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At least 901 records · Page 50Linked to original sources

bioRxiv2018

DSCAM-AS1 promotes tumor growth of breast cancer by reducing miR-204-5p and upregulating RRM2

We intended to analyze the effects of DSCAM-AS1, miR-204-5p and RRM2 on breast cancer (BC) cells growth. Microarray analysis and qRT-PCR were employed to determine DSCAM-AS1 and miR-204-5p expression in BC. Luciferase reporter assay and cell transfection assay were applied to examine the target relationship between DSCAM-AS1, miR-204-5p and MMR2. CCK-8 assay, transwell assay and flow cytometry were used to detect cell proliferation, invasion and apoptosis of breast cancer cells. The expression of DSCAM-AS1, miR-204-5p and MMR2 were confirmed by Western Blot. We also conducted In vivo assay to verify the effect of DSCAM-AS1 on tumor formation.DSCAM-AS1 was up-regulated, while miR-204-5p was down-regulated in BC tissues and cells. Meanwhile, DSCAM-AS1 directly targeted miR-204-5p. DSCAM-AS1 promoted the proliferation and invasion of BC cells and restrained cell apoptosis by reducing miR-204-5p and inhibiting miR-204-5p expression. RRM2 was up-regulated in BC cells, and miR-204-5p inhibited RRM2 expression by targeting RRM2. Overexpression of RRM2 stimulated proliferation and cell invasion and impeded apoptosis of BC cells. In vivo experiments showed that knockdown of DSCAM-AS1 decreased the tumorigenesis of BC cells, increased the expression of miR-204-5p while inhibited RRM2 expression.DSCAM-AS1 promoted proliferation and impaired apoptosis of BC cells by reducing miR-204-5p and enhancing RRM2 expression. DSCAM-AS1/miR-204-5p/RRM2 may serve as novel therapeutic targets for BC.\n\nSummary statementMicroarray analysis and qRT-PCR were employed to determine DSCAM-AS1 and miR-204-5p expression in BC. DSCAM-AS1 promoted proliferation and impaired apoptosis of BC cells by reducing miR-204-5p and enhancing RRM2 expression.

biochemistry↗

bioRxiv2018

Pat1 promotes processing body assembly by enhancing the phase separation of the DEAD-box ATPase Dhh1 and RNA

Processing bodies (PBs) are cytoplasmic mRNP granules that assemble via liquid-liquid phase separation and are implicated in the decay or storage of mRNAs. How PB assembly is regulated in cells remains unclear. We recently identified the ATPase activity of the DEAD-box protein Dhh1 as a key regulator of PB dynamics and demonstrated that Not1, an activator of the Dhh1 ATPase and member of the CCR4-NOT deadenylase complex inhibits PB assembly in vivo [Mugler et al., 2016]. Here, we show that the PB component Pat1 antagonizes Not1 and promotes PB assembly via its direct interaction with Dhh1. Intriguingly, in vivo PB dynamics can be recapitulated in vitro, since Pat1 enhances the phase separation of Dhh1 and RNA into liquid droplets, whereas Not1 reverses Pat1-Dhh1-RNA condensation. Overall, our results uncover a function of Pat1 in promoting the multimerization of Dhh1 on mRNA, thereby aiding the assembly of large multivalent mRNP granules that are PBs.

biochemistry↗

bioRxiv2018

Proton co-transport and voltage dependence enforce unidirectional metal transport in an Nramp transporter

Secondary transporters harness electrochemical energy to move substrate through structurally-enforced co-substrate \"coupling\". We untangle the \"proton-metal coupling\" behavior by a Natural resistance-associated macrophage protein (Nramp) transporter into two distinct phenomena: {Delta}pH stimulation of metal transport and metal stimulation of proton co-transport. Surprisingly, metal type dictates co-transport stoichiometry, leading to manganese-proton symport but cadmium uniport. Additionally, the membrane potential affects both the kinetics and thermodynamics of metal transport. A conserved salt-bridge network near the metal-binding site imparts voltage dependence and enables proton co-transport, properties that allow this Nramp transporter to maximize metal uptake and prevent deleterious back-transport of acquired metals. We provide a new mechanistic model for Nramp metal-proton symport in which, in addition to substrate gradients determining directionality as in canonical secondary transport, synergy between protein structure and physiological voltage enforces unidirectional substrate movement. Our results illustrate a functional advantage that arises from deviations from the traditional model of symport.

