Ubiquitin Phosphorylation at Thr12 Modulates the DNA Damage Response.

The ubiquitin system regulates the DNA damage response (DDR) by modifying histone H2A at Lys15 (H2AK15ub) and triggering downstream signaling events. Here, we find that phosphorylation of ubiquitin at Thr12 (pUbT12) controls the DDR by inhibiting the function of 53BP1, a key factor for DNA double-strand break repair by non-homologous end joining (NHEJ). Detectable as a chromatin modification on H2AK15ub, pUbT12 accumulates in nuclear foci and is increased upon DNA damage. Mutating Thr12 prevents the removal of ubiquitin from H2AK15ub by USP51 deubiquitinating enzyme, leading to a pronounced accumulation of ubiquitinated chromatin. Chromatin modified by pUbT12 is inaccessible to 53BP1 but permissive to the homologous recombination (HR) proteins RNF169, RAD51, and the BRCA1/BARD1 complex. Phosphorylation of ubiquitin at Thr12 in the chromatin context is a new histone mark, H2AK15pUbT12, that regulates the DDR by hampering the activity of 53BP1 at damaged chromosomes.

Bcd1p controls RNA loading of the core protein Nop58 during C/D box snoRNP biogenesis.

Biogenesis of eukaryotic box C/D small nucleolar ribonucleoproteins (C/D snoRNPs) is guided by conserved trans-acting factors that act collectively to assemble the core proteins SNU13/Snu13, NOP58/Nop58, NOP56/Nop56, FBL/Nop1, and box C/D small nucleolar RNAs (C/D snoRNAs), in human and in yeast, respectively. This finely elaborated process involves the sequential interplay of snoRNP-related proteins and RNA through the formation of transient pre-RNP complexes. BCD1/Bcd1 protein is essential for yeast cell growth and for the specific accumulation of box C/D snoRNAs. In this work, chromatin, RNA, and protein immunoprecipitation assays revealed the ordered loading of several snoRNP-related proteins on immature and mature snoRNA species. Our results identify Bcd1p as an assembly factor of C/D snoRNP biogenesis that is likely recruited cotranscriptionally and that directs the loading of the core protein Nop58 on RNA.

Biophysical characterization of human-cell-expressed, full-length κI O18/O8, AL-09, λ6a, and Wil immunoglobulin light chains.

Immunoglobulin light chain (AL) amyloidosis involves the deposition of insoluble monoclonal AL protein fibrils in the extracellular space of different organs leading to dysfunction and death. Development of methods to efficiently express and purify AL proteins with acceptable standards of homogeneity and structural integrity has become critical to understand the in vitro and in vivo aspects of AL protein aggregation, and thus the disease progression. In this study, we report the biophysical characterization of His-tagged and untagged versions of AL full-length (FL) κI and λ6 subgroup proteins and their mutants expressed from the Expi293F human cell line. We used an array of biophysical and biochemical methods to analyze the structure and stability of the monomers, oligomerization states, and thermodynamic characteristics of the purified FL proteins and how they compare with the bacterially expressed FL proteins. Our results demonstrate that the tagged and untagged versions of FL proteins have comparable stability to proteins expressed in bacterial cells but exhibit multiple unfolding transitions and reversibility. Non-reducing SDS-PAGE and analytical ultracentrifugation analysis showed presence of monomers and dimers, with an insignificant amount of higher-order oligomers, in the purified fraction of all proteins. Overall, the FL proteins were expressed with sufficient yields for biophysical studies and can replace bacterial expression systems.

Dimerization-dependent serine protease activity of FAM111A prevents replication fork stalling at topoisomerase 1 cleavage complexes.

