Susan Tsutakawa

Susan Tsutakawa
Lawrence Berkeley National Laboratory | LBL

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76
Publications
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Publications

Publications (76)
Chapter
Structures provide a critical breakthrough step for biological analyses, and small angle X-ray scattering (SAXS) is a powerful structural technique to study dynamic DNA repair proteins. As toxic and mutagenic repair intermediates need to be prevented from inadvertently harming the cell, DNA repair proteins often chaperone these intermediates throug...
Article
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All tumors have DNA mutations, and a predictive understanding of those mutations could inform clinical treatments. However, 40% of the mutations are variants of unknown significance (VUS), with the challenge being to objectively predict whether a VUS is pathogenic and supports the tumor or whether it is benign. To objectively decode VUS, we mapped...
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We present a Chemistry and Structure Screen Integrated Efficiently (CASSIE) approach (named for Greek prophet Cassandra) to design inhibitors for cancer biology and pathogenesis. CASSIE provides an effective path to target master keys to control the repair-replication interface for cancer cells and SARS CoV-2 pathogenesis as exemplified here by spe...
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The biological and functional significance of selected CASP14 targets are described by the authors of the structures. The authors highlight the most relevant features of the target proteins and discuss how well these features were reproduced in the respective submitted predictions. The overall ability to predict three-dimensional structures of prot...
Article
Stalled DNA replication fork restart after stress as orchestrated by ATR kinase, BLM helicase, and structure specific nucleases enables replication, cell survival, and genome stability. Here we unveil human exonuclease V (EXO5) as an ATR-regulated DNA structure specific nuclease and BLM partner for replication fork restart. We find that elevated EX...
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Papain-like protease (PLpro) from SARS-CoV-2 plays essential roles in the replication cycle of the virus. In particular, it preferentially interacts with and cleaves human interferon-stimulated gene 15 (hISG15) to suppress the innate immune response of the host. We used small-angle X-ray and neutron scattering combined with computational techniques...
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The RNA transcription complex (RTC) from the virus, SARS-CoV-2, is responsible for recognizing and processing RNA for two principal purposes. The RTC copies viral RNA for propagation into new virus and for ribosomal transcription of viral proteins. To accomplish these activities the RTC mechanism must also conform to a large number of imperatives i...
Article
The general transcription factor TFIIH contains three ATP-dependent catalytic activities. TFIIH functions in nucleotide excision repair primarily as a DNA helicase and in Pol II transcription initiation as a dsDNA translocase and protein kinase. During initiation, the XPB/Ssl2 subunit of TFIIH couples ATP hydrolysis to dsDNA translocation facilitat...
Article
The canonical DNA glycosylase role is global base damage repair but includes functions in epigenetic gene regulation, immune response modulation, replication, and transcription. In this issue of Structure, Eckenroth et al. (2020) present the NEIL2 glycosylase structure. Its catalytic domain flexibility differentiates it from most other glycosylases...
Preprint
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The general transcription factor TFIIH contains three ATP-dependent catalytic activities. TFIIH functions in nucleotide excision repair primarily as a DNA helicase and in Pol II transcription initiation as a dsDNA translocase and protein kinase. During initiation, the XPB/Ssl2 subunit of TFIIH couples ATP hydrolysis to dsDNA translocation facilitat...
Article
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DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a key member of the phosphatidylinositol-3 kinase-like (PIKK) family of protein kinases with critical roles in DNA-double strand break repair, transcription, metastasis, mitosis, RNA processing, and innate and adaptive immunity. The absence of DNA-PKcs from many model organisms has led to...
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Critical for transcription initiation and bulky lesion DNA repair, TFIIH provides an exemplary system to connect molecular mechanisms to biological outcomes due to its strong genetic links to different specific human diseases. Recent advances in structural and computational biology provide a unique opportunity to re-examine biologically relevant mo...
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Xeroderma pigmentosum group G (XPG) protein is both a functional partner in multiple DNA damage responses (DDR) and a pathway coordinator and structure-specific endonuclease in nucleotide excision repair (NER). Different mutations in the XPG gene ERCC5 lead to either of two distinct human diseases: Cancer-prone xeroderma pigmentosum (XP-G) or the f...
