151 results found
Zhang X, Gao F, Ye F, et al., 2024, Structural basis of s54 displacement and promoter escape in bacterial transcription, Proceedings of the National Academy of Sciences of USA, ISSN: 0027-8424
Gene transcription is a fundamental cellular process carried out by RNA polymerase (RNAP). Transcription initiation is highly regulated and in bacteria, transcription initiation is mediated by sigma (σ) factors. σ recruits RNAP to the promoter DNA region, located upstream oftranscription start site (TSS), and facilitates open complex formation, where double stranded DNA is opened up into a transcription bubble and template strand DNA is positioned inside RNAP for initial RNA synthesis. During initial transcription, RNAP remains bound to sigma andupstream DNA, presumably with an enlarging transcription bubble. The release of RNAP fromupstream DNA is required for promoter escape and processive transcription elongation. Bacteria sigma factors can be broadly separated into two classes with the majority belonging to the σ70 class, represented by the σ70 that regulates housekeeping genes. σ54 forms a class on its own and regulates stress response genes. Extensive studies on σ70 have revealed the molecularmechanisms of σ70 dependent process while how σ54 transitions from initial transcription to elongation is currently unknown. Here we present a series of cryo electron microscopy structures of the RNAP-σ54 initial transcribing complexes with progressively longer RNA, which reveal structural changes that lead to promoter escape. Our data show that initially, the transcription bubble enlarges, DNA strands scrunch, reducing the interactions between σ54 and DNA strands in the transcription bubble. RNA extension and further DNA scrunching help to release RNAP from σ54 and upstream DNA, enabling the transition to elongation.
Yates LA, Zhang X, 2023, Phosphoregulation of the checkpoint kinase Mec1ATR, DNA REPAIR, Vol: 129, ISSN: 1568-7864
Belan O, Greenhough L, Kuhlen L, et al., 2023, Visualization of direct and diffusion-assisted RAD51 nucleation by full-length human BRCA2 protein, Molecular Cell, Vol: 83, Pages: 2925-2940.e8, ISSN: 1097-2765
Homologous recombination (HR) is essential for error-free repair of DNA double-strand breaks, perturbed replication forks (RFs), and post-replicative single-stranded DNA (ssDNA) gaps. To initiate HR, the recombination mediator and tumor suppressor protein BRCA2 facilitates nucleation of RAD51 on ssDNA prior to stimulation of RAD51 filament growth by RAD51 paralogs. Although ssDNA binding by BRCA2 has been implicated in RAD51 nucleation, the function of double-stranded DNA (dsDNA) binding by BRCA2 remains unclear. Here, we exploit single-molecule (SM) imaging to visualize BRCA2-mediated RAD51 nucleation in real time using purified proteins. We report that BRCA2 nucleates and stabilizes RAD51 on ssDNA either directly or through an unappreciated diffusion-assisted delivery mechanism involving binding to and sliding along dsDNA, which requires the cooperative action of multiple dsDNA-binding modules in BRCA2. Collectively, our work reveals two distinct mechanisms of BRCA2-dependent RAD51 loading onto ssDNA, which we propose are critical for its diverse functions in maintaining genome stability and cancer suppression.
Liang P, Lister K, Yates L, et al., 2023, Phosphoregulation of DNA repair via the Rad51 auxiliary factor Swi5-Sfr1, Journal of Biological Chemistry, Vol: 299, Pages: 1-15, ISSN: 0021-9258
Homologous recombination (HR) is a major pathway for the repair of DNA double-strand breaks, the most severe form of DNA damage. The Rad51 protein is central to HR, but multiple auxiliary factors regulate its activity. The heterodimeric Swi5-Sfr1 complex is one such factor. It was previously shown that two sites within the intrinsically disordered domain of Sfr1 are critical for the interaction with Rad51. Here, we show that phosphorylation of five residues within this domain regulates the interaction of Swi5-Sfr1 with Rad51. Biochemical reconstitutions demonstrated that a phosphomimetic mutant version of Swi5-Sfr1 is defective in both the physical and functional interaction with Rad51. This translated to a defect in DNA repair, with the phosphomimetic mutant yeast strain phenocopying the previously established interaction mutant. Interestingly, a strain in which Sfr1 phosphorylation was blocked also displayed sensitivity to DNA damage. Taken together, we propose that controlled phosphorylation of Sfr1 is important for the role of Swi5-Sfr1 in promoting Rad51-dependent DNA repair.
