Reconstitution of Rad53 Activation by Mec1 through Adaptor Protein Mrc1

Division of Biological Sciences, University of California, San Diego, La Jolla, California 92093-0653, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 06/2009; 284(28):18593-604. DOI: 10.1074/jbc.M109.018242
Source: PubMed

ABSTRACT Upon DNA replication stress, stalled DNA replication forks serve as a platform to recruit many signaling proteins, leading to the activation of the DNA replication checkpoint. Activation of Rad53, a key effector kinase in the budding yeast Saccharomyces cerevisiae, is essential for stabilizing DNA replication forks during replication stress. Using an activity-based assay for Rad53, we found that Mrc1, a replication fork-associated protein, cooperates with Mec1 to activate Rad53 directly. Reconstitution of Rad53 activation using purified Mec1 and Mrc1 showed that the addition of Mrc1 stimulated a more than 70-fold increase in the ability of Mec1 to activate Rad53. Instead of increasing the catalytic activity of Mec1, Mrc1 was found to facilitate the phosphorylation of Rad53 by Mec1 via promotion of a stronger enzyme-substrate interaction between them. Further, the conserved C-terminal domain of Mrc1 was found to be required for Rad53 activation. These results thus provide insights into the role of the adaptor protein Mrc1 in activating Rad53 in the DNA replication checkpoint.

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    • "In response to replication stress Mrc1 acts as a signal transducer mediating full Rad53 kinase activation (Alcasabas et al., 2001). By analogy with the paradigmatic checkpoint transducer Rad9, Mrc1 is thought to act as a scaffold promoting Rad53 transautophosphorylation events (Pellicioli and Foiani, 2005; Chen and Zhou, 2009; Berens and Toczyski, 2012). Mec1 phosphorylates Rad53 in a Mrc1-dependent manner, and full kinase activity is achieved when different Rad53 molecules hyperphosphorylate each other before being released to reach their targets (Pellicioli and Foiani, 2005). "
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    ABSTRACT: Conditions challenging replication fork progression, collectively referred to as replication stress, represent a major source of genomic instability and are associated to cancer onset. The replication checkpoint, a specialized branch of the DNA damage checkpoint, monitors fork problems, and triggers a cellular response aimed at preserving genome integrity. Here, we review the mechanisms by which the replication checkpoint monitors and responds to replication stress, focusing on the checkpoint-mediated pathways contributing to protect replication fork integrity. We discuss how cells achieve checkpoint signaling inactivation once replication stress is overcome and how a failure to timely revert checkpoint-mediated changes in cellular physiology might impact on replication dynamics and genome integrity. We also highlight the checkpoint function as an anti-cancer barrier preventing cells malignant transformation following oncogene-induced replication stress.
    Frontiers in Genetics 03/2013; 4:26. DOI:10.3389/fgene.2013.00026
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    • "Both the adaptors are able to recruit the checkpoint kinases Rad53 and Dun1, via their FHA domains, to trigger their activation. In particular, Mrc1 interacts with Tof1 and Csm3, associates with DNA replication forks [51,52,53] and has been shown to promote Rad53 phosphorylation by Mec1 directly under DNA replication stress conditions [54]. In agreement with the biochemical findings, the co-localization of Mrc1 and Mec1 is sufficient to promote Rad53 activation [55]. "
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    ABSTRACT: In order to preserve genome integrity, extrinsic or intrinsic DNA damages must be repaired before they accumulate in cells and trigger other mutations and genome rearrangements. Eukaryotic cells are able to respond to different genotoxic stresses as well as to single DNA double strand breaks (DSBs), suggesting highly sensitive and robust mechanisms to detect lesions that trigger a signal transduction cascade which, in turn, controls the DNA damage response (DDR). Furthermore, cells must be able to distinguish natural chromosomal ends from DNA DSBs in order to prevent inappropriate checkpoint activation, DDR and chromosomal rearrangements. Since the original discovery of RAD9, the first DNA damage checkpoint gene identified in Saccharomyces cerevisiae, many genes that have a role in this pathway have been identified, including MRC1, MEC3, RAD24, RAD53, DUN1, MEC1 and TEL1. Extensive studies have established most of the genetic basis of the DNA damage checkpoint and uncovered its different functions in cell cycle regulation, DNA replication and repair, and telomere maintenance. However, major questions concerning the regulation and functions of the DNA damage checkpoint remain to be answered. First, how is the checkpoint activity coupled to DNA replication and repair? Second, how do cells distinguish natural chromosome ends from deleterious DNA DSBs? In this review we will examine primarily studies performed using Saccharomyces cerevisiae as a model system.
    12/2012; 2(4):505-23. DOI:10.3390/biom2040505
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    • "If colocalization of Mec1 and Mrc1 is the only requirement for the reduced but significant level of checkpoint activation we observe in the ddc1∆ dpb11-1 tel1∆ strain, then Mec1 recruitment to a stalled fork is the only molecular event required to activate the checkpoint. In vitro results suggest that this model is possible, since Mrc1 purified from bacteria can promote Mec1 phosphorylation of Rad53 even in the absence of Mec1 activators (Chen and Zhou, 2009). Moreover, some Rad53 phosphorylation is seen in the replication-checkpoint mimic in the absence of a LacO array, suggesting that the Ddc2-LacI/Mrc1-LacI heterodimer is sufficient to promote Rad53 phosphorylation (Figures 1A and 2A). "
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    ABSTRACT: When DNA is damaged or DNA replication goes awry, cells activate checkpoints to allow time for damage to be repaired and replication to complete. In Saccharomyces cerevisiae, the DNA damage checkpoint, which responds to lesions such as double-strand breaks, is activated when the lesion promotes the association of the sensor kinase Mec1 and its targeting subunit Ddc2 with its activators Ddc1 (a member of the 9-1-1 complex) and Dpb11. It has been more difficult to determine what role these Mec1 activators play in the replication checkpoint, which recognizes stalled replication forks, since Dpb11 has a separate role in DNA replication itself. Therefore we constructed an in vivo replication-checkpoint mimic that recapitulates Mec1-dependent phosphorylation of the effector kinase Rad53, a crucial step in checkpoint activation. In the endogenous replication checkpoint, Mec1 phosphorylation of Rad53 requires Mrc1, a replisome component. The replication-checkpoint mimic requires colocalization of Mrc1-LacI and Ddc2-LacI and is independent of both Ddc1 and Dpb11. We show that these activators are also dispensable for Mec1 activity and cell survival in the endogenous replication checkpoint but that Ddc1 is absolutely required in the absence of Mrc1. We propose that colocalization of Mrc1 and Mec1 is the minimal signal required to activate the replication checkpoint.
    Molecular biology of the cell 02/2012; 23(6):1058-67. DOI:10.1091/mbc.E11-10-0852 · 4.47 Impact Factor
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