Biomedical subjects

J N Mark Glover

Publications and source records attributed to J N Mark Glover.

At least 19 recordsLinked to original sources

PubMed2025

Pathogenic variants in MAEA disrupt DNA replication fork stability and are associated with developmental abnormalities in humans.

Replication stress (RS) poses a threat to genome stability and drives genomic rearrangements. The homologous recombination (HR) pathway repairs stalled replication forks (RFs) and prevents such instability. Through an E3 ubiquitin ligase screen aimed at identifying regulators of RAD51, we identified macrophage erythroblast attacher (MAEA), a core component of C-terminal to Lish (CTLH) E3 ubiquitin ligase complex, as a regulator of the HR pathway. Loss of MAEA impairs RAD51 recruitment at stalled RFs, leading to increased sensitivity to RS-inducing agents and excessive degradation of nascent DNA strands. Mechanistically, MAEA associates with and mediates the ubiquitylation of Ku80, enabling its removal from RF ends and facilitating the loading of RAD51. Notably, MAEA deficiency is associated with a developmental disorder involving microcephaly, craniofacial abnormalities, ocular defects, and heart malformations. Functional assays show that disease-linked MAEA variants (R34C, E349G, Y394D, and M396R) are defective in RS response. These findings establish MAEA as an essential factor in RF protection and genome integrity.

Humans↗

PubMed2006

Protonation-mediated structural flexibility in the F conjugation regulatory protein, TraM.

TraM is essential for F plasmid-mediated bacterial conjugation, where it binds to the plasmid DNA near the origin of transfer, and recognizes a component of the transmembrane DNA transfer complex, TraD. Here we report the 1.40 A crystal structure of the TraM core tetramer (TraM58-127). TraM58-127 is a compact eight-helical bundle, in which the N-terminal helices from each protomer interact to form a central, parallel four-stranded coiled-coil, whereas each C-terminal helix packs in an antiparallel arrangement around the outside of the structure. Four protonated glutamic acid residues (Glu88) are packed in a hydrogen-bonded arrangement within the central four-helix bundle. Mutational and biophysical analyses indicate that this protonated state is in equilibrium with a deprotonated tetrameric form characterized by a lower helical content at physiological pH and temperature. Comparison of TraM to its Glu88 mutants predicted to stabilize the helical structure suggests that the protonated state is the active form for binding TraD in conjugation.

Amino Acid Sequence↗

PubMed2006

BRCA1 DNA-binding activity is stimulated by BARD1.

The breast- and ovarian-specific tumor suppressor BRCA1 has been implicated in numerous cellular processes, including transcription, ubiquitination, and DNA repair. Its tumor suppression activity is tightly linked to that of BARD1, a protein that heterodimerizes with BRCA1. It has been previously shown that BRCA1 binds to DNA, an interesting functional observation in light of the genetic data linking BRCA1 to DNA repair pathways. In this work, we reexamine the DNA-binding properties of BRCA1, comparing them with the DNA-binding properties of the BRCA1/BARD1 heterodimer. Because nuclear BRCA1 exists as a heterodimer with BARD1, it is likely that in vitro studies of the heterodimer will provide a more accurate model of physiologic conditions. Our results indicate that whereas BARD1 cannot directly bind DNA, it does enhance DNA binding by BRCA1. This is a surprising observation as both DNA-binding domains are distal to the BARD1-interacting RING domain of BRCA1. Further analysis of the dimerization reveals that the BRCA1/BARD1 interaction is not limited to the amino-terminal RING domains of each protein. The carboxyl terminus of BRCA1 contributes significantly to the stability of the heterodimer. We also show that the presence of BARD1 has a secondary effect, as autoubiquitination of BRCA1/BARD1 heterodimers additionally enhances the affinity of BRCA1 for DNA. Together, these data suggest that BRCA1 and BARD1 heterodimerization is stabilized via domains not previously thought to interact and that BARD1 acts in both ubiquitination-dependent and ubiquitination-independent ways to influence the role of BRCA1 in DNA repair.

Animals↗

PubMed2006

Crystal structure of a core spliceosomal protein interface.