biochemistry↗

bioRxiv2018

Extracellular Adenine Nucleotide and Adenosine Metabolism in Calcific Aortic Valve Disease

Extracellular nucleotide catabolism contributes to immunomodulation, cell differentiation and tissue mineralization by controlling nucleotide and adenosine concentrations and its purinergic effects. Disturbances of purinergic signaling in valves may lead to its calcification. This study aimed to investigate the side-specific changes in extracellular nucleotide and adenosine metabolism in the aortic valve during calcific aortic valve disease (CAVD) and to identify the individual enzymes that are involved in these pathways as well as their cellular origin.\n\nStenotic aortic valves were characterized by reduced levels of extracellular ATP removal and impaired production of adenosine. Respectively, already reduced levels of extracellular adenosine were immediately degraded further due to the elevated rate of adenosine deamination. For the first time, we revealed that this metabolic pattern was observed only on the fibrosa surface of stenotic valve that is consistent with the mineral deposition on the aortic side of the valve. Furthermore, we demonstrated that non-stenotic valves expressed mostly ecto-nucleoside triphosphate diphosphohydrolase 1 (eNTPD1) and ecto-5nucleotidase (e5NT), while stenotic valves ecto-nucleotide pyrophosphatase/ phosphodiesterase 1, alkaline phosphatase and ecto-adenosine deaminase (eADA). On the surface of endothelial cells, isolated from non-stenotic valves, high activities of eNTPD1 and e5NT were found. Whereas, in valvular interstitial cells, eNPP1 activity was also detected. Stenotic valve immune infiltrate was an additional source of eADA. We demonstrated the presence of A1, A2a and A2b adenosine receptors in both, non-stenotic and stenotic valves with diminished expression of A2a and A2b in the former.\n\nExtracellular nucleotide and adenosine metabolism that involves complex ecto-enzyme pathways and adenosine receptor signaling were adversely modified in CAVD. In particular, diminished activities of eNTPD1 and e5NT with the increase in eADA that originated from valvular endothelial and interstitial cells as well as from immune inflitrate may affect aortic valve extracellular nucleotide concentrations to favor a proinflammatory milieu, highlighting a potential mechanism and target for CAVD therapy.

biochemistry↗

bioRxiv2018

Identification of Small Molecule Modulators of Diguanylate Cyclase by FRET-based High-Throughput-Screening

The bacterial second messenger cyclic diguanosine monophosphate (c-di-GMP) is a key regulator of cellular motility, the cell cycle, and biofilm formation with its resultant antibiotic tolerance, which may make chronic infections difficult to treat. Therefore, diguanylate cyclases, which regulate the spatiotemporal production of c-di-GMP, may be attractive drug targets to control biofilm formation that is part of chronic infections. In this paper, we present a FRET-based biochemical high-throughput screening approach coupled with detailed structure-activity studies to identify synthetic small molecule modulators of the diguanylate cyclase, DgcA, from Caulobacter crescentus. We identified a set of 7 small molecules that in the low {micro}M range regulate DgcA enzymatic activity. Subsequent structure activity studies on selected scaffolds revealed a remarkable diversity of modulatory behaviors, including slight chemical substitutions that revert the effects from allosteric enzyme inhibition to activation. The compounds identified represent novel chemotypes and are potentially developable into chemical genetic tools for the dissection of c-di-GMP signaling networks and alteration of c-di-GMP associated phenotypes. In sum, our studies underline the importance for detailed mechanism of action studies for inhibitors of c-di-GMP signaling and demonstrate the complex interplay between synthetic small molecules and the regulatory mechanisms that control the activity of diguanylate cyclases.

biochemistry↗

bioRxiv2018

A quantitative and predictive model for RNA binding by human Pumilio proteins

High-throughput methodologies have enabled routine generation of RNA target sets and sequence motifs for RNA-binding proteins (RBPs). Nevertheless, quantitative approaches are needed to capture the landscape of RNA/RBP interactions responsible for cellular regulation. We have used the RNA-MaP platform to directly measure equilibrium binding for thousands of designed RNAs and to construct a predictive model for RNA recognition by the human Pumilio proteins PUM1 and PUM2. Despite prior findings of linear sequence motifs, our measurements revealed widespread residue flipping and instances of positional coupling. Application of our thermodynamic model to published in vivo crosslinking data reveals quantitative agreement between predicted affinities and in vivo occupancies. Our analyses suggest a thermodynamically driven, continuous Pumilio binding landscape that is negligibly affected by RNA structure or kinetic factors, such as displacement by ribosomes. This work provides a quantitative foundation for dissecting the cellular behavior of RBPs and cellular features that impact their occupancies.