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Small Angle X‐ray Scattering (SAXS) measures comprehensive distance information on a protein's structure, which can constrain and guide computational structure prediction algorithms. Here we evaluate structure predictions of 11 monomeric and oligomeric proteins for which SAXS data were collected and provided to predictors in the 13th round of the C...
Article
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Transcription preinitiation complexes (PICs) are vital assemblies whose function underlies the expression of protein-encoding genes. Cryo-EM advances have begun to uncover their structural organization. Nevertheless, functional analyses are hindered by incompletely modeled regions. Here we integrate all available cryo-EM data to build a practically...
Article
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Experimental data offers empowering constraints for structure prediction. These constraints can be used to filter equivalently scored models or more powerfully within optimization functions toward prediction. In CASP12, Small Angle X-ray Scattering (SAXS) and Cross-Linking Mass Spectrometry (CLMS) data, measured on an exemplary set of novel fold ta...
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Original article: Nature Communications 8 Article number: 15855 (2017); Published: 27 June 2017; Updated: 7 August 2017
Data
S hFEN1 recognizes its substrate primarily through dsDNA Cartoon schematic of DNA in hFEN1-D86N structure. Spheres on DNA mark where protein is within 4 Å of template strand (brown), 5'-flap nts in dsDNA (gold), 5'-flap nts in ssDNA (orange), and 3'-flap strand (pink). Contacting protein residues (not depicted) are labeled by residue number at thei...
Data
hFEN positive charges rotate dsDNA into a catalytic position on the active site metals guided by coordinated Tyr40 side chain rotation as shown by morphing between hFEN1-R100A and hFEN1-D86N substrate structures Morphing between overlaid hFEN1-R100A and hFEN1-D86N substrate complexes to illustrate change in gateway helix 4 and cap helices and rotat...
Data
Supplementary Figures, Supplementary Table and Supplementary References
Data
hFEN1 active site DNA complex with attacking water as shown by hFEN1-D86N-substrate Close-up view of the hFEN1-D86N active site, with attacking water. The second metal from an overlaid WT-product complex (PDB code 3Q8K) is depicted as a gray sphere. Movie is related to Figure 3.
Article
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DNA replication and repair enzyme Flap Endonuclease 1 (FEN1) is vital for genome integrity, and FEN1 mutations arise in multiple cancers. FEN1 precisely cleaves single-stranded (ss) 50-flaps one nucleotide into duplex (ds) DNA. Yet, how FEN1 selects for but does not incise the ss 50-flap was enigmatic. Here we combine crystallographic, biochemical...
Article
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Human flap endonuclease 1 (FEN1) and related structure-specific 5’nucleases precisely identify and incise aberrant DNA structures during replication, repair and recombination to avoid genomic instability. Yet, it is unclear how the 5’nuclease mechanisms of DNA distortion and protein ordering robustly mediate efficient and accurate substrate recogni...
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Covalent DNA-protein crosslinks (DPCs) are toxic DNA lesions that interfere with essential chromatin transactions, such as replication and transcription. Little was known about DPC-specific repair mecha- nisms until the recent identification of a DPC-pro- cessing protease in yeast. The existence of a DPC protease in higher eukaryotes is inferred fr...
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A central question in protein evolution is the extent to which naturally occurring proteins sample the space of folded structures accessible to the polypeptide chain. Repeat proteins composed of multiple tandem copies of a modular structure unit are widespread in nature and have critical roles in molecular recognition, signalling, and other essenti...
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Protecting, reversing, and remodeling stalled replication forks are critical to genome stability and require coordinating DNA replication, remodeling, and repair. In this issue, Kile et al. (2015) find that unexpected HLTF specificity for DNA's 3'-hydroxyl tail helps control these biological functions. Copyright © 2015 Elsevier Inc. All rights rese...
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Proliferating cell nuclear antigen (PCNA) is a pivotal replication protein, which also controls cellular responses to DNA damage. Posttranslational modification of PCNA by SUMO and ubiquitin modulate these responses. How the modifiers alter PCNA-dependent DNA repair and damage tolerance pathways is largely unknown. We used hybrid methods to identif...