The precise excision of introns from precursor mRNAs (pre-mRNAs) in eukaryotes is accomplished by the spliceosome, a complex assembly containing five small nuclear ribonucleoprotein (snRNP) particles. Human p14, a component of the spliceosomal U2 and U11/U12 snRNPs, has been shown to associate directly with the pre-mRNA branch adenosine early in spliceosome assembly and within the fully assembled spliceosome. Here we report the 2.5-A crystal structure of a complex containing p14 and a peptide derived from the p14-associated U2 snRNP component SF3b155. p14 contains an RNA recognition motif (RRM), the surface of which is largely occluded by a C-terminal alpha-helix and a portion of the SF3b155 peptide. An analysis of RNA.protein crosslinking to wild-type and mutant p14 shows that the branch adenosine directly interacts with a conserved aromatic within a pocket on the surface of the complex. This result, combined with a comparison of the structure with known RRMs and pseudoRRMs as well as model-building by using the electron cryomicroscopy structure of a spliceosomal U11/U12 di-snRNP, suggests that p14.SF3b155 presents a noncanonical surface for RNA recognition at the heart of the mammalian spliceosome.

Adenosine↗

PubMed2006

Insights into the molecular basis of human hereditary breast cancer from studies of the BRCA1 BRCT domain.

The C-terminal, BRCT repeats of BRCA1 are essential for the tumor suppressor function of this protein. Here we review structural and functional studies of this domain. Both repeats adopt similar folds and pack in an intimate, head-to-tail manner. The domain binds phosphorylated targets such as the DNA damage-associated kinase BACH1, with a specificity for pSer-X-X-Phe motifs. Structural studies reveal that the N-terminal repeat is responsible for pSer binding while a groove at the interface of the two repeats recognizes the Phe. Missense variants identified in breast cancer screening programs often disrupt these interactions and these molecular defects may lead to an increased cancer risk.

BRCA1 Protein↗

PubMed2006

Principles of protein-DNA recognition revealed in the structural analysis of Ndt80-MSE DNA complexes.

The Saccharomyces cerevisiae transcription factor Ndt80 selectively binds a DNA consensus sequence (the middle sporulation element [MSE]) to activate gene expression after the successful completion of meiotic recombination. Here we report the X-ray crystal structures of Ndt80 bound to ten distinct MSE variants. Comparison of these structures with the structure of Ndt80 bound to a consensus MSE reveals structural principles that determine the DNA binding specificity of this transcription factor. The 5' GC-rich end of the MSE contains distinct 5'-YpG-3' steps that are recognized by arginine side chains through a combination of hydrogen bonding and cation-pi interactions. The 3' AT-rich region is recognized via minor groove contacts that sterically exclude the N2 atom of GC base pairs. The conformation of the AT-rich region is fixed by interactions with the protein that favor recognition of poly(A)-poly(T) versus mixed AT sequences through an avoidance of major groove steric clashes at 5'-ApT-3' steps.

Base Composition↗

PubMed2005

Structure of the BRCT repeat domain of MDC1 and its specificity for the free COOH-terminal end of the gamma-H2AX histone tail.

MDC1 (mediator of DNA damage checkpoint protein 1) regulates the recognition and repair of DNA double strand breaks in mammalian cells through its interactions with nuclear foci containing the COOH-terminally phosphorylated form of the histone variant, H2AX. Here we demonstrate that the tandem BRCT repeats of MDC1 directly bind to the phosphorylated tail of H2AX-Ser(P)-Gln-Glu-Tyr, in a manner that is critically dependent on the free carboxylate group of the COOH-terminal Tyr residue. We have determined the x-ray crystal structure of the MDC1 BRCT repeats at 1.45 Angstroms resolution. By a comparison with the structure of the BRCA1 BRCT bound to a phosphopeptide, we suggest that two arginine residues in MDC1, Arg(1932) and Arg(1933) may recognize the COOH terminus of the peptide as well as the penultimate Glu of H2AX, while Gln(2013) may provide additional specificity for the COOH-terminal Tyr.

Adaptor Proteins, Signal Transducing↗

PubMed2005

The molecular architecture of the mammalian DNA repair enzyme, polynucleotide kinase.