FAM111A, a serine protease, plays roles in DNA replication and antiviral defense. Missense mutations in the catalytic domain cause hyper-autocleavage and are associated with genetic disorders with developmental defects. Despite the enzyme's biological significance, the molecular architecture of the FAM111A serine protease domain (SPD) is unknown. Here, we show that FAM111A is a dimerization-dependent protease containing a narrow, recessed active site that cleaves substrates with a chymotrypsin-like specificity. X-ray crystal structures and mutagenesis studies reveal that FAM111A dimerizes via the N-terminal helix within the SPD. This dimerization induces an activation cascade from the dimerization sensor loop to the oxyanion hole through disorder-to-order transitions. Dimerization is essential for proteolytic activity in vitro and for facilitating DNA replication at DNA-protein crosslink obstacles in cells, while it is dispensable for autocleavage. These findings underscore the role of dimerization in FAM111A's function and highlight the distinction in its dimerization dependency between substrate cleavage and autocleavage.

An autoinhibited state of 53BP1 revealed by small molecule antagonists and protein engineering.

The recruitment of 53BP1 to chromatin, mediated by its recognition of histone H4 dimethylated at lysine 20 (H4K20me2), is important for DNA double-strand break repair. Using a series of small molecule antagonists, we demonstrate a conformational equilibrium between an open and a pre-existing lowly populated closed state of 53BP1 in which the H4K20me2 binding surface is buried at the interface between two interacting 53BP1 molecules. In cells, these antagonists inhibit the chromatin recruitment of wild type 53BP1, but do not affect 53BP1 variants unable to access the closed conformation despite preservation of the H4K20me2 binding site. Thus, this inhibition operates by shifting the conformational equilibrium toward the closed state. Our work therefore identifies an auto-associated form of 53BP1-autoinhibited for chromatin binding-that can be stabilized by small molecule ligands encapsulated between two 53BP1 protomers. Such ligands are valuable research tools to study the function of 53BP1 and have the potential to facilitate the development of new drugs for cancer therapy.

An autoinhibited state of 53BP1 revealed by small molecule antagonists and protein engineering.

The recruitment of 53BP1 to chromatin, mediated by its recognition of histone H4 dimethylated at lysine 20 (H4K20me2), is important for DNA double-strand break repair. Using a series of small molecule antagonists, we demonstrate a conformational equilibrium between an open and a pre-existing lowly populated closed state of 53BP1 in which the H4K20me2 binding surface is buried at the interface between two interacting 53BP1 molecules. In cells, these antagonists inhibit the chromatin recruitment of wild type 53BP1, but do not affect 53BP1 variants unable to access the closed conformation despite preservation of the H4K20me2 binding site. Thus, this inhibition operates by shifting the conformational equilibrium toward the closed state. Our work therefore identifies an auto-associated form of 53BP1 - autoinhibited for chromatin binding - that can be stabilized by small molecule ligands encapsulated between two 53BP1 protomers. Such ligands are valuable research tools to study the function of 53BP1 and have the potential to facilitate the development of new drugs for cancer therapy.

The box C/D snoRNP assembly factor Bcd1 interacts with the histone chaperone Rtt106 and controls its transcription dependent activity.

Biogenesis of eukaryotic box C/D small nucleolar ribonucleoproteins initiates co-transcriptionally and requires the action of the assembly machinery including the Hsp90/R2TP complex, the Rsa1p:Hit1p heterodimer and the Bcd1 protein. We present genetic interactions between the Rsa1p-encoding gene and genes involved in chromatin organization including RTT106 that codes for the H3-H4 histone chaperone Rtt106p controlling H3K56ac deposition. We show that Bcd1p binds Rtt106p and controls its transcription-dependent recruitment by reducing its association with RNA polymerase II, modulating H3K56ac levels at gene body. We reveal the 3D structures of the free and Rtt106p-bound forms of Bcd1p using nuclear magnetic resonance and X-ray crystallography. The interaction is also studied by a combination of biophysical and proteomic techniques. Bcd1p interacts with a region that is distinct from the interaction interface between the histone chaperone and histone H3. Our results are evidence for a protein interaction interface for Rtt106p that controls its transcription-associated activity.

NMR assignment and solution structure of the external DII domain of the yeast Rvb2 protein.

