The many roads to checkpoint activation

The genome of eukaryotic organisms is under constant surveillance by the DNA damage checkpoint (DDC) signaling network. In budding yeast, the sensor kinase Mec1^hATR and the downstream kinase Rad53^hCHK2 are the main checkpoint kinases in the DDC network. Mec1 and Rad53 play crucial roles in the preservation of genomic integrity and cell viability, as they regulate key effector proteins involved in processes such as DNA replication, repair, transcription and cell cycle control. Over the past 15 y, significant progress has been made in understanding how checkpoint kinases are activated, but are we close to a full mechanistic understanding of DDC activation and regulation? According to a recent paper in the October 15, 2012 edition of Cell Cycle by Wang et al.,¹ the answer is: not really.

The work by Wang et al. addresses the mechanism of signal transduction from Mec1 to Rad53, which is a critical step in the activation of the downstream portion of the DDC network. In budding yeast, the Rad9 adaptor protein is a key player in this step, functioning to recruit Rad53 molecules to sites of DNA damage where Mec1 is located.² How Rad9 recognizes sites of DNA lesions is a central question that has attracted the attention of several laboratories studying DDC signaling. It is now clear that Rad9 can be recruited to sites of DNA damage through redundant mechanisms that rely on histone modifications. The TUDOR domain of Rad9 recognizes histone H3K79 methylated by the Dot1 methylase, and the BRCT domain of Rad9 binds to histone H2A phosphorylated by Mec1 at S129 (H2A^pS129).^3,4 Additionally, Rad9 can be recruited by the BRCT domain-containing protein Dpb11^hTopBP1, which directly interacts with the 9-1-1 clamp and Mec1 at sites of DNA damage.⁵ The Rad9-Dpb11 interaction is mediated by Cdc28 (yeast CDK)-dependent phosphorylation of Rad9, which is subsequently recognized by the BRCT domains of Dpb11. How CDK-dependent phosphorylation of Rad9 is recognized by Dpb11 has been the focus of recent investigations.^6,7 An initial report by Granata et al. revealed that CDK-dependent phosphorylation of Rad9 on serine 11 (S11) is important for the interaction.⁶ In contrast, a more recent report from Pfander and Diffley indicated that the CDK-dependent phosphorylation of Rad9 on S462 and T474 plays a preponderant role in mediating the Rad9-Dpb11 interaction.⁷ In the October 15, 2012 edition of Cell Cycle, Wang et al. provide new insights that may reconcile these two previous reports. Using a series of point mutations aimed at disrupting different CDK consensus phosphorylation sites (over 17 phosphosites were mutated in different combinations), the authors show that multiple CDK phosphosites on Rad9 can function in a redundant manner to promote Rad53 activation. While the authors confirm that S462 and T474 are indeed important, their data show that other CDK phosphosites also contribute to the Rad9-Dpb11 interaction, revealing additional layers of redundancy in how Rad9 can be recruited.

Work by several laboratories has unveiled multiple modes of Rad9 recruitment, prompting the question of why there is so much redundancy toward Rad53 activation. It is clear that these different mechanisms of Rad9 recruitment are under distinct spatial regulation. While H3K79 methylation is widespread on chromatin and does not appear to be responsive to DNA damage, H2A^pS129 is mostly induced by DNA damage at regions of a few kilobases surrounding the lesions.^3,8 On the other hand, recruitment of Dpb11 is mostly dependent on the 9-1-1 clamp that is likely loaded specifically at sites of lesions.⁵ These observations suggest that each mode enables recruitment at distinct proximities to the site of lesion. It is tempting to speculate that in addition to providing “back-up” mechanisms, these different modes of recruitment cooperatively help cells fine-tune the levels and dynamics of Rad53 activation. To test this hypothesis, it would be important to use genotoxic conditions that more closely mimic physiological levels of stress. While relatively high doses of genotoxins are useful in identifying key players in the DNA damage response, they may mask many of the regulatory aspects of the response. Based on the work by Wang et al., it is possible to envision that different types or levels of DNA damage may lead to distinct patterns of Rad9 phosphorylation, creating distinct levels of interaction with Dpb11 and adding even more layers of regulation toward Rad53 activation.

Wang G, Tong X, Weng S, Zhou H. Multiple phosphorylation of Rad9 by CDK is required for DNA damage checkpoint activation. Cell Cycle. 2012;11:3792–800. doi: 10.4161/cc.21987.