Mammalian polynucleotide kinase (PNK) is a key component of both the base excision repair (BER) and nonhomologous end-joining (NHEJ) DNA repair pathways. PNK acts as a 5'-kinase/3'-phosphatase to create 5'-phosphate/3'-hydroxyl termini, which are a necessary prerequisite for ligation during repair. PNK is recruited to repair complexes through interactions between its N-terminal FHA domain and phosphorylated components of either pathway. Here, we describe the crystal structure of intact mammalian PNK and a structure of the PNK FHA bound to a cognate phosphopeptide. The kinase domain has a broad substrate binding pocket, which preferentially recognizes double-stranded substrates with recessed 5' termini. In contrast, the phosphatase domain efficiently dephosphorylates single-stranded 3'-phospho termini as well as double-stranded substrates. The FHA domain is linked to the kinase/phosphatase catalytic domain by a flexible tether, and it exhibits a mode of target selection based on electrostatic complementarity between the binding surface and the phosphothreonine peptide.

Amino Acid Motifs↗

PubMed2005

Structural basis for phosphorylation-dependent signaling in the DNA-damage response.

The response of eukaryotic cells to DNA damage requires a multitude of protein-protein interactions that mediate the ordered repair of the damage and the arrest of the cell cycle until repair is complete. Two conserved protein modules, BRCT and forkhead-associated (FHA) domains, play key roles in the DNA-damage response as recognition elements for nuclear Ser/Thr phosphorylation induced by DNA-damage-responsive kinases. BRCT domains, first identified at the C-terminus of BRCA1, often occur as multiple tandem repeats of individual BRCT modules. Our recent structural and functional work has revealed how BRCT repeats recognize phosphoserine protein targets. It has also revealed a secondary binding pocket at the interface between tandem repeats, which recognizes the amino-acid 3 residues C-terminal to the phosphoserine. We have also studied the molecular function of the FHA domain of the DNA repair enzyme, polynucleotide kinase (PNK). This domain interacts with threonine-phosphorylated XRCC1 and XRCC4, proteins responsible for the recruitment of PNK to sites of DNA-strand-break repair. Our studies have revealed a flexible mode of recognition that allows PNK to interact with numerous negatively charged substrates.

Amino Acid Sequence↗

PubMed2004

Structural basis of phosphopeptide recognition by the BRCT domain of BRCA1.

The BRCT repeats in BRCA1 are essential for its tumor suppressor activity and interact with phosphorylated protein targets containing the sequence pSer-X-X-Phe, where X indicates any residue. The structure of the tandem BRCA1 BRCT repeats bound to an optimized phosphopeptide reveals that the N-terminal repeat harbors a conserved BRCT phosphoserine-binding pocket, while the interface between the repeats forms a hydrophobic groove that recognizes the phenylalanine. Crystallographic and biochemical data suggest that the structural integrity of both binding sites is essential for peptide recognition. The diminished peptide-binding capacity observed for cancer-associated BRCA1-BRCT variants may explain the enhanced cancer risks associated with these mutations.

Amino Acid Sequence↗

PubMed2004

Dimerization of CtIP, a BRCA1- and CtBP-interacting protein, is mediated by an N-terminal coiled-coil motif.

CtIP is a transcriptional co-regulator that binds a number of proteins involved in cell cycle control and cell development, such as CtBP (C terminus-binding protein), BRCA1 (breast cancer-associated protein-1), and LMO4 (LIM-only protein-4). The only recognizable structural motifs within CtIP are two putative coiled-coil domains located near the N and C termini of the protein. We now show that the N-terminal coiled coil (residues 45-160), but not the C-terminal coiled coil, mediates homodimerization of CtIP in mammalian 293T cells. The N-terminal coiled coil did not facilitate binding to LMO4 and BRCA1 proteins in these cells. A protease-resistant domain (residues 27-168) that minimally encompasses the putative N-terminal coiled coil was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. This region is predicted to contain two smaller coiled-coil regions. The CtIP-(45-160) dimerization domain is helical and dimeric, indicating that the domain does form a coiled coil. The two smaller domains, CtIP-(45-92) and CtIP-(93-160), also formed dimers of lower binding affinity, but with less helical content than the longer peptide. The hydrodynamic radius of CtIP-(45-160) is the same as those of CtIP-(45-92) and CtIP-(93-160), implying that CtIP-(45-160) does not form a single long coiled coil, but a more compact structure involving homodimerization of the two smaller coiled coils, which fold back as a four-helix bundle or other compact structure. These results suggest a specific model for CtIP homodimerization via its N terminus and contribute to an improved understanding of how this protein might assemble other factors required for its role as a transcriptional corepressor.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Recognition of 5'-YpG-3' sequences by coupled stacking/hydrogen bonding interactions with amino acid residues.