Zhang X, Yates L, Morgan M, 2023, A DNA damage-induced phosphorylation circuit enhances Mec1ATR Ddc2ATRIP recruitment to Replication Protein A, Proceedings of the National Academy of Sciences of USA, Vol: 120, Pages: 1-10, ISSN: 0027-8424
The cell cycle checkpoint kinase Mec1ATR and its integral partner Ddc2ATRIP are vital for the DNA damage and replication stress response. Mec1–Ddc2 “senses” single-stranded DNA (ssDNA) by being recruited to the ssDNA binding Replication Protein A (RPA) via Ddc2. In this study, we show that a DNA damage–induced phosphorylation circuit modulates checkpoint recruitment and function. We demonstrate that Ddc2–RPA interactions modulate the association between RPA and ssDNA and that Rfa1-phosphorylation aids in the further recruitment of Mec1–Ddc2. We also uncover an underappreciated role for Ddc2 phosphorylation that enhances its recruitment to RPA-ssDNA that is important for the DNA damage checkpoint in yeast. The crystal structure of a phosphorylated Ddc2 peptide in complex with its RPA interaction domain provides molecular details of how checkpoint recruitment is enhanced, which involves Zn2+. Using electron microscopy and structural modeling approaches, we propose that Mec1–Ddc2 complexes can form higher order assemblies with RPA when Ddc2 is phosphorylated. Together, our results provide insight into Mec1 recruitment and suggest that formation of supramolecular complexes of RPA and Mec1–Ddc2, modulated by phosphorylation, would allow for rapid clustering of damage foci to promote checkpoint signaling.
Ye Y, Zhang X, Jin J, et al., 2023, A Multiport Current Flow Controller for Meshed Multiterminal DC Grids, IEEE TRANSACTIONS ON POWER ELECTRONICS, Vol: 38, Pages: 5479-5489, ISSN: 0885-8993
Zhang X, Ye F, Gao F, et al., 2022, Mechanisms of DNA opening revealed in AAA+ transcription complex structures, Science Advances, Vol: 8, Pages: 1-12, ISSN: 2375-2548
Gene transcription is carried out by RNA polymerase (RNAP) and requires the conversion of the initial closed promoter complex, where DNA is double stranded, to a transcription-competent open promoter complex, where DNA is opened up. In bacteria, RNAP relies on σ factors for its promoter specificities. Using a special form of sigma factor (σ54), which forms a stable closed complex and requires its activator that belongs to the AAA+ ATPases (ATPases associated with diverse cellular activities), we obtained cryo–electron microscopy structures of transcription initiation complexes that reveal a previously unidentified process of DNA melting opening. The σ54 amino terminus threads through the locally opened up DNA and then becomes enclosed by the AAA+ hexameric ring in the activator-bound intermediate complex. Our structures suggest how ATP hydrolysis by the AAA+ activator could remove the σ54 inhibition while helping to open up DNA, using σ54 amino-terminal peptide as a pry bar.
Kotta-Loizou I, Giuliano MG, Jovanovic M, et al., 2022, The RNA repair proteins RtcAB regulate transcription activator RtcR via its CRISPR-associated Rossmann fold domain, iScience, Vol: 25, ISSN: 2589-0042
CRISPR-associated Rossmann fold (CARF) domain signaling underpins modulation of CRISPR-Cas nucleases; however, the RtcR CARF domain controls expression of two conserved RNA repair enzymes, cyclase RtcA and ligase RtcB. Here, we demonstrate that RtcAB are required for RtcR-dependent transcription activation and directly bind to RtcR CARF. RtcAB catalytic activity is not required for complex formation with CARF, but is essential yet not sufficient for RtcRAB-dependent transcription activation, implying the need for an additional RNA repair-dependent activating signal. This signal differs from oligoadenylates, a known ligand of CARF domains, and instead appears to originate from the translation apparatus: RtcB repairs a tmRNA that rescues stalled ribosomes and increases translation elongation speed. Taken together, our data provide evidence for an expanded range for CARF domain signaling, including the first evidence of its control via in trans protein-protein interactions, and a feed-forward mechanism to regulate RNA repair required for a functioning translation apparatus.
