SUMMARY HSP90 is a molecular chaperone that associates with numerous substrate proteins called clients. It plays many important roles in human biology and medicine, but determinants of client recognition by HSP90 have remained frustratingly elusive. We systematically and quantitatively surveyed most human kinases, transcription factors, and E3 ligases for interaction with HSP90 and its cochaperone CDC37. Unexpectedly, many more kinases than transcription factors bound HSP90. CDC37 interacted with kinases, but not with transcription factors or E3 ligases. HSP90::kinase interactions varied continuously over a 100-fold range and provided a platform to study client protein recognition. In wild-type clients, HSP90 did not bind particular sequence motifs, but rather associated with intrinsically unstable kinases. Stabilization of the kinase in either its active or inactive conformation with diverse small molecules decreased HSP90 association. Our results establish HSP90 client recognition as a combinatorial process: CDC37 provides recognition of the kinase family, whereas thermodynamic parameters determine client binding within the family.

SUMMARY How disease-associated mutations impair protein activities in the context of biological networks remains mostly undetermined. Although a few renowned alleles are well characterized, functional information is missing for over 100,000 disease-associated variants. Here we functionally profile several thousand missense mutations across a spectrum of Mendelian disorders using various interaction assays. The majority of disease-associated alleles exhibit wild-type chaperone binding profiles, suggesting they preserve protein folding or stability. While common variants from healthy individuals rarely affect interactions, two-thirds of disease-associated alleles perturb protein-protein interactions, with half corresponding to “edgetic” alleles affecting only a subset of interactions while leaving most other interactions unperturbed. With transcription factors, many alleles that leave protein-protein interactions intact affect DNA binding. Different mutations in the same gene leading to different interaction profiles often result in distinct disease phenotypes. Thus disease-associated alleles that perturb distinct protein activities rather than grossly affecting folding and stability are relatively widespread.

The ADP-ribosylation of proteins is an important posttranslational modification that occurs in a variety of biological processes, including DNA repair, transcription, chromatin biology and long-term memory formation. Yet no protein modules are known that specifically recognize the ADP-ribose nucleotide. We provide biochemical and structural evidence that macro domains are high-affinity ADP-ribose binding modules. Our structural analysis reveals a conserved ligand binding pocket among the macro domain fold. Consistently, distinct human macro domains retain their ability to bind ADP-ribose. In addition, some macro domain proteins also recognize poly-ADP-ribose as a ligand. Our data suggest an important role for proteins containing macro domains in the biology of ADP-ribose.

Damaged DNA templates provide an obstacle to the replication fork and can cause genome instability. In eukaryotes, tolerance to damaged DNA is mediated largely by the RAD6 pathway involving ubiquitylation of the DNA polymerase processivity factor PCNA. Whereas monoubiquitylation of PCNA mediates error-prone translesion synthesis (TLS), polyubiquitylation triggers an error-free pathway. Both branches of this pathway are believed to occur in S phase in order to ensure replication completion. However, we found that limiting TLS or the error-free pathway to the G2/M phase of the cell-cycle efficiently promote lesion tolerance. Thus, our findings indicate that both branches of the DNA damage tolerance pathway operate effectively after chromosomal replication, outside S phase. We therefore propose that the RAD6 pathway acts on single-stranded gaps left behind newly restarted replication forks.

Background: PCNA mono-ubiquitination at stalled replication forks recruits translesion synthesis polymerases for fork restart. Results:The mono-ADP-ribosyltransferase PARP10 interacts with PCNA through a PIP-box. PARP10 knockdown results in DNA damage hypersensitivity and defective translesion synthesis. Conclusion: PARP10 participates in PCNA-dependent DNA damage tolerance. Significance: This is the first time that post-translational modification by mono-ADP-ribosylation is implicated in DNA repair.