Biology subjects

Sixma, T. K.

Publications and source records attributed to Sixma, T. K..

8 recordsLinked to original sources

bioRxiv2026

A ubiquitin chain-feeding mechanism for BRCA1-A

The BRCA1-A complex is a multi-subunit, metallo-deubiquitinating enzyme (metallo-DUB) involved in genome maintenance. BRCA1-A displays strict specificity for K63-linked ubiquitin, with a strong preference for long chains, but the mechanistic basis for this selectivity has remained unclear. To address this, we developed a novel activity-based probe that is specific for metallo-DUBs and mimics di- or polyubiquitin chains of any linkage (di- and poly-ubiquitinATA). We solved cryoEM structures of BRCA1-A bound to K63-linked probe chains of various length, capturing multiple conformational and catalytic states. The structures reveal how allosteric regulation of catalysis occurs within the complex and how BRCA1-A uses auxiliary ubiquitin-binding sites to engage substrate by avidity and to trigger processive cleavage. Crucially, avidity and processivity can only apply to long polyubiquitin chains, explaining BRCA1-As substrate preference. Together, these results establish BRCA1-A as a chain-shortening DUB specialised for trimming extended K63-linked polyubiquitin chains.

biochemistry↗

bioRxiv2024

USP1/UAF1 targets polyubiquitinated PCNA with an exo-cleavage mechanism that enriches for monoubiquitinated PCNA

DNA damage tolerance (DDT) is an important pathway that allows our cells to bypass DNA lesions during replication. DDT is orchestrated by ubiquitination of PCNA: Monoubiquitination (PCNA-Ub) initiates recruitment of TLS polymerases but also serves as substrate for K63-linked polyubiquitination that leads to HR-mediated bypass mechanisms. Recent work on USP1/UAF1 inhibition revealed that K48-linked chains are also formed on PCNA, resulting in its proteasomal degradation. USP1/UAF1 is established as deubiquitinating enzyme (DUB) for PCNA-Ub, but little is known about deubiquitination of chains on PCNA. Here we show that USP1/UAF1 cleaves both K48 and K63-linked ubiquitin chains on PCNA efficiently, using an exo-cleavage mechanism. Kinetic analysis reveals that USP1/UAF1 prefers cleaving the ubiquitin-ubiquitin bond over cleavage of the ubiquitin-PCNA isopeptide bond and therefore treats poly- and monoubiquitinated PCNA as different substrates. A cryo-EM structure of USP1/UAF1 with a K63-diubiquitin and structure-based mutagenesis reveals that its mechanistic preference is maintained in evolution. Its kinetic mechanism results in relative enrichment of monoubiquitinated PCNA that could initially promote TLS over HR-like bypass. Taken together, these results suggest that USP1/UAF1 could be important in temporary protection of PCNA against K48- and K63-linked polyubiquitination and highlight this DUB as a potential regulator of DDT pathway choice.

biochemistry↗

bioRxiv2024

STK19 drives Transcription-Coupled Repair by stimulating repair complex stability, Pol II ubiquitylation and TFIIH recruitment

DNA damage forms a major obstacle for gene transcription by RNA polymerase II (Pol II). Transcription-coupled nucleotide excision repair (TC-NER) efficiently eliminates transcription-blocking lesions (TBLs), thereby safeguarding accurate transcription, preserving correct cellular function and counteracting aging. TC-NER initiation involves the recognition of lesion-stalled Pol II by CSB, which recruits the CRL4CSA E3 ubiquitin ligase complex and UVSSA. TBL-induced ubiquitylation of Pol II at lysine 1268 of the RPB1 subunit by CRL4CSA serves as a critical TC-NER checkpoint, governing Pol II stability and initiating TBL excision by TFIIH recruitment. However, the precise regulatory mechanisms of the CRL4CSA E3 ligase activity and TFIIH recruitment remain elusive. Here, we reveal Inactive Serine/Threonine Kinase 19 (STK19) as a novel TC-NER factor, that is essential for correct TBL removal repair and subsequent transcription restart. Cryo-EM studies demonstrate that STK19 is an integral part of the Pol II-TC-NER complex, bridging CSA with UVSSA, RPB1 and downstream DNA. Live-cell imaging and interaction studies show that STK19 stimulates TC-NER complex stability and CRL4CSA activity, resulting in efficient Pol II ubiquitylation and correct UVSSA and TFIIH binding. These findings underscore the crucial role of STK19 as a core component of the TC-NER machinery and its key involvement in the cellular responses to DNA damage that interfere with transcription.