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Protein Phosphatase 2A (PP2A) is a major Ser/Thr phosphatase involved in the regulation of various cellular processes. PP2A assembles into diverse trimeric holoenzymes, which consist of a scaffolding (A) subunit, a catalytic (C) subunit and various regulatory (B) subunits. Here we report a 2.0 Å crystal structure of the free B''/PR70 subunit and a...
Article
Flap endonuclease 1 (FEN1) and XPG are essential 5′ nuclease superfamily endonucleases in DNA replication and repair. FEN1 incises in the dsDNA region adjacent to 5′ flaps, while XPG incises in the dsDNA region adjacent to DNA bubbles. We have used a hybrids method analysis combining crystallography, Small Angle X-ray Scattering (SAXS), Electron Mi...
Article
The bacterial recombinase RecA mediates homologous recombination and recombinational DNA repair through homology search and strand exchange. During homologous recombination, RecA binds an incoming single-stranded DNA (ssDNA) in its primary binding site, site I, to form a nucleoprotein filament whose crystal structure is known. The structure of the...
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The recent innovation of collecting X-ray scattering from solutions containing purified macromolecules in high-throughput has yet to be truly exploited by the biological community. Yet, this capability is becoming critical given that the growth of sequence and genomics data is significantly outpacing structural biology results. Given the huge misma...
Article
RECQL5 is a member of the highly conserved RecQ family of DNA helicases involved in DNA repair. RECQL5 interacts with RNA polymerase II (Pol II) and inhibits transcription of protein-encoding genes by an unknown mechanism. We show that RECQL5 contacts the Rpb1 jaw domain of Pol II at a site that overlaps with the binding site for the transcription...
Article
NEIL1, one of the five mammalian DNA glycosylases that excise oxidized DNA base lesions in the human genome to initiate base excision repair (BER), contains an intrinsically disordered C-terminal domain (CTD; ~100 residues), not conserved in its E. coli prototype Nei. Although dispensable for NEIL1's lesion excision and AP lyase activities, this se...
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Non-coding apurinic/apyrimidinic (AP) sites in DNA form spontaneously and as DNA base excision repair intermediates are the most common toxic and mutagenic in vivo DNA lesion. For repair, AP sites must be processed by 5' AP endonucleases in initial stages of base repair. Human APE1 and bacterial Nfo represent the two conserved 5' AP endonuclease fa...
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By coupling the protection and organization of single-stranded DNA (ssDNA) with recruitment and alignment of DNA processing factors, replication protein A (RPA) lies at the heart of dynamic multi-protein DNA processing machinery. Nevertheless, how RPA coordinates biochemical functions of its eight domains remains unknown. We examined the structural...
Article
1135 rFVIIIFc is a recombinant fusion protein comprising human B domain-deleted (BDD) coagulation factor VIII covalently attached to the Fc domain of human immunoglobulin G1 (IgG1). rFVIIIFc is currently in a phase 3 clinical study as a novel long-lasting FVIII molecule designed to potentially reduce dosing frequency and provide prolonged protectio...
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The DNA-repair enzyme Tdp2 resolves 5'-phosphotyrosyl DNA adducts and mediates resistance to anticancer drugs that target covalent topoisomerase-DNA complexes. Tdp2 also participates in key signaling pathways during development and tumorigenesis and cleaves a protein-RNA linkage during picornavirus replication. The crystal structure of zebrafish Td...
Article
Propagation and maintenance of the cellular genome are among the most fundamental cellular processes, encompassing pathways associated with DNA replication, damage response, and repair. Replication Protein A (RPA), the primary single-stranded DNA-binding protein (SSB) in eukaryotes, serves to protect ssDNA generated during these events and to recru...
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Processing of Okazaki fragments to complete lagging strand DNA synthesis requires coordination among several proteins. RNA primers and DNA synthesised by DNA polymerase α are displaced by DNA polymerase δ to create bifurcated nucleic acid structures known as 5'-flaps. These 5'-flaps are removed by Flap Endonuclease 1 (FEN), a structure-specific nuc...
