DNA Substrate Binding Research Articles

Revised mechanism of hydroxyurea-induced cell cycle arrest and an improved alternative

Replication stress describes endogenous and exogenous challenges to DNA replication in the S-phase. Stress during this critical process causes helicase-polymerase decoupling at replication forks, triggering the S-phase checkpoint, which orchestrates global replication fork stalling and delayed entry into G2. The replication stressor most often used to induce the checkpoint response in yeast is hydroxyurea (HU), a clinically used chemotherapeutic. The primary mechanism of S-phase checkpoint activation by HU has thus far been considered to be a reduction of deoxynucleotide triphosphate synthesis by inhibition of ribonucleotide reductase (RNR), leading to helicase-polymerase decoupling and subsequent activation of the checkpoint, facilitated by the replisome-associated mediator Mrc1. In contrast, we observe that HU causes cell cycle arrest in budding yeast independent of both the Mrc1-mediated replication checkpoint response and the Psk1-Mrc1 oxidative signaling pathway. We demonstrate a direct relationship between HU incubation and reactive oxygen species (ROS) production in yeast and human cells and show that antioxidants restore growth of yeast in HU. We further observe that ROS strongly inhibits the in vitro polymerase activity of replicative polymerases (Pols), Pol α, Pol δ, and Pol ε, causing polymerase complex dissociation and subsequent loss of DNA substrate binding, likely through oxidation of their integral iron-sulfur (Fe-S) clusters. Finally, we present "RNR-deg," a genetically engineered alternative to HU in yeast with greatly increased specificity of RNR inhibition, allowing researchers to achieve fast, nontoxic, and more readily reversible checkpoint activation compared to HU, avoiding harmful ROS generation and associated downstream cellular effects that may confound interpretation of results.

Structure of Escherichia coli exonuclease VII

Exonuclease VII (ExoVII) is a ubiquitous bacterial nuclease. Encoded by the xseA and xseB genes, ExoVII participates in multiple nucleic acid-dependent pathways including the processing of multicopy single-stranded DNA and the repair of covalent DNA-protein crosslinks (DPCs). Although many biochemical properties of ExoVII have been defined, little is known about its structure/function relationships. Here, we use cryoelectron microscopy (cryoEM) to determine that Escherichia coli ExoVII comprises a highly elongated XseA4·XseB24 holo-complex. Each XseA subunit dimerizes through a central extended α-helical segment decorated by six XseB subunits and a C-terminal, domain-swapped β-barrel element; two XseA2·XseB12 subcomplexes further associate using N-terminal OB (oligonucleotide/oligosaccharide-binding) folds and catalytic domains to form a spindle-shaped, catenated octaicosamer. The catalytic domains of XseA, which adopt a nuclease fold related to 3-dehydroquinate dehydratases, are sequestered in the center of the complex and accessible only through large pores formed between XseA tetramers. The architectural organization of ExoVII, combined with biochemical studies, indicate that substrate selectivity is controlled by steric access to its nuclease elements and that tetramer dissociation results from substrate DNA binding. Despite a lack of sequence and fold homology, the physical organization of ExoVII is reminiscent of Mre11·Rad50/SbcCD ATP (adenosine triphosphate)-dependent nucleases used in the repair of double-stranded DNA breaks, including those formed by DPCs through aberrant topoisomerase activity, suggesting that there may have been convergent evolutionary pressure to contend with such damage events.

PreAcrs: a machine learning framework for identifying anti-CRISPR proteins

BackgroundAnti-CRISPR proteins are potent modulators that inhibit the CRISPR-Cas immunity system and have huge potential in gene editing and gene therapy as a genome-editing tool. Extensive studies have shown that anti-CRISPR proteins are essential for modifying endogenous genes, promoting the RNA-guided binding and cleavage of DNA or RNA substrates. In recent years, identifying and characterizing anti-CRISPR proteins has become a hot and significant research topic in bioinformatics. However, as most anti-CRISPR proteins fall short in sharing similarities to those currently known, traditional screening methods are time-consuming and inefficient. Machine learning methods could fill this gap with powerful predictive capability and provide a new perspective for anti-CRISPR protein identification.ResultsHere, we present a novel machine learning ensemble predictor, called PreAcrs, to identify anti-CRISPR proteins from protein sequences directly. Three features and eight different machine learning algorithms were used to train PreAcrs. PreAcrs outperformed other existing methods and significantly improved the prediction accuracy for identifying anti-CRISPR proteins.ConclusionsIn summary, the PreAcrs predictor achieved a competitive performance for predicting new anti-CRISPR proteins in terms of accuracy and robustness. We anticipate PreAcrs will be a valuable tool for researchers to speed up the research process. The source code is available at: https://github.com/Lyn-666/anti_CRISPR.git.

Structural insight into replicative helicase loading in Escherichia coli.