Hao M, Ye F, Jovanovic M, et al., 2022, Structures of Class I and Class II transcription complexes reveal the molecular basis of RamA-dependent transcription activation, Advanced Science, Vol: 9, Pages: 1-10, ISSN: 2198-3844
Transcription activator RamA is linked to multidrug resistance of Klebsiella pneumoniae through controlling genes that encode efflux pumps (acrA) and porin-regulating antisense RNA (micF). In bacteria, σ70, together with activators, controls the majority of genes by recruiting RNA polymerase (RNAP) to the promoter regions. RNAP and σ70 form a holoenzyme that recognizes -35 and -10 promoter DNA consensus sites. Many activators bind upstream from the holoenzyme and can be broadly divided into two classes. RamA acts as a class I activator on acrA and class II activator on micF, respectively. The authors present biochemical and structural data on RamA in complex with RNAP-σ70 at the two promoters and the data reveal the molecular basis for how RamA assembles and interacts with core RNAP and activates transcription that contributes to antibiotic resistance. Further, comparing with CAP/TAP complexes reveals common and activator-specific features in activator binding and uncovers distinct roles of the two C-terminal domains of RNAP α subunit.
Zhang X, Blundell T, Tainer JA, 2021, The renaissance in biophysics and molecular biology enabled by the interface of DNA repair and replication with cancer, PROGRESS IN BIOPHYSICS & MOLECULAR BIOLOGY, Vol: 163, Pages: 1-4, ISSN: 0079-6107
Williams RM, Zhang X, 2021, Roles of ATM and ATR in DNA double strand breaks and replication stress, PROGRESS IN BIOPHYSICS & MOLECULAR BIOLOGY, Vol: 163, Pages: 109-119, ISSN: 0079-6107
Kaneko Y, Shimoda K, Ayala R, et al., 2021, p97 and p47 function in membrane tethering in cooperation with FTCD during mitotic Golgi reassembly, EMBO JOURNAL, Vol: 40, ISSN: 0261-4189
Zhang X, Carver A, 2021, Rad51 filament dynamics and its antagonistic modulators, Seminars in Cell and Developmental Biology, Vol: 113, Pages: 3-13, ISSN: 1084-9521
Rad51 recombinase is the central player in homologous recombination, the faithful repair pathway for double-strand breaks and key event during meiosis. Rad51 forms nucleoprotein filaments on single-stranded DNA, exposed by a double-strand break. These filaments are responsible for homology search and strand invasion, which lead to homology-directed repair. Due to its central roles in DNA repair and genome stability, Rad51 is modulated by multiple factors and post-translational modifications. In this review, we summarize our current understanding of the dynamics of Rad51 filaments, the roles of other factors and their modes of action in modulating key stages of Rad51 filaments: formation, stability and disassembly.
Williams RM, Zhang X, 2021, Roles of ATM and ATR in DNA double strand breaks and replication stress, PROGRESS IN BIOPHYSICS & MOLECULAR BIOLOGY, Vol: 161, Pages: 27-38, ISSN: 0079-6107
Tannous EA, Yates LA, Zhang X, et al., 2021, Mechanism of auto-inhibition and activation of Mec1ATR checkpoint kinase, Nature Structural and Molecular Biology, Vol: 28, Pages: 50-61, ISSN: 1545-9985
In response to DNA damage or replication fork stalling, the basal activity of Mec1ATR is stimulated in a cell-cycle-dependent manner, leading to cell-cycle arrest and the promotion of DNA repair. Mec1ATR dysfunction leads to cell death in yeast and causes chromosome instability and embryonic lethality in mammals. Thus, ATR is a major target for cancer therapies in homologous recombination-deficient cancers. Here we identify a single mutation in Mec1, conserved in ATR, that results in constitutive activity. Using cryo-electron microscopy, we determine the structures of this constitutively active form (Mec1(F2244L)-Ddc2) at 2.8 Å and the wild type at 3.8 Å, both in complex with Mg2+-AMP-PNP. These structures yield a near-complete atomic model for Mec1-Ddc2 and uncover the molecular basis for low basal activity and the conformational changes required for activation. Combined with biochemical and genetic data, we discover key regulatory regions and propose a Mec1 activation mechanism.