molecular biology↗

bioRxiv2023

Surprising variety in the USP deubiquitinase catalytic mechanism

The USP family of deubiquitinases (DUBs) controls many ubiquitin-dependent signaling events. This generates therapeutic potential, with active-site inhibitors in preclinical and clinical studies. Understanding of the USP active site was so far primarily guided by USP7 data, where the catalytic triad consists of cysteine, histidine and a third residue (first critical residue), which polarizes the histidine through a hydrogen bond. A conserved aspartate (second critical residue) is directly adjacent to this first critical residue. Here we study the roles of these critical residues in a subset of USPs and reveal a remarkable variety in function. While USP7 relies on the first critical residue for catalysis, this residue is dispensable in USP1, USP15, USP40 and USP48. Instead, their second critical residue is vital for catalysis. Interestingly, without their respective vital residue USP7, USP15 and USP40 can still perform nucleophilic attack. The diverging catalytic mechanisms of USP1 and USP7 are independent of substrate and retained in cells for USP1. The unexpected variety of catalytic mechanisms in this well-conserved protein family may generate opportunities for selective targeting of individual USPs. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/550302v2_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1f3fdbeorg.highwire.dtl.DTLVardef@197f836org.highwire.dtl.DTLVardef@28b588org.highwire.dtl.DTLVardef@a62a2b_HPS_FORMAT_FIGEXP M_FIG C_FIG SynopsisO_LIThe roles of the highly conserved critical residues in USP active sites are poorly understood. Here we show that these two residues have varying importance for catalysis between different USPs. C_LIO_LIExcept for USP7, the majority of USPs does not rely on the canonical third catalytic residue (first critical residue). Instead, the USPs tested rely primarily on the highly conserved second critical residue. C_LIO_LIIn some USPs, either critical residues can accommodate nucleophilic attack (USP7, USP40, USP15). USP1 and USP48 are unable to perform the nucleophilic attack without the second critical residue. C_LI

biochemistry↗

bioRxiv2021

Chaperoning of the histone octamer by the acidic domain of DNA repair factor APLF

Nucleosome assembly requires the coordinated deposition of histone complexes H3-H4 and H2A-H2B to form a histone octamer on DNA. In the current paradigm, specific histone chaperones guide the deposition of first H3-H4 and then H2A-H2B(1-5). Here, we show that the acidic domain of DNA repair factor APLF (APLFAD) can assemble the histone octamer in a single step, and deposit it on DNA to form nucleosomes. The crystal structure of the APLFAD-histone octamer complex shows that APLFAD tethers the histones in their nucleosomal conformation. Mutations of key aromatic anchor residues in APLFAD affect chaperone activity in vitro and in cells. Together, we propose that chaperoning of the histone octamer is a mechanism for histone chaperone function at sites where chromatin is temporarily disrupted. one sentence summaryHistone chaperone APLF assembles histones H2A-H2B/H3-H4 into histone octamers to deposit them onto DNA and form nucleosomes.

biochemistry↗

bioRxiv2021

S18-phosphorylation of USP7 regulates interaction with TCEAL4 that defines specific complexes and potentially distinct functions.

USP7 is a nuclear deubiquitylase (DUB) with multiple cancer-associated substrates for which selective inhibitors are available, yet it remains unclear how the pleiotropic effects of USP7 are regulated. We report that S18-phosphorylation does not influence USP7 catalytic activity but instead confers selectivity for protein interactions. In particular, non-S18-phosphorylatable USP7 preferentially interacts with USP11 and TRIM27, together with TCEAL1 and TCEAL4 whose functions are unknown. Intriguingly, USP7 can interact with two cellular forms of TCEAL4, but USP11 only interacts with a lower abundance K142 mono-ubiquitylated form (TCEAL4-Ub), which can scaffold a complex containing both DUBs. Whilst USP11 and TCEAL4 are both USP7 substrates, TCEAL4-Ub levels are specifically maintained by USP11 with their levels positively correlated in cancer cell lines. Together these data illustrate how USP7 phosphorylation and TCEAL4 ubiquitylation combine to define distinct USP7 complexes. As TCEAL4 itself interacts with proteins involved in ubiquitylation and various forms of DNA regulation, these complexes may direct cellular activity of USP7.

cell biology↗

bioRxiv2021

Proteomic analysis identifies novel binding partners of BAP1

BRCA1-associated protein 1 (BAP1) is a tumor suppressor and its loss can result in mesothelioma, uveal and cutaneous melanoma, clear cell renal cell carcinoma and bladder cancer. BAP1 is a deubiquitinating enzyme of the UCH class that has been implicated in various cellular processes like cell growth, cell cycle progression, ferroptosis and ER metabolic stress response. ASXL proteins activate BAP1 by forming the polycomb repressive deubiquitinase (PR-DUB) complex which acts on H2AK119ub1. Besides the ASXL proteins, BAP1 is known to interact with an established set of additional proteins. Here, we identify novel BAP1 interacting proteins in the cytoplasm by expressing GFP-tagged BAP1 in an endogenous BAP1 deficient cell line using affinity purification followed by mass spectrometry (AP-MS) analysis. Among these novel interacting proteins are Histone acetyltransferase 1 (HAT1) and all subunits of the heptameric coat protein complex I (COPI) that is involved in vesicle formation and protein cargo binding and sorting. We validate that the HAT1 and COPI interactions occur at endogenous levels but find that this interaction with COPI is not mediated through the C-terminal KxKxx cargo sorting signals of the COPI complex.

biochemistry↗

bioRxiv2018

The unstructured linker arms of MutL enable GATC site incision beyond roadblocks during initiation of DNA mismatch repair

DNA mismatch repair (MMR) maintains genome stability through repair of DNA replication errors. In Escherichia coli, initiation of MMR involves recognition of the mismatch by MutS, recruitment of MutL, activation of endonuclease MutH and DNA strand incision at a hemimethylated GATC site. Here we studied the mechanism of communication that couples mismatch recognition to daughter strand incision. We investigated the effect of catalytically-deficient Cas9 as well as stalled RNA polymerase as roadblocks placed on DNA in between the mismatch and GATC site in ensemble and single molecule nanomanipulation incision assays. The MMR proteins were observed to incise GATC sites beyond a roadblock, albeit with reduced efficiency. This residual incision is completely abolished upon shortening the disordered linker regions of MutL. These results indicate that roadblock bypass can be fully attributed to the long, disordered linker regions in MutL and establish that communication during MMR initiation occurs along the DNA backbone.

biochemistry↗