DNA replication is an essential, precisely regulated process that occurs once in a cell cycle. In the Gram-negative bacterium Escherichia coli, the replicative helicase EcDnaB and the helicase loader EcDnaC play key roles in the initiation step at the replication origin, oriC. EcDnaB and EcDnaC form a heterododecamer, in which hexameric EcDnaB is bound to hexameric EcDnaC. Using genetic, biochemical and structural biology approaches, many groups have probed the mechanism of replicative helicase loading, using helicases and helicase loaders from various species. Recent X-ray crystallography and cryogenic electron microscopy (cryo-EM) structural studies of the EcDnaB-EcDnaC complex revealed that the interaction of DnaC with DnaB triggers distortion accumulation on the closed ring of hexameric DnaB, inducing DnaB subunits to adopt the open helical form for replication progression. The high-resolution crystal structure of the DnaB-DnaC complex solved by Nagata et al. contributed to a better understanding of the conformational rearrangement of the DnaB ring. In addition to the structural alterations in DnaB subunits by DnaC, the binding of single-stranded DNA (ssDNA) substrates alters the ATP- and ADP-bound forms of DnaB and DnaC. These studies have proposed mechanisms by which DnaC regulates helicase loading onto ssDNA.

Nse5/6 inhibits the Smc5/6 ATPase and modulates DNA substrate binding

Eukaryotic cells employ three SMC (structural maintenance of chromosomes) complexes to control DNA folding and topology. The Smc5/6 complex plays roles in DNA repair and in preventing the accumulation of deleterious DNA junctions. To elucidate how specific features of Smc5/6 govern these functions, we reconstituted the yeast holo‐complex. We found that the Nse5/6 sub‐complex strongly inhibited the Smc5/6 ATPase by preventing productive ATP binding. This inhibition was relieved by plasmid DNA binding but not by short linear DNA, while opposing effects were observed without Nse5/6. We uncovered two binding sites for Nse5/6 on Smc5/6, based on an Nse5/6 crystal structure and cross‐linking mass spectrometry data. One binding site is located at the Smc5/6 arms and one at the heads, the latter likely exerting inhibitory effects on ATP hydrolysis. Cysteine cross‐linking demonstrated that the interaction with Nse5/6 anchored the ATPase domains in a non‐productive state, which was destabilized by ATP and DNA. Under similar conditions, the Nse4/3/1 module detached from the ATPase. Altogether, we show how DNA substrate selection is modulated by direct inhibition of the Smc5/6 ATPase by Nse5/6.

Structural and Functional Characterization of a Unique AP Endonuclease From Deinococcus radiodurans.

Various endogenous and exogenous agents cause DNA damage, including apurinic/apyrimidinic (AP) sites. Due to their cytotoxic effects, AP sites are usually cleaved by AP endonuclease through the base excision repair (BER) pathway. Deinococcus radiodurans, an extraordinary radiation-resistant bacterium, is known as an ideal model organism for elucidating DNA repair processes. Here, we have investigated a unique AP endonuclease (DrXth) from D. radiodurans and found that it possesses AP endonuclease, 3′-phosphodiesterase, 3′-phosphatase, and 3′–5′ exonuclease but has no nucleotide incision repair (NIR) activity. We also found that Mg2+ and Mn2+ were the preferred divalent metals for endonuclease and exonuclease activities, respectively. In addition, DrXth were crystallized and the crystals diffracted to 1.5 Å. Structural and biochemical analyses demonstrated that residue Gly198 is the key residue involved in the substrate DNA binding and cleavage. Deletion of the drxth gene in D. radiodurans caused elevated sensitivity to DNA damage agents and increased spontaneous mutation frequency. Overall, our results indicate that DrXth is an important AP endonuclease involved in BER pathway and functions in conjunction with other DNA repair enzymes to maintain the genome stability.

Observation of processive telomerase catalysis using high-resolution optical tweezers.

Telomere maintenance by telomerase is essential for continuous proliferation of human cells and is vital for the survival of stem cells and 90% of cancer cells. To compensate for telomeric DNA lost during DNA replication, telomerase processively adds GGTTAG repeats to chromosome ends by copying the template region within its RNA subunit. Between repeat additions, the RNA template must be recycled. How telomerase remains associated with substrate DNA during this critical translocation step remains unknown. Using a newly developed single-molecule telomerase activity assay utilizing high-resolution optical tweezers, we demonstrate that stable substrate DNA binding at an anchor site within telomerase facilitates the processive synthesis of telomeric repeats. The product DNA synthesized by telomerase can be recaptured by the anchor site or fold into G-quadruplex structures. Our results provide detailed mechanistic insights into telomerase catalysis, a process of critical importance in aging and cancer.