Xiang X, Zhang X, Gu Y, et al., 2020, Analysis and investigation of internal AC Frequency to minimize AC current magnitude and reactive power circulation in chain-link modular multilevel direct DC-DC converters, IEEE Transactions on Circuits and Systems Part 1: Regular Papers, Vol: 67, Pages: 5586-5599, ISSN: 1549-8328
Chain-link modular multilevel direct dc-dc converters (CLMMCs) have attracted much interest recently in for dc power systems because they achieve higher device utilization, lower power losses and they are physically more compact than the alternative front-to-front modular multilevel dc-ac-dc converters (FFMMCs). The CLMMCs rely on circulating an internal ac current to manage energy balance of the sub-module (SM) stacks but this current inevitably contributes to extra current stresses for circuit components and can also lead to excess reactive power circulation within the converter. This paper presents a circuit analysis that there exists a frequency value for the internal ac components that may minimize the current stresses and avoid excessive reactive power circulation. For illustration, the circuit analysis is applied to one of the base formats, the buck-boost CLMMC as an example. The key relationship between the CLMMC and the standard dc-ac modular multilevel converter is explored and established. The equivalent circuits for their dc and ac components with consideration of SM capacitance and SM voltage ripples are created and analyzed in detail, and a full derivation is provided for the specific ac frequency. From this example, this analytical method is extended and applied to other base formats of CLMMCs. The theoretical analysis and results are verified by a set of full-scale simulation examples and down-scaled experiments on a laboratory prototype.
Williams R, Yates L, Zhang X, 2020, Structures and regulations of ATM and ATR, master kinases in genome integrity, Current Opinion in Structural Biology, Vol: 61, Pages: 98-105, ISSN: 0959-440X
Homologous recombination (HR) is a faithful repair mechanism for double stranded DNA breaks. Two highly homologous master kinases, the tumour suppressors ATM and ATR (Tel1 and Mec1 in yeast), coordinate cell cycle progression with repair during HR. Despite their importance, our molecular understanding of these apical coordinators has been limited, in part due to their large sizes. With the recent development in cryo-electron microscopy, significant advances have been made in structural characterisation of these proteins in the last two years. These structures, combined with new biochemical studies, now provide a more detailed understanding of how a low basal activity is maintained and how activation may occur. In this review, we summarize recent advances in the structural and molecular understanding of these key components in HR, compare the common and distinct features of these kinases and suggest aspects of structural components that are likely to be involved in regulating its activity.
Zhang X, Blundell TL, 2020, Editorial overview: Macromolecular assemblies, CURRENT OPINION IN STRUCTURAL BIOLOGY, Vol: 61, Pages: VI-VIII, ISSN: 0959-440X
Gao F, Danson AE, Ye F, et al., 2020, Bacterial enhancer binding proteins - AAA+ proteins in transcription activation, Biomolecules, Vol: 10, ISSN: 2218-273X
Bacterial enhancer-binding proteins (bEBPs) are specialised transcriptional activators. bEBPs are hexameric AAA+ ATPases and use ATPase activities to remodel RNA polymerase (RNAP) complexes that contain the major variant sigma factor, σ54 to convert the initial closed complex to the transcription competent open complex. Earlier crystal structures of AAA+ domains alone have led to proposals of how nucleotide-bound states are sensed and propagated to substrate interactions. Recently, the structure of the AAA+ domain of a bEBP bound to RNAP-σ54-promoter DNA was revealed. Together with structures of the closed complex, an intermediate state where DNA is partially loaded into the RNAP cleft and the open promoter complex, a mechanistic understanding of how bEBPs use ATP to activate transcription can now be proposed. This review summarises current structural models and the emerging understanding of how this special class of AAA+ proteins utilises ATPase activities to allow σ54-dependent transcription initiation.