biochemistry↗

bioRxiv2018

Structures in multiple conformations reveal distinct transition metal and proton pathways in an Nramp transporter

Nramp family transporters--expressed in organisms from bacteria to humans--enable uptake of essential divalent transition metals via an alternating-access mechanism that includes proton co-transport. We present high-resolution structures of Deinococcus radiodurans (Dra)Nramp at complementary stages of its transport cycle to provide a thorough description of the Nramp transport cycle by identifying the key intramolecular rearrangements and changes to the metal coordination sphere. Strikingly, while metal transport requires cycling from outward-to inward-open states, efficient proton transport still occurs in outward-locked (but not inward-locked) DraNramp. We propose a model in which metal and proton enter the transporter via the same external pathway to the binding site, but follow separate routes to the cytoplasm, thus resolving the electrostatic dilemma of using a cation co-substrate to drive a cation primary substrate. Our results illustrate the flexibility of the LeuT fold to support a broad range of co-substrate coupling and conformational change mechanisms.

biochemistry↗

bioRxiv2018

A freeze-and-thaw induced-fragment of the microtubule-associated protein Tau in rat brain extracts: implications for the study of neurodegenerative diseases

Tau is a microtubule-associated protein responsible for controlling the stabilization of microtubules in neurons. Tau function is regulated by phosphorylation. However, in some neurological diseases Tau becomes aberrantly hyperphosphorylated, which contributes to the pathogenesis of neurological diseases, known as tauopathies. Western blotting (WB) has been widely employed to determine Tau levels in neurological disease models. However, Tau quantification by WB should be interpreted with care, as this approach has been recognized as prone to produce artifactual results if not properly performed. In this study, our goal was to evaluate the influence of a freeze-and-thaw cycle, a common procedure preceding WB, to the integrity of Tau in brain homogenates from rats, 3xTg-AD mice and human samples. Homogenates were prepared in ice-cold RIPA buffer supplemented with protease/phosphatase inhibitors. Immediately after centrifugation, an aliquot of the extracts was analyzed via WB to quantify total and phosphorylated Tau levels. The remaining aliquots were stored for at least 2 weeks at either -20{degrees}C or -80{degrees}C and then subjected to WB. Extracts from rodent brains submitted to freeze-and-thaw presented a ~25 kDa fragment immunoreactive to anti-Tau antibodies. An in-gel digestion followed by mass spectrometry analysis in excised bands revealed this ~25 kDa species corresponds to a Tau fragment. Freeze-and-thaw-induced Tau proteolysis was detected even when extracts were stored at -80{degrees}C. This phenomenon was not observed in human samples at any storage condition tested. Based on these findings, we strongly recommend the use of fresh extracts of brain samples in molecular analysis of Tau levels in rodents.

biochemistry↗

bioRxiv2018

HIV-1 Protease Uses Bi-Specific S2/S2’ Subsites To Optimize Cleavage of Two Classes of Target Sites

Retroviral proteases (PR) have a unique specificity that allows cleavage of sites with or without a P1 proline. A P1 proline is required at the MA/CA cleavage site due to its role in a post-cleavage conformational change in the capsid protein. However, the HIV-1 PR prefers to have large hydrophobic amino acids flanking the scissile bond, suggesting PR recognizes two different classes of substrate sequences. We analyzed the cleavage rate of over 150 iterations of six different HIV-1 cleavage sites to explore rate determinants of cleavage. We found that cleavage rates are strongly influenced by the two amino acids flanking the amino acids at the scissile bond (P2-P1/P1-P2), with two complementary sets of rules. When P1 is proline, the P2 side chain interacts with a polar region in the S2 subsite of the PR, while the P2 amino acid interacts with a hydrophobic region of the S2 subsite. When P1 is not proline, the orientations of the P2 and P2 side chains with respect to the scissile bond are reversed; P2 residues interact with a hydrophobic face of the S2 subsite while the P2 amino acid usually engages hydrophilic amino acids in the S2 subsite. These results reveal that the HIV-1 PR has evolved bi-functional S2 and S2 subsites to accommodate the steric effects imposed by a P1 proline on the orientation of P2 and P2 substrate side chains. These results also suggest a new strategy for inhibitor design to engage the multiple specificities in these subsites.