Mechanism of Filamentation-Induced Allosteric Activation of the SgrAI Endonuclease

Filament formation by enzymes is increasingly recognized as an important phenomenon with potentially unique regulatory properties and biological roles. SgrAI is an allosterically regulated type II restriction endonuclease that forms filaments with enhanced DNA cleavage activity and altered sequence specificity. Here, we present the cryoelectron microscopy (cryo-EM) structure of the filament of SgrAI in its activated configuration. The structural data illuminate the mechanistic origin of hyperaccelerated DNA cleavage activity and suggests how indirect DNA sequence readout within filamentous SgrAI may enable recognition of substantially more nucleotide sequences than its low-activity form, thereby altering and partially relaxing its DNA sequence specificity. Together, substrate DNA binding, indirect readout, and filamentation simultaneously enhance SgrAI's catalytic activity and modulate substrate preference. This unusual enzyme mechanism may have evolved to perform the specialized functions of bacterial innate immunity in rapid defense against invading phage DNA without causing damage to the host DNA.

Thermodynamics of the DNA Repair Process by Endonuclease VIII

In the present work, a thermodynamic analysis of the interaction between endonuclease VIII (Endo VIII) and model DNA substrates containing damaged nucleotides, such as 5,6-dihydrouridine and 2-hydroxymethyl-3-hydroxytetrahydrofuran (F-site), was performed. The changes in the fluorescence intensity of the 1,3-diaza-2-oxophenoxazine (tC°) residue located in the complementary chain opposite to the specific site were recorded in the course of the enzyme-substrate interaction. The kinetics was analyzed by the stopped-flow method at different temperatures. The changes of standard Gibbs free energy, enthalpy, and entropy of sequential steps of DNA substrate binding, as well as activation enthalpy and entropy for the transition complex formation of the catalytic stage, were calculated. The comparison of the kinetic and thermodynamic data characterizing the conformational transitions of enzyme and DNA in the course of their interaction made it possible to specify the nature of the molecular processes occurring at the stages of substrate binding, recognition of the damaged base, and its removal from DNA.

Microheterogeneity of Topoisomerase IA/IB and Their DNA-Bound States.

Topoisomerases are important complex enzymes that modulate DNA topology to maintain chromosome superstructure and integrity. These enzymes are involved in many cellular processes that resolve specific DNA superstructures and intermediates. The low abundance combined with the biological heterogeneity of relevant intermediates of topoisomerases makes their structural information not readily accessible using traditional structural biology tools (e.g., NMR and X-ray crystallography). In the present work, a second-generation trapped ion mobility spectrometry–mass spectrometry (TIMS–MS) was used to study Escherichia coli topoisomerase IA (EcTopIA) and variola virus topoisomerase IB (vTopIB) as well as their complexes with a single-stranded DNA and a stem-loop DNA under native conditions. The higher trapping efficiency and extended mass range of the new, convex TIMS geometry allowed for the separation and identification of multiple conformational states for the two topoisomerases and their DNA complexes. Inspection of the conformational space of EcTopIA and vTopIB in complex with DNA showed that upon DNA binding, the number of conformational states is significantly reduced, suggesting that the DNA binding selects for a narrow range of conformers restricted by the interaction with the DNA substrate. The large microheterogeneity observed for the two DNA binding proteins suggests that they can have multiple biological functions. This work highlights the potential of TIMS–MS for the structural investigations of intrinsically disordered proteins (e.g., DNA binding proteins) as a way to gain a better understanding of the mechanisms involved in DNA substrate recognition, binding, and assembly of the catalytically active enzyme–DNA complex.

Structural insights into DNA degradation by human mitochondrial nuclease MGME1.

Mitochondrial nucleases play important roles in accurate maintenance and correct metabolism of mtDNA, the own genetic materials of mitochondria that are passed exclusively from mother to child. MGME1 is a highly conserved DNase that was discovered recently. Mutations in MGME1-coding gene lead to severe mitochondrial syndromes characterized by external ophthalmoplegia, emaciation, and respiratory failure in humans. Unlike many other nucleases that are distributed in multiple cellular organelles, human MGME1 is a mitochondria-specific nuclease; therefore, it can serve as an ideal target for treating related syndromes. Here, we report one HsMGME1-Mn2+ complex and three different HsMGME1-DNA complex structures. In combination with in vitro cleavage assays, our structures reveal the detailed molecular basis for substrate DNA binding and/or unwinding by HsMGME1. Besides the conserved two-cation-assisted catalytic mechanism, structural analysis of HsMGME1 and comparison with homologous proteins also clarified substrate binding and cleavage directionalities of the DNA double-strand break repair complexes RecBCD and AddAB.

Dynamics of Scabin toxin. A proposal for the binding mode of the DNA substrate.

Scabin is a mono-ADP-ribosyltransferase enzyme and is a putative virulence factor produced by the plant pathogen, Streptomyces scabies. Previously, crystal structures of Scabin were solved in the presence and absence of substrate analogues and inhibitors. Herein, experimental (hydrogen-deuterium exchange), simulated (molecular dynamics), and theoretical (Gaussian Network Modeling) approaches were systematically applied to study the dynamics of apo-Scabin in the context of a Scabin·NAD+·DNA model. MD simulations revealed that the apo-Scabin solution conformation correlates well with the X-ray crystal structure, beyond the conformation