Stach L, Morgan RM, Makhlouf L, et al., 2020, Crystal structure of the catalytic D2 domain of the AAA+ ATPase p97 reveals a putative helical split-washer-type mechanism for substrate unfolding, FEBS Letters, Vol: 594, Pages: 933-943, ISSN: 0014-5793
Several pathologies have been associated with the AAA+ ATPase p97, an enzyme essential to protein homeostasis. Heterozygous polymorphisms in p97 have been shown to cause neurological disease, while elevated proteotoxic stress in tumours has made p97 an attractive cancer chemotherapy target. The cellular processes reliant on p97 are well described. High‐resolution structural models of its catalytic D2 domain, however, have proved elusive, as has the mechanism by which p97 converts the energy from ATP hydrolysis into mechanical force to unfold protein substrates. Here, we describe the high‐resolution structure of the p97 D2 ATPase domain. This crystal system constitutes a valuable tool for p97 inhibitor development and identifies a potentially druggable pocket in the D2 domain. In addition, its P61 symmetry suggests a mechanism for substrate unfolding by p97.
Yates L, Williams R, Hailemariam S, et al., 2020, Cryo-EM structure of nucleotide-bound Tel1ATM unravels the molecular basis of inhibition and structural rationale for disease-associated mutations, Structure, Vol: 28, Pages: 96-104.e3, ISSN: 0969-2126
Yeast Tel1 and its highly conserved human orthologue ATM are large protein kinases centralto the maintenance of genome integrity. Mutations in ATM are found in ataxia-telangiectasia(A-T) patients and ATM is one of the most frequently mutated genes in many cancers. Usingcryo electron microscopy, we present the structure of Tel1 in a nucleotide-bound state. Ourstructure reveals molecular details of key residues surrounding the nucleotide binding site andprovides a structural and molecular basis for its intrinsically low basal activity. We show thatthe catalytic residues are in a productive conformation for catalysis, but the PIKK-regulatorydomain-Insert (PRD-I) restricts peptide-substrate access and the N-lobe is in an openconformation, thus explaining the requirement for Tel1 activation. Structural comparisons withother PIKKs suggest a conserved and common allosteric activation mechanism. Our work alsoprovides a structural rationale for many mutations found in A-T and cancer.
Ye F, Kotta-Loizou I, Jovanovic M, et al., 2019, Structural basis of transcription inhibition by the DNA mimic protein Ocr of bacteriophage T7, eLife, Vol: 9, ISSN: 2050-084X
Abstract Bacteriophage T7 infects Escherichia coli and evades the host defence system. The Ocr protein of T7 was shown to exist as a dimer mimicking DNA and to bind to host restriction enzymes, thus preventing the degradation of the viral genome by the host. Here we report that Ocr can also inhibit host transcription by directly binding to bacterial RNA polymerase (RNAP) and competing with the recruitment of RNAP by sigma factors. Using cryo electron microscopy, we determined the structures of Ocr bound to RNAP. The structures show that an Ocr dimer binds to RNAP in the cleft, where key regions of sigma bind and where DNA resides during transcription synthesis, thus providing a structural basis for the transcription inhibition. Our results reveal the versatility of Ocr in interfering with host systems and suggest possible strategies that could be exploited in adopting DNA mimicry as a basis for forming novel antibiotics. Impact statement DNA mimicry Ocr protein, a well-studied T7 phage protein that inhibits host restriction enzymes, can also inhibit host transcription through competing with sigma factors in binding to RNA polymerase.
