Structure and function of the Rad9-binding region of the DNA-damage checkpoint adaptor TopBP1

TopBP1 is a scaffold protein that coordinates activation of the DNA-damage-checkpoint response by coupling binding of the 9-1-1 checkpoint clamp at sites of ssDNA, to activation of the ATR-ATRIP checkpoint kinase complex. We have now determined the crystal structure of the N-terminal region of human TopBP1, revealing an unexpected triple-BRCT domain structure. The arrangement of the BRCT domains differs significantly from previously described tandem BRCT domain structures, and presents two distinct sites for binding phosphopeptides in the second and third BRCT domains. We show that the site in the second but not third BRCT domain in the N-terminus of TopBP1, provides specific interaction with a phosphorylated motif at pSer387 in Rad9, which can be generated by CK2

Phosphorylation-dependent assembly and coordination of the DNA damage checkpoint apparatus by Rad4TopBP1

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

Structural Insights into Recognition of MDC1 by TopBP1 in DNA Replication Checkpoint Control

SummaryActivation of the DNA replication checkpoint by the ATR kinase requires protein interactions mediated by the ATR-activating protein, TopBP1. Accumulation of TopBP1 at stalled replication forks requires the interaction of TopBP1 BRCT5 with the phosphorylated SDT repeats of the adaptor protein MDC1. Here, we present the X-ray crystal structures of the tandem BRCT4/5 domains of TopBP1 free and in complex with a MDC1 consensus pSDpT phosphopeptide. TopBP1 BRCT4/5 adopts a variant BRCT-BRCT packing interface and recognizes its target peptide in a manner distinct from that observed in previous tandem BRCT- peptide structures. The phosphate-binding pocket and positively charged residues in a variant loop in BRCT5 present an extended binding surface for the negatively charged MDC1 phosphopeptide. Mutations in this surface reduce binding affinity and recruitment of TopBP1 to γH2AX foci in cells. These studies reveal a different mode of phosphopeptide binding by BRCT domains in the DNA damage response

BRCT domains of the DNA damage checkpoint proteins TOPBP1/Rad4 display distinct specificities for phosphopeptide ligands

TOPBP1 and its fission yeast homologue Rad4, are critical players in a range of DNA replication, repair and damage signalling processes. They are composed of multiple BRCT domains, some of which bind phosphorylated motifs in other proteins. They thus act as multi-point adaptors bringing proteins together into functional combinations, dependent on post-translational modifications downstream of cell cycle and DNA damage signals. We have now structurally and/or biochemically characterised a sufficient number of high-affinity complexes for the conserved N-terminal region of TOPBP1 and Rad4 with diverse phospho-ligands, including human RAD9 and Treslin, and Schizosaccharomyces pombe Crb2 and Sld3, to define the determinants of BRCT domain specificity. We use this to identify and characterise previously unknown phosphorylationdependent TOPBP1/Rad4-binding motifs in human RHNO1 and the fission yeast homologue of MDC1, Mdb1. These results provide important insights into how multiple BRCT domains within TOPBP1/Rad4 achieve selective and combinatorial binding of their multiple partner proteins

Phosphopeptide interactions with BRCA1 BRCT domains: More than just a motif.

BRCA1 BRCT domains function as phosphoprotein-binding modules for recognition of the phosphorylated protein-sequence motif pSXXF. While the motif interaction interface provides strong anchor points for binding, protein regions outside the motif have recently been found to be important for binding affinity. In this review, we compare the available structural data for BRCA1 BRCT domains in complex with phosphopeptides in order to gain a more complete understanding of the interaction between phosphopeptides and BRCA1-BRCT domains.We thank Dr Takashi Ochi for helpful discussion and comments on the manuscript. QW and TLB are funded by the Wellcome Trust (Grant: 093167/Z/10/Z). HJ thanks UCB and the Biotechnology and Biological Sciences Research Council (BBSRC) for a CASE Studentship.This is the final published version. It first appeared at http://dx.doi.org/10.1016/j.pbiomolbio.2015.02.00

BRCT domains: A little more than kin, and less than kind

AbstractBRCT domains are versatile protein modular domains found as single units or as multiple copies in more than 20 different proteins in the human genome. Interestingly, most BRCT-containing proteins function in the same biological process, the DNA damage response network, but show specificity in their molecular interactions. BRCT domains have been found to bind a wide array of ligands from proteins, phosphorylated linear motifs, and DNA. Here we discuss the biology of BRCT domains and how a domain-centric analysis can aid in the understanding of signal transduction events in the DNA damage response network

Rad17 Plays a Central Role in Establishment of the Interaction between TopBP1 and the Rad9-Hus1-Rad1 Complex at Stalled Replication Forks

Rad17 is critical for the ATR-dependent activation of Chk1 during checkpoint responses. It is known that Rad17 loads the Rad9-Hus1-Rad1 (9-1-1) complex onto DNA. We show that Rad17 also mediates the interaction of 9-1-1 with the ATR-activating protein TopBP1 in Xenopus egg extracts. Studies with Rad17 mutants indicate that binding of ATP to Rad17 is essential for the association of 9-1-1 and TopBP1. Furthermore, hydrolysis of ATP by Rad17 is necessary for the loading of 9-1-1 onto DNA and the elevated, checkpoint-dependent accumulation of TopBP1 on chromatin. Significantly, a mutant 9-1-1 complex that cannot bind TopBP1 has a normal capacity to promote elevated accumulation of TopBP1 on chromatin. Taken together, we propose the following mechanism. First, Rad17 loads 9-1-1 onto DNA. Second, TopBP1 accumulates on chromatin in a manner that depends on both Rad17 and 9-1-1. Finally, 9-1-1 and TopBP1 dock in a Rad17-dependent manner before activation of Chk1

14-3-3 Proteins, FHA Domains and BRCT Domains in the DNA Damage Response

The DNA damage response depends on the concerted activity of protein serine/threonine kinases and modular phosphoserine/threonine-binding domains to relay the damage signal and recruit repair proteins. The PIKK family of protein kinases, which includes ATM/ATR/DNA-PK, preferentially phosphorylate Ser-Gln sites, while their basophilic downstream effecter kinases, Chk1/Chk2/MK2 preferentially phosphorylate hydrophobic-X-Arg-X-X-Ser/Thr-hydrophobic sites. A subset of tandem BRCT domains act as phosphopeptide binding modules that bind to ATM/ATR/DNA-PK substrates after DNA damage. Conversely, 14-3-3 proteins interact with substrates of Chk1/Chk2/MK2. FHA domains have been shown to interact with substrates of ATM/ATR/DNA-PK and CK2. In this review we consider how substrate phsophorylation together with BRCT domains, FHA domains and 14-3-3 proteins function to regulate ionizing radiation-induced nuclear foci and help to establish the G2/M checkpoint. We discuss the role of MDC1 a molecular scaffold that recruits early proteins to foci, such as NBS1 and RNF8, through distinct phosphodependent interactions. In addition, we consider the role of 14-3-3 proteins and the Chk2 FHA domain in initiating and maintaining cell cycle arrest