biochemistry↗

bioRxiv2018

Structural basis for independent origins of new catalytic machineries in plant AAAD proteins

Radiation of the plant pyridoxal 5-phosphate (PLP)-dependent aromatic L-amino acid decarboxylase (AAAD) family has yielded an array of paralogous enzymes exhibiting divergent substrate preferences and catalytic mechanisms. Plant AAADs catalyze either the decarboxylation or decarboxylation-dependent oxidative deamination of aromatic L-amino acids to produce aromatic monoamines or aromatic acetaldehydes, respectively. These compounds serve as key precursors for the biosynthesis of several important classes of plant natural products, including indole alkaloids, benzylisoquinoline alkaloids, hydroxycinnamic acid amides, phenylacetaldehyde-derived floral volatiles, and tyrosol derivatives. Here, we present the crystal structures of four functionally distinct plant AAAD paralogs. Through structural and functional analyses, we identify variable structural features of the substrate-binding pocket that underlie the divergent evolution of substrate selectivity toward indole, phenyl, or hydroxyphenyl amino acids in plant AAADs. Moreover, we describe two mechanistic classes of independently arising mutations in AAAD paralogs leading to the convergent evolution of the derived aldehyde synthase activity. Applying knowledge learned from this study, we successfully engineered a shortened benzylisoquinoline alkaloid pathway to produce (S)-norcoclaurine in yeast. This work highlights the pliability of the AAAD fold that allows change of substrate selectivity and access to alternative catalytic mechanisms with only a few mutations. SignificancePlants biosynthesize their own proteinogenic aromatic L-amino acids, namely L-phenylalanine, L-tyrosine and L-tryptophan, not only for building proteins but also for the production of a plethora of aromatic-amino-acid-derived natural products. Pyridoxal 5-phosphate (PLP)-dependent aromatic L-amino acid decarboxylase (AAAD) family enzymes play important roles in channeling various aromatic L-amino acids into diverse downstream specialized metabolic pathways. Through comparative structural analysis of four functionally divergent plant AAAD proteins together with biochemical characterization and molecular dynamics simulations, we reveal the structural and mechanistic basis for the rich divergent and convergent evolutionary development within the plant AAAD family. Knowledge learned from this study aids our ability to engineer high-value aromatic-L-amino-acid-derived natural product biosynthesis in heterologous chassis organisms.

biochemistry↗

bioRxiv2018

An inhibitor-interaction intermediate of HIV-1 protease, revealed by Isothermal Titration Calorimetry and NMR spectroscopy

Some of drug-resistant mutants of HIV-1 protease (PR), such as a clinically-relevant drug- resistant PR mutant (Flap+(I54V)) containing L10I, G48V, I54V and V82A mutations, produce significant changes in the balance between entropy and enthalpy of the drug-PR interactions, compared to the wild-type (WT) PR. Here, to gain a comprehensive understanding of the entropy-enthalpy compensation effects, we compared nuclear magnetic resonance (NMR), fluorescence spectroscopy and isothermal titration calorimetry (ITC) data of a WT PR with Flap+(I54V) and related mutants: (1) Flap+(I54V); (2) Flap+(I54A) which evolves from Flap+(I54V) in the continued presence of inhibitor yet does not exhibit entropy-enthalpy compensation; and (3) Flap+(I54), a control mutant that contains only L10I, G48V and V82A mutations. Our data indicate that WT and Flap+(I54A) show enthalpy-driven inhibitor-interaction, while Flap+(I54) and Flap+(I54V) exhibit entropy-driven inhibitor interaction. Interestingly, Flap+(I54A) exhibited significantly slower heat flow in the competitive ITC experiment with a strong binder, darunavir, and a weak binder, acetyl-pepstatin, but did not exhibit such slow heat flow in the direct inhibitor-titration experiments. NMR confirmed replacement of the weak binder by the strong binder in a competitive manner. This difference in the heat flow of the competitive binding experiment compared to the direct experiment can only be explained by assuming an inhibitor-bound intermediate pathway. A similar, but attenuated, tendency for slow heat flow was also detected in the competitive experiment with WT. Overall, our data suggests that an inhibitor-bound intermediate affects the entropy-enthalpy compensation of inhibitor-PR interaction.