We report the nearly complete 1H, 15N and 13C resonance assignment and the solution structure of the external DII domain of the yeast Rvb2 protein, a member of the AAA+ATPase superfamily.

Mechanism of 53BP1 activity regulation by RNA-binding TIRR and a designer protein.

Dynamic protein interaction networks such as DNA double-strand break (DSB) signaling are modulated by post-translational modifications. The DNA repair factor 53BP1 is a rare example of a protein whose post-translational modification-binding function can be switched on and off. 53BP1 is recruited to DSBs by recognizing histone lysine methylation within chromatin, an activity directly inhibited by the 53BP1-binding protein TIRR. X-ray crystal structures of TIRR and a designer protein bound to 53BP1 now reveal a unique regulatory mechanism in which an intricate binding area centered on an essential TIRR arginine residue blocks the methylated-chromatin-binding surface of 53BP1. A 53BP1 separation-of-function mutation that abolishes TIRR-mediated regulation in cells renders 53BP1 hyperactive in response to DSBs, highlighting the key inhibitory function of TIRR. This 53BP1 inhibition is relieved by TIRR-interacting RNA molecules, providing proof-of-principle of RNA-triggered 53BP1 recruitment to DSBs.

The RPAP3-Cterminal domain identifies R2TP-like quaternary chaperones.

R2TP is an HSP90 co-chaperone that assembles important macro-molecular machineries. It is composed of an RPAP3-PIH1D1 heterodimer, which binds the two essential AAA+ATPases RUVBL1/RUVBL2. Here, we resolve the structure of the conserved C-terminal domain of RPAP3, and we show that it directly binds RUVBL1/RUVBL2 hexamers. The human genome encodes two other proteins bearing RPAP3-C-terminal-like domains and three containing PIH-like domains. Systematic interaction analyses show that one RPAP3-like protein, SPAG1, binds PIH1D2 and RUVBL1/2 to form an R2TP-like complex termed R2SP. This co-chaperone is enriched in testis and among 68 of the potential clients identified, some are expressed in testis and others are ubiquitous. One substrate is liprin-α2, which organizes large signaling complexes. Remarkably, R2SP is required for liprin-α2 expression and for the assembly of liprin-α2 complexes, indicating that R2SP functions in quaternary protein folding. Effects are stronger at 32 °C, suggesting that R2SP could help compensating the lower temperate of testis.

Functional and Structural Insights of the Zinc-Finger HIT protein family members Involved in Box C/D snoRNP Biogenesis.

Zf­HIT family members share the zf­HIT domain (ZHD), which is characterized by a fold in "treble-clef" through interleaved CCCC and CCHC ZnF motifs that both bind a zinc atom. Six proteins containing ZHD are present in human and three in yeast proteome, all belonging to multimodular RNA/protein complexes involved in gene regulation, chromatin remodeling, and snoRNP assembly. An interesting characteristic of the cellular complexes that ensure these functions is the presence of the RuvBL1/2/Rvb1/2 ATPases closely linked with zf­HIT proteins. Human ZNHIT6/BCD1 and its counterpart in yeast Bcd1p were previously characterized as assembly factors of the box C/D snoRNPs. Our data reveal that the ZHD of Bcd1p is necessary but not sufficient for yeast growth and that the motif has no direct RNA-binding capacity but helps Bcd1p maintain the box C/D snoRNAs level in steady state. However, we demonstrated that Bcd1p interacts nonspecifically with RNAs depending on their length. Interestingly, the ZHD of Bcd1p is functionally interchangeable with that of Hit1p, another box C/D snoRNP assembly factor belonging to the zf­HIT family. This prompted us to use NMR to solve the 3D structures of ZHD from yeast Bcd1p and Hit1p to highlight the structural similarity in the zf­HIT family. We identified structural features associated with the requirement of Hit1p and Bcd1p ZHD for cell growth and box C/D snoRNA stability under heat stress. Altogether, our data suggest an important role of ZHD could be to maintain functional folding to the rest of the protein, especially under heat stress conditions.