Danson AE, Jovanovic M, Buck M, et al., 2019, Mechanisms of σ<SUP>54</SUP>-Dependent Transcription Initiation and Regulation, JOURNAL OF MOLECULAR BIOLOGY, Vol: 431, Pages: 3960-3974, ISSN: 0022-2836
Ebright RH, Werner F, Zhang X, 2019, RNA Polymerase Reaches 60: Transcription Initiation, Elongation, Termination, and Regulation in Prokaryotes, JOURNAL OF MOLECULAR BIOLOGY, Vol: 431, Pages: 3945-3946, ISSN: 0022-2836
Daza-Martin M, Starowicz K, Jamshad M, et al., 2019, Isomerization of BRCA1-BARD1 promotes replication fork protection, NATURE, Vol: 571, Pages: 521-+, ISSN: 0028-0836
Yates LA, Williams RM, Hailemariam S, et al., 2019, Structure of nucleotide-bound Tel1<sup>ATM</sup> reveals the molecular basis of inhibition and structural rationale for disease mutations
<jats:sec><jats:title>SUMMARY</jats:title><jats:p>Yeast Tel1 and its highly conserved human orthologue ATM are large protein kinases central to the maintenance of genome integrity. Mutations in ATM are found in ataxia-telangiectasia (A-T) patients and ATM is one of the most frequently mutated genes in many cancers. Using cryo electron microscopy, we present the structure of Tel1 in a nucleotide-bound state. Our structure reveals molecular details of key residues surrounding the nucleotide binding site and provides a structural and molecular basis for its intrinsically low basal activity. We show that the catalytic residues are in a productive conformation for catalysis, but the PIKK-regulatory domain-Insert (PRD-I) restricts peptide-substrate access and the N-lobe is in an open conformation, thus explaining the requirement for Tel1 activation. Structural comparisons with other PIKKs suggest a conserved and common allosteric activation mechanism. Our work also provides a structural rationale for many mutations found in A-T and cancer.</jats:p></jats:sec>
Danson A, Jovanovic M, Buck M, et al., 2019, Mechanisms of s54-dependent transcription initiation and regulation, Journal of Molecular Biology, ISSN: 0022-2836
Cellular RNA polymerase is a multi-subunit macromolecular assembly responsible for gene transcription, a highly regulated process conserved from bacteria to humans. In bacteria, sigma factors are employed to mediate gene-specific expression in response to a variety of environmental conditions. The major variant σ factor, σ54, has a specific role in stress responses. Unlike σ70-dependent transcription, which often can spontaneously proceed to initiation, σ54-dependent transcription requires an additional ATPase protein for activation. As a result, structures of a number of distinct functional states during the dynamic process of transcription initiation have been captured using the σ54 system with both x-ray crystallography and cryo electron microscopy, furthering our understanding of σ54-dependent transcription initiation and DNA opening. Comparisons with σ70 and eukaryotic polymerases reveal unique and common features during transcription initiation.
Yates LA, Aramayo RJ, Pokhrel N, et al., 2018, A structural and dynamic model for the assembly of Replication Protein A on single-stranded DNA, Nature Communications, Vol: 9, ISSN: 2041-1723
Replication Protein A (RPA), the major eukaryotic single stranded DNA-binding protein, binds to exposed ssDNA to protect it from nucleases, participates in a myriad of nucleic acid transactions and coordinates the recruitment of other important players. RPA is a heterotrimer and coats long stretches of single-stranded DNA (ssDNA). The precise molecular architecture of the RPA subunits and its DNA binding domains (DBDs) during assembly is poorly understood. Using cryo electron microscopy we obtained a 3D reconstruction of the RPA trimerisation core bound with ssDNA (∼55 kDa) at ∼4.7 Å resolution and a dimeric RPA assembly on ssDNA. FRET-based solution studies reveal dynamic rearrangements of DBDs during coordinated RPA binding and this activity is regulated by phosphorylation at S178 in RPA70. We present a structural model on how dynamic DBDs promote the cooperative assembly of multiple RPAs on long ssDNA.
Glyde R, Ye F, Jovanovic M, et al., 2018, Structures of bacterial RNA polymerase complexes reveal mechanisms of DNA loading and transcription initiation, Molecular Cell, Vol: 70, Pages: 1111-1120.e3, ISSN: 1097-2765
Gene transcription is carried out by multi-subunit RNA polymerases (RNAP).Transcription initiation is a dynamic multi-step process that involves the opening of the double stranded DNA to form a transcription bubble and delivery of the template strand deep into the RNAP for RNA synthesis. Applying cryo electron microscopy to a unique transcription system using 54 (N), the major bacterial variant sigma factor, we capture a new intermediate state at 4.1 Å where promoter DNA is caught at the entrance of the RNAP cleft. Combining with new structures of the open promotercomplex and an initial de novo transcribing complex at 3.4 and 3.7 Å respectively, our studies reveal the dynamics of DNA loading and mechanism of transcription bubble stabilisation that involves coordinated, large scale conformational changes of the universally conserved features within RNAP and DNA. In addition, our studies reveal a novel mechanism of strand separation by 54.
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