Article
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Processivity clamps such as proliferating cell nuclear antigen (PCNA) and the checkpoint sliding clamp Rad9/Rad1/Hus1 (9-1-1) act as versatile scaffolds in the coordinated recruitment of proteins involved in DNA replication, cell-cycle control, and DNA repair. Association and handoff of DNA-editing enzymes, such as flap endonuclease 1 (FEN1), with...
Article
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Flap endonucleases (FENs), essential for DNA replication and repair, recognize and remove RNA or DNA 5'-flaps. Related to FEN specificity for substrates with free 5'-ends, but controversial, is the role of the helical arch observed in varying conformations in substrate-free FEN structures. Conflicting models suggest either 5'-flaps thread through t...
Article
Detailed structural, mutational, and biochemical analyses of human FEN1/DNA complexes have revealed the mechanism for recognition of 5' flaps formed during lagging strand replication and DNA repair. FEN1 processes 5' flaps through a previously unknown, but structurally elegant double-stranded (ds) recognition/single stranded (ss) incision mechanism...
Article
Structure-specific 5'-nucleases form a superfamily of evolutionarily conserved phosphodiesterases that catalyse a precise incision of a diverse range of DNA and RNA substrates in a sequence-independent manner. Superfamily members, such as flap endonucleases, exonuclease 1, DNA repair protein XPG, endonuclease GEN1 and the 5'-3'-exoribonucleases, pl...
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PCNA ubiquitination in response to DNA damage leads to the recruitment of specialized translesion polymerases to the damage locus. This constitutes one of the initial steps in translesion synthesis (TLS)--a critical pathway for cell survival and for maintenance of genome stability. The recent crystal structure of ubiquitinated PCNA (Ub-PCNA) sheds...
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Bacterial conjugation is regulated by two-component repression comprising the antisense RNA FinP, and its protein co-factor FinO. FinO mediates base-pairing of FinP to the 5′-untranslated region (UTR) of traJ mRNA, which leads to translational inhibition of the transcriptional activator TraJ and subsequent down regulation of conjugation genes. Yet,...
Article
Flap endonuclease (FEN1), essential for DNA replication and repair, removes RNA and DNA 5' flaps. FEN1 5' nuclease superfamily members acting in nucleotide excision repair (XPG), mismatch repair (EXO1), and homologous recombination (GEN1) paradoxically incise structurally distinct bubbles, ends, or Holliday junctions, respectively. Here, structural...
Article
Adaptive immune systems have recently been recognized in prokaryotic organisms where, in response to viral infection, they incorporate short fragments of invader-derived DNA into loci called clustered regularly interspaced short palindromic repeats (CRISPRs). In subsequent infections, the CRISPR loci are transcribed and processed into guide sequenc...
Article
Replication protein A (RPA) is the primary eukaryotic single-stranded DNA (ssDNA) binding protein utilized in diverse DNA transactions in the cell. RPA is a heterotrimeric protein with seven globular domains connected by flexible linkers, which enable substantial interdomain motion that is essential to its function. Small angle X-ray scattering (SA...
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We present an efficient pipeline enabling high-throughput analysis of protein structure in solution with small angle X-ray scattering (SAXS). Our SAXS pipeline combines automated sample handling of microliter volumes, temperature and anaerobic control, rapid data collection and data analysis, and couples structural analysis with automated archiving...
Article
The BARD1 N-terminal RING domain binds BRCA1 while the BARD1 C-terminal ankyrin and tandem BRCT repeat domains bind CstF-50 to modulate mRNA processing and RNAP II stability in response to DNA damage. Here we characterize the BARD1 structural biochemistry responsible for CstF-50 binding. The crystal structure of the BARD1 BRCT domain uncovers a deg...
Article
MAP2Ks are dual-specificity protein kinases functioning at the center of three-tiered MAP kinase modules. The structure of the kinase domain of the MAP2K MEK6 with phosphorylation site mimetic aspartic acid mutations (MEK6/DeltaN/DD) has been solved at 2.3 angstroms resolution. The structure reveals an autoinhibited elongated ellipsoidal dimer. The...