The combined biochemical and structural study of hundreds of protein-DNA complexes has indicated that sequence-specific interactions are mediated by two mechanisms termed direct and indirect readout. Direct readout involves direct interactions between the protein and base-specific atoms exposed in the major and minor grooves of DNA. For indirect readout, the protein recognizes DNA by sensing conformational variations in the structure dependent on nucleotide sequence, typically through interactions with the phosphodiester backbone. Based on our recent structure of Ndt80 bound to DNA in conjunction with a search of the existing PDB database, we propose a new method of sequence-specific recognition that utilizes both direct and indirect readout. In this mode, a single amino acid side-chain recognizes two consecutive base-pairs. The 3'-base is recognized by canonical direct readout, while the 5'-base is recognized through a variation of indirect readout, whereby the conformational flexibility of the particular dinucleotide step, namely a 5'-pyrimidine-purine-3' step, facilitates its recognition by the amino acid via cation-pi interactions. In most cases, this mode of DNA recognition helps explain the sequence specificity of the protein for its target DNA.

Arginine↗

PubMed2004

Interactions between BRCT repeats and phosphoproteins: tangled up in two.

The C-terminal region of the breast-cancer-associated protein BRCA1 contains a pair of tandem BRCA1 C-terminal (BRCT) repeats that are essential for the tumour suppressor function of the protein. Similar repeat sequences have been identified in many proteins that seem to mediate cellular mechanisms for dealing with DNA damage. The BRCT domain in BRCA1 has been recently shown to constitute a module for recognizing phosphorylated (phospho-) peptides, with a recognition groove that spans both BRCT repeats. The fact that many other BRCT-containing proteins have phospho-peptide binding activity suggests that BRCT repeats might mediate phosphorylation-dependent protein-protein interactions in processes that are central to cell-cycle checkpoint and DNA repair functions.

Adaptor Proteins, Signal Transducing↗

PubMed2003

FinO is an RNA chaperone that facilitates sense-antisense RNA interactions.

The protein FinO represses F-plasmid conjugative transfer by facilitating interactions between the mRNA of the major F-plasmid transcriptional activator, TraJ, and an antisense RNA, FinP. FinO is known to bind stem-loop structures in both FinP and traJ RNAs; however, the mechanism by which FinO facilitates sense-antisense pairing is poorly understood. Here we show that FinO acts as an RNA chaperone to promote strand exchange and duplexing between minimal RNA targets derived from FinP. This strongly suggests that FinO may function to destabilize internal secondary structures within FinP and traJ RNAs that would otherwise act as a kinetic trap to sense-antisense pairing. The energy for FinO-catalyzed base-pair destabilization does not arise from ATP hydrolysis but appears to be supplied directly from FinO RNA binding free energy. An analysis of the activities of mutants that are specifically deficient in strand exchange but not RNA-binding activity demonstrates that strand exchange is essential to the ability of FinO to mediate sense-antisense RNA recognition, and that this function also plays a role in repression of conjugation in vivo.

Base Sequence↗

PubMed2003

Detection of protein folding defects caused by BRCA1-BRCT truncation and missense mutations.

Most cancer-associated BRCA1 mutations identified to date result in the premature translational termination of the protein, highlighting a crucial role for the C-terminal, BRCT repeat region in mediating BRCA1 tumor suppressor function. However, the molecular and genetic effects of missense mutations that map to the BRCT region remain largely unknown. Using a protease-based assay, we directly assessed the sensitivity of the folding of the BRCT domain to an extensive set of truncation and single amino acid substitutions derived from breast cancer screening programs. The protein can tolerate truncations of up to 8 amino acids, but further deletion results in drastic BRCT folding defects. This molecular phenotype can be correlated with an increased susceptibility to disease. A cross-validated computational assessment of the BRCT mutation data base suggests that as much as half of all BRCT missense mutations contribute to BRCA1 loss of function and disease through protein-destabilizing effects. The coupled use of proteolytic methods and computational predictive methods to detect mutant BRCA1 conformations at the protein level will augment the efficacy of current BRCA1 screening protocols, especially in the absence of clinical data that can be used to discriminate deleterious BRCT missense mutations from benign polymorphisms.

