Biology subjects

Rennie, M. L.

Publications and source records attributed to Rennie, M. L..

6 recordsLinked to original sources

bioRxiv2026

Identification of a FANCD2-interacting protein motif (DIP-box) linking DNA Damage Response processes

Repair of DNA damage is critical to genome integrity and the DNA clamp, FANCD2, is a major player in the response to interstrand crosslinks tying the two DNA strands together. It has long been suggested FANCD2 recruits other factors to coordinate repair these crosslinks, however it remains unclear if FANCD2 directly mediates this and how this is achieved. Here we show that FANCD2 has a conserved acidic region that directly recognises established DNA repair proteins containing a short linear motif. We discover new candidate interactors engaging with FANCD2 in a similar mode that are involved in histone modification, and RNA processing. Our data demonstrate FANCD2 is a hub for protein-protein interactions providing a structural explanation for FANCD2s central role in repair of DNA interstrand crosslinks. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/707417v2_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@10d0f59org.highwire.dtl.DTLVardef@6acac2org.highwire.dtl.DTLVardef@125a277org.highwire.dtl.DTLVardef@1cd4194_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Structural and biochemical insights on the mechanism of action of the clinical USP1 inhibitor, KSQ-4279

DNA damage triggers cell signalling cascades that mediate repair. This signalling is frequently dysregulated in cancers. The proteins that mediate this signalling are potential targets for therapeutic intervention. Ubiquitin-specific protease 1 (USP1) is one such target, with small molecule inhibitors already in clinical trials. Here we use biochemical assays and cryo-electron microscopy (cryo-EM) to study the clinical USP1 inhibitor, KSQ-4279 (RO7623066), and compare this to the well-established, tool compound, ML323. We find that KSQ-4279 binds to the same cryptic site of USP1 as ML323 but disrupts the protein structure in subtly different ways. Inhibitor binding drives a substantial increase in thermal stability of USP1, which may be mediated through the inhibitors filling a hydrophobic tunnel in USP1. Our results contribute to the understanding of the mechanism of action of USP1 inhibitors at the molecular level.

biochemistry↗

bioRxiv2022

Structural and biochemical basis of FANCI-FANCD2 interdependent ubiquitination

The Fanconi Anaemia pathway operates for the repair of interstrand crosslinks and the maintenance of genomic stability upon replication stalling. Di-monoubiquitination of the FANCI-FANCD2 (ID2) complex is a central and crucial step in this pathway. Evidence suggests that FANCD2 ubiquitination precedes FANCI ubiquitination, and that both the FANCD2-ubiquitinated (ID2Ub) and the di- monoubiquitinated (IUbD2Ub) complex clamp on DNA. However, FANCD2 is deubiquitinated at a faster rate than FANCI, which can result in a FANCI-ubiquitinated ID2 complex (IUbD2). Here, we present a 4.1 [A] cryo-EM structure of IUbD2 complex bound to double-stranded DNA. We show that this complex, like ID2Ub and IUbD2Ub, is also in the closed ID2 conformation and clamps on DNA. While the target lysine of FANCD2 (K561) is partially buried in the non-ubiquitinated ID2-DNA complex, it becomes fully exposed in the IUbD2-DNA structure, and thus can be ubiquitinated at a faster rate. The IUbD2-DNA complex cannot easily revert to the non-ubiquitinated ID2 state, due to USP1-UAF1-resistance, conferred by the presence of DNA and FANCD2. ID2Ub-DNA, on the other hand, can be efficiently deubiquitinated by USP1-UAF1, unless further ubiquitination on FANCI occurs. FANCI ubiquitination also progresses at a faster rate in ID2Ub-DNA over ID2-DNA complex, and results in partial DNA-dependent protection from FANCD2 deubiquitination. Taken together, our results suggest that, while FANCD2 ubiquitination promotes FANCI ubiquitination, FANCI ubiquitination in turn maintains FANCD2 ubiquitination by two mechanisms: it prevents excessive FANCD2 deubiquitination within an IUbD2Ub-DNA complex, and it enables re-ubiquitination of FANCD2 within a transient, closed-on-DNA, IUbD2 complex.

biochemistry↗

bioRxiv2022

Cryo-EM reveals a mechanism of USP1 inhibition through a cryptic binding site

Repair of DNA damage is critical to genomic integrity and frequently disrupted in cancers. USP1, a nucleus-localized deubiquitinase, lies at the interface of multiple DNA repair pathways and is a promising drug target for certain cancers. Although multiple inhibitors of this enzyme, including one in phase I clinical trials, have been established, their binding mode is unknown. Here we use cryo-Electron Microscopy to study an assembled enzyme-substrate-inhibitor complex of USP1 and the well-established inhibitor, ML323. Achieving 2.5 [A] resolution, we discover an unusual binding mode in which the inhibitor displaces part of the hydrophobic core of USP1. The consequent conformational changes in the secondary structure lead to subtle rearrangements in the active site that underlie the mechanism of inhibition. These structures provide a platform for structure-based drug design targeting USP1. One Sentence SummaryUSP1, a cancer target, is inhibited by ML323 displacing part of the protein fold, allosterically disrupting the active site.

biochemistry↗

bioRxiv2020

Structural basis of FANCD2 deubiquitination by USP1-UAF1

Ubiquitin-Specific Protease 1 (USP1), together with the cofactor UAF1, acts during DNA repair processes to specifically to remove mono-ubiquitin signals. The mono-ubiquitinated FANCI-FANCD2 heterodimer is one such substrate and is involved in the repair of DNA interstrand crosslinks via the Fanconi Anemia pathway. Here we determine structures of human USP1-UAF1 with and without ubiquitin, and bound to mono-ubiquitinated FANCI-FANCD2 substrate. Crystal structures of USP1-UAF1 reveal plasticity in USP1 and key differences to USP12-UAF1 and USP46-UAF1. A cryoEM reconstruction of USP1-UAF1 in complex mono-ubiquitinated FANCI-FANCD2, highlights a highly orchestrated deubiquitination process with USP1-UAF1 driving conformational changes in the substrate. An extensive interface between UAF1 and FANCI, confirmed by mutagenesis and biochemical assays, provides a molecular explanation for their requirement despite neither being directly involved in catalysis. Overall, our data provide molecular details of USP1-UAF1 regulation and substrate recognition.

biochemistry↗

bioRxiv2020

Differential functions of FANCI and FANCD2 ubiquitination stabilize ID2 complex on DNA

The Fanconi Anemia (FA) pathway is a dedicated pathway for the repair of DNA interstrand crosslinks, and which is additionally activated in response to other forms of replication stress. A key step in the activation of the FA pathway is the monoubiquitination of each of the two subunits (FANCI and FANCD2) of the ID2 complex on specific lysine residues. However, the molecular function of these modifications has been unknown for nearly two decades. Here we find that ubiquitination of FANCD2 acts to increase ID2s affinity for double stranded DNA via promoting/stabilizing a large-scale conformational change in the complex, resulting in a secondary "Arm" ID2 interphase encircling DNA. Ubiquitination of FANCI, on the other hand, largely protects the ubiquitin on FANCD2 from USP1/UAF deubiquitination, with key hydrophobic residues of FANCIs ubiquitin being important for this protection. In effect, both of these post-translational modifications function to stabilise a conformation in which the ID2 complex encircles DNA.

biochemistry↗