biochemistry↗

bioRxiv2018

Crystal Structure of a Natural Light-Gated Anion Channelrhodopsin

The anion channelrhodopsin GtACR1 from the alga Guillardia theta is a potent neuron-inhibiting optogenetics tool. Presented here, its X-ray structure at 2.9 [A] reveals a tunnel traversing the protein from its extracellular surface to a large cytoplasmic cavity. The tunnel is lined primarily by small polar and aliphatic residues essential for anion conductance. A disulfide-immobilized extracellular cap facilitates channel closing and the ion path is blocked mid-membrane by its photoactive retinylidene chromophore and further by a cytoplasmic side constriction. The structure also reveals a novel photoactive site configuration that maintains the retinylidene Schiff base protonated when the channel is open. These findings suggest a new channelrhodopsin mechanism, in which the Schiff base not only controls gating, but also serves as a direct mediator for anion flux.

biochemistry↗

bioRxiv2018

Introduction of Kinesin-13 family specific residues increases the microtubule end residence time of a Kinesin-1

Kinesins that regulate microtubule dynamics, such as the Kinesin-13 MCAK, require the ability to recognise the microtubule end. All microtubule regulating kinesins studied to date have this ability and thus exhibit increased microtubule end residence times. In contrast, purely translocating kinesins such as Kinesin-1 do not need to recognise the microtubule end for their function. The residues K524, E525 and R528 in the 4 helix of the Kinesin-13, MCAK, are critical for microtubule end recognition. Here, we show that introducing these Kinesin-13 family-specific residues into a Kinesin-1 increases its microtubule-end residence time up to 4-fold. However, this increase in end residence is not sufficient to confer microtubule depolymerisation activity to a Kinesin-1.\n\nSignificance StatementThe introduction of Kinesin-13 family specific residues from the 4 helix of the microtubule depolymerising kinesin, MCAK, into a Kinesin-1, increases the microtubule-end residence time between 2 and 4-fold. This demonstrates both the significance of these residues in modulating microtubule end residence, and the capacity to tune kinesin function by modifying the microtubule binding face of the motor domain using protein engineering. Increasing the microtubule end residence time in this way is not sufficient to confer depolymerisation activity to a Kinesin-1. Thus, indicating that the ability to recognise and reside at the microtubule end is not the only determinant of depolymerisation activity. The Kinesin-13 motor domain may also possess the ability to actively break interactions between tubulin subunits.

biochemistry↗

bioRxiv2018

Structural instability of IκB kinase β promotes autophagic degradation through enhancement of Keap1 binding

IKK{beta}, an essential kinase of NF-[kcy]B signaling, is composed of an N-terminal kinase domain (KD) and a C-terminal scaffolding domain, containing a ubiquitin-like domain (ULD). The Hsp90 chaperon has special responsibility for folding of protein kinases including IKK{beta}. Here, we found that Hsp90 inhibition induced IKK{beta} degradation, which is partially mediated by Keap1. Geldanamycin (GA), a Hsp90 inhibitor, enhances association of IKK{beta} with Keap1 through the binding site in KD, and translocates IKK{beta} to detergent-insoluble fractions leading its autophagic degradation. An electrophile tBHQ suppressed Keap1-mediated proteasomal Nrf2 degradation but not autophagic IKK{beta} degradation. Substitution mutation of Leu353 to Ala in the ULD destabilizes IKK{beta}, enhances its association with Keap1, translocates it to detergent-insoluble fractions, and causes its autophagic degradation. These results suggest that Keap1 is involved in the degradation of structural destabilized IKK{beta} and negative regulation of NF-[kcy]B under proteotoxic stress.

