Structure of the TELO2-TTI1-TTI2 complex and its function in TOR recruitment to the R2TP chaperone

The R2TP (RUVBL1-RUVBL2-RPAP3-PIH1D1) complex, in collaboration with HSP90, functions as a chaperone for the assembly and stability of protein complexes, including RNA polymerases, snRNPs and PI3 kinase-like kinases (PIKK) such as TOR and SMG1. PIKK stabilisation depends on an additional complex of TELO2, TTI1 and TTI2 (TTT), whose structure and function are poorly understood. We have now determined the cryo-EM structure of the human R2TP-TTT complex that together with biochemical experiments reveals the mechanism of TOR recruitment to the R2TP-TTT chaperone. The HEAT-repeat TTT complex binds the kinase domain of TOR, without blocking its activity, and delivers TOR to the R2TP chaperone. In addition, TTT regulates the R2TP chaperone by inhibiting RUVBL1-RUVBL2 ATPase activity and by modulating the conformation and interactions of the PIH1D1 and RPAP3 components of R2TP. Together, our results show how TTT couples the recruitment of TOR to R2TP with the regulation of this chaperone system.

Both a Unique Motif at the C Terminus and an N-Terminal HEAT Repeat Contribute to G-Quadruplex Binding and Origin Regulation by the Rif1 Protein

ABSTRACT Rif1 is a key factor for spatiotemporal regulation of DNA replication. Rif1 suppresses origin firing in the mid-late replication domains by generating replication-suppressive chromatin architecture and by recruiting a protein phosphatase. In fission yeast, the function of Hsk1, a kinase important for origin firing, can be bypassed by rif1Δ due to the loss of origin suppression. Rif1 specifically binds to G-quadruplex (G4) in vitro. Here, we show both conserved N-terminal HEAT repeats and C-terminal nonconserved segments are required for origin suppression. The N-terminal 444 amino acids and the C-terminal 229 amino acids can each mediate specific G4 binding, although high-affinity G4 binding requires the presence of both N- and C-terminal segments. The C-terminal 91 amino acids, although not able to bind to G4, can form a multimer. Furthermore, genetic screening led to identification of two classes of rif1 point mutations that can bypass Hsk1, one that fails to bind to chromatin and one that binds to chromatin. These results illustrate functional domains of Rif1 and indicate importance of both the N-terminal HEAT repeat segment and C-terminal G4 binding/oligomerization domain as well as other functionally unassigned segments of Rif1 in regulation of origin firing.

Structural basis of HEAT-kleisin interactions in the human condensin I subcomplex

ABSTRUCTCondensin I is a multi-protein complex that plays an essential role in mitotic chromosome assembly and segregation in eukaryotes. It is composed of five subunits: two SMC (SMC2 and SMC4), a kleisin (CAP-H) and two HEAT-repeat (CAP-D2 and -G) subunits. Although it has been shown that balancing acts of the two HEAT-repeat subunits enable this complex to support dynamic assembly of chromosomal axes in vertebrate cells, its underlying mechanisms remain poorly understood. Here, we report the crystal structure of a human condensin I subcomplex comprising hCAP-G and hCAP-H. hCAP-H binds to the concave surfaces of a harp-shaped HEAT repeat domain of hCAP-G. A physical interaction between hCAP-G and hCAP-H is indeed essential for mitotic chromosome assembly recapitulated in Xenopus egg cell-free extracts. Furthermore, this study reveals that the human CAP-G-H subcomplex has the ability to interact with not only a double-stranded DNA, but also a single-stranded DNA, implicating potential, functional divergence of the vertebrate condensin I complex in mitotic chromosome assembly.

Multiple interactions between Scc1 and Scc2 activate cohesin’s DNA dependent ATPase and replace Pds5 during loading

SummaryIn addition to sharing with condensin an ability to organize DNA into chromatids, cohesin regulates enhancer-promoter interactions and confers sister chromatid cohesion. Association with chromosomes is regulated by hook-shaped HEAT repeat proteins that Associate With its Kleisin (Scc1) subunit (HAWKs), namely Scc3, Pds5, and Scc2. Unlike Pds5, Scc2 is not a stable cohesin constituent but, as shown here, transiently displaces Pds5 during loading. Scc1 mutations that compromise its interaction with Scc2 adversely affect cohesin’s ATPase activity, loading, and translocation while Scc2 mutations that alter how the ATPase responds to DNA abolish loading despite cohesin’s initial association with loading sites. Lastly, Scc2 mutations that permit loading in the absence of Scc4 increase Scc2’s association with chromosomal cohesin and reduce that of Pds5. We suggest that cohesin switches between two states, one with Pds5 bound to Scc1 that is not able to hydrolyse ATP efficiently but is capable of release from chromosomes and another in which Scc2, transiently replacing Pds5, stimulates the ATP hydrolysis necessary for loading and translocation away from loading sites.