
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↵4 Corresponding author. E-MAIL selledge{at}bcm.tmc.edu; FAX (713) 798-8717.
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- Accepted May 22, 2003.
- Received March 27, 2003.
- Cold Spring Harbor Laboratory Press