biochemistry↗

bioRxiv2018

CHEMOMETRIC APPROACHES FOR DEVELOPING INFRARED NANOSENSORS TO IMAGE ANTHRACYCLINES

Generation, identification, and validation of optical probes to image molecular targets in a biological milieu remains a challenge. Synthetic molecular recognition approaches leveraging the intrinsic near-infrared fluorescence of single-walled carbon nanotubes is a promising approach for chronic biochemical imaging in tissues. However, generation of nanosensors for selective imaging of molecular targets requires a heuristic approach. Here, we present a chemometric platform for rapidly screening libraries of candidate single-walled carbon nanotube nanosensors against biochemical analytes to quantify fluorescence response to small molecules including vitamins, neurotransmitters, and chemotherapeutics. We further show this approach can be leveraged to identify biochemical analytes that selectively modulate the intrinsic near-infrared fluorescence of candidate nanosensors. Chemometric analysis thus enables identification of nanosensor-analyte hits and also nanosensor fluorescence signaling modalities such as wavelength-shifts that are optimal for translation to biological imaging. Through this approach, we identify and characterize a nanosensor for the chemotherapeutic anthracycline doxorubicin, which provides an up to 17 nm fluorescence red-shift and exhibits an 8 {micro}M limit of detection, compatible with peak circulatory concentrations of doxorubicin common in therapeutic administration. We demonstrate selectivity of this nanosensor over dacarbazine, a chemotherapeutic commonly co-injected with DOX. Lastly, we demonstrate nanosensor tissue compatibility for imaging of doxorubicin in muscle tissue by incorporating nanosensors into the mouse hindlimb and measuring nanosensor response to exogenous DOX administration. Our results motivate chemometric approaches to nanosensor discovery for chronic imaging of drug partitioning into tissues and towards real-time monitoring of drug accumulation.

biochemistry↗

bioRxiv2018

Accurate Determination of Human CPR Conformational Equilibrium by smFRET using Dual Orthogonal Non-Canonical Amino Acid Labeling

Conjugation of fluorescent dyes to proteins - a prerequisite for the study of conformational dynamics by single molecule Forster resonance energy transfer (smFRET) - can lead to substantial changes of the dyes photophysical properties, ultimately biasing the quantitative determination of inter-dye distances. In particular the popular cyanine dyes and their derivatives, which are by far the most used dyes in smFRET experiments, exhibit such behavior. To overcome this, a general strategy to site-specifically equip proteins with FRET pairs by chemo-selective reactions using two distinct non-canonical amino acids simultaneously incorporated through genetic code expansion in Escherichia coli was developed. Applied to human NADPH- cytochrome P450 reductase (CPR), the importance of homogenously labeled samples for accurate determination of FRET efficiencies was demonstrated. Furthermore, the effect of NADP+ on the ionic strength dependent modulation of the conformational equilibrium of CPR was unveiled. Given its generality and accuracy, the presented methodology establishes a new benchmark to decipher complex molecular dynamics on single molecules.

biochemistry↗

bioRxiv2018

Temperature-responsive competitive inhibition of CRISPR-Cas9

CRISPR-Cas immune systems utilize RNA-guided nucleases to protect bacteria from bacteriophage infection. Bacteriophages have in turn evolved inhibitory anti-CRISPR (Acr) proteins, including six inhibitors (AcrIIA1-6) that can block DNA cutting and genome editing by type II-A CRISPR-Cas9 enzymes. We show here that AcrIIA2 and its homologue, AcrIIA2b, prevent Cas9 binding to DNA by occluding protein residues required for DNA binding. Cryo-EM-determined structures of AcrIIA2 or AcrIIA2b bound to S. pyogenes Cas9 reveal a mode of competitive inhibition of DNA binding that is distinct from other known Acrs. Differences in the temperature dependence of Cas9 inhibition by AcrIIA2 and AcrIIA2b arise from differences in both inhibitor structure and the local inhibitor-binding environment on Cas9. These findings expand the natural toolbox for regulating CRISPR-Cas9 genome editing temporally, spatially and conditionally.

biochemistry↗

bioRxiv2018

Human CtIP forms a tetrameric dumbbell-shaped particle which binds and bridges complex DNA end structures for double-strand break repair

CtIP is involved in the resection of double-stranded DNA breaks during the S and G2 phases of the cell cycle for repair by homologous recombination. Acting in concert with the MRN complex, it plays a particularly important role in handling complex DNA end structures by localised nucleolytic processing of DNA termini in preparation for longer range resection. Here we show that human CtIP is a tetrameric protein adopting a dumbbell architecture in which DNA binding domains are connected by long coiled-coils. The protein complex binds two short DNA duplexes with high affinity and bridges DNA molecules in trans. DNA binding is potentiated by dephosphorylation and is not specific for DNA end structures per se. However, the affinity for linear DNA molecules is increased if the DNA terminates with more complex structures including forked ssDNA overhangs and model nucleoprotein conjugates. This work provides a biochemical and structural basis for the function of CtIP at complex DNA breaks.

biochemistry↗