Phosphorylation-Dependent Assembly and Coordination of the DNA Damage Checkpoint Apparatus by Rad4TopBP1

Abstract

The BRCT-domain protein Rad4(TopBP1) facilitates activation of the DNA damage checkpoint in Schizosaccharomyces pombe by physically coupling the Rad9-Rad1-Hus1 clamp, the Rad3(ATR) -Rad26(ATRIP) kinase complex, and the Crb2(53BP1) mediator. We have now determined crystal structures of the BRCT repeats of Rad4(TopBP1), revealing a distinctive domain architecture, and characterized their phosphorylation-dependent interactions with Rad9 and Crb2(53BP1). We identify a cluster of phosphorylation sites in the N-terminal region of Crb2(53BP1) that mediate interaction with Rad4(TopBP1) and reveal a hierarchical phosphorylation mechanism in which phosphorylation of Crb2(53BP1) residues Thr215 and Thr235 promotes phosphorylation of the noncanonical Thr187 site by scaffolding cyclin-dependent kinase (CDK) recruitment. Finally, we show that the simultaneous interaction of a single Rad4(TopBP1) molecule with both Thr187 phosphorylation sites in a Crb2(53BP1) dimer is essential for establishing the DNA damage checkpoint.

Facile manipulation of protein localization in fission yeast through binding of GFP-binding protein to GFP

“…A 13-kDa soluble protein derived from a llama heavy chain antibody (called GFP-binding protein or GBP) has been recently developed for protein targeting in vivo (Rothbauer et al, 2006(Rothbauer et al, , 2008 and rapidly applied to cultured mammalian cells and various model organisms, including fly, worm, Nicotiana, Arabidopsis and S. pombe (Deng and Moseley, 2013;Dodgson et al, 2013;Grallert et al, 2013;Maier et al, 2013;Neumuller et al, 2012;Qu et al, 2013;Rothbauer et al, 2008;Schornack et al, 2009;Sonneville et al, 2012;Tao et al, 2014;Ye et al, 2012). This single-domain antibody features has high binding affinity to GFP as well as to some GFP variants, such as yellow fluorescent protein (YFP), with a stoichiometric ratio of 1:1, but not to cyan fluorescent protein (CFP) or any derivatives of DsRed, such as mRFP, mCherry or mOrange (Kirchhofer et al, 2010;Kubala et al, 2010;Rothbauer et al, 2008).…”

Multi-BRCT scaffolds use distinct strategies to support genome maintenance

“…23 It shows that although each BRCT repeat adopts a classical fold, the relative orientation of the 2 repeats within BRCT1-2 (perpendicular) is distinct from that in BRCT3-4 (anti-parallel), and both are distinct from canonical tBRCTs (parallel). Other unique features include the engagement of both BRCT1 and 2 with phospho-peptide, allowing association with a Crb2 dimer, and the interaction of BRCT4, but not BRCT3, with a phospho-peptide of Rad9.…”

“…1). 22,23,24,25 Both adaptors transmit signals from the apical checkpoint kinase, Mec1 (S. p. Rad3), to a downstream effector kinase, Rad53 (S. p. Cds1). 26 Using budding yeast as an example, Mec1 and its partner Ddc2 localize to DNA lesion sites via interactions with RPA coated single strand DNA (ssDNA) (Fig.…”

Dimerization Mediated by a Divergent Forkhead-associated Domain Is Essential for the DNA Damage and Spindle Functions of Fission Yeast Mdb1

“…To test this idea, we adopted an approach of constructing an artificial heterodimer (30). We fused a monomeric GFP-binding protein (GBP) to Mdb1(105-624), so that it can form a heterodimer with GFP-tagged Mdb1(105-624).…”