Amino Acid Sequence↗

PubMed2003

Characterizing the structural features of RNA/RNA interactions of the F-plasmid FinOP fertility inhibition system.

F-like plasmid transfer is mediated by the FinOP fertility inhibition system. Expression of the F positive regulatory protein, TraJ, is controlled by the action of the antisense RNA, FinP, and the RNA-binding protein FinO. FinO binds to and protects FinP from degradation and promotes duplex formation between FinP and traJ mRNA, leading to repression of both traJ expression and conjugative F transfer. FinP antisense RNA secondary structure is composed of two stem-loops separated by a 4-base single-stranded spacer and flanked on each side by single-stranded tails. Here we show that disruption of the expected Watson-Crick base pairing between the loops of FinP stem-loop I and its cognate RNA binding partner, traJ mRNA stem-loop Ic, led to a moderate reduction in the rate of duplex formation in vitro. In vivo, alterations of the anti-ribosome binding site region in the loop of FinP stem-loop I reduced the ability of the mutant FinP to mediate fertility inhibition and to inhibit TraJ expression when expressed in trans at an elevated copy number. Alterations of intermolecular complementarity between the stems of these RNAs reduced the rate of duplex formation. Our results suggest that successful interaction between stem-loop I of FinP and stem-loop Ic of traJ mRNA requires that base pairing must proceed from an initial loop-loop interaction through the top portion of the stems for stable duplex formation to occur.

Base Sequence↗

PubMed2002

Structural consequences of a cancer-causing BRCA1-BRCT missense mutation.

The integrity of the carboxyl-terminal BRCT repeat region is critical for BRCA1 tumor suppressor function; however, the molecular details of how a number of clinically derived BRCT missense mutations affect BRCA1 function remain largely unknown. Here we assess the structural response of the BRCT tandem repeat domain to a well characterized, cancer-associated single amino acid substitution, Met-1775 --> Arg-1775. The structure of BRCT-M1775R reveals that the mutated side chain is extruded from the protein hydrophobic core, thereby altering the protein surface. Charge-charge repulsion, rearrangement of the hydrophobic core, and disruption of the native hydrogen bonding network at the interface between the two BRCT repeats contribute to the conformational instability of BRCT-M1775R. Destabilization and global unfolding of the mutated BRCT domain at physiological temperatures explain the pleiotropic molecular and genetic defects associated with the BRCA1-M1775R protein.

Amino Acid Sequence↗

PubMed2002

Structure of the sporulation-specific transcription factor Ndt80 bound to DNA.

Progression through the middle phase of sporulation in Saccharomyces cerevisiae is promoted by the successful completion of recombination at the end of prophase I. Completion of meiotic recombination allows the activation of the sporulation-specific transcription factor Ndt80, which binds to a specific DNA sequence, the middle sporulation element (MSE), and activates approximately 150 genes to enable progression through meiosis. Here, we isolate the DNA-binding domain of Ndt80 and determine its crystal structure both free and in complex with an MSE-containing DNA. The structure reveals that Ndt80 is a member of the Ig-fold family of transcription factors. The structure of the DNA-bound form, refined at 1.4 A, reveals an unexpected mode of recognition of 5'-pyrimidine- guanine-3' dinucleotide steps by arginine residues that simultaneously recognize the 3'-guanine base through hydrogen bond interactions and the 5'-pyrimidine through stacking/van der Waals interactions. Analysis of the DNA-binding affinities of MSE mutants demonstrates the central importance of these interactions, and of the AT-rich portion of the MSE. Functional similarities between Ndt80 and the Caenorhabditis elegans p53 homolog suggest an evolutionary link between Ndt80 and the p53 family.

Amino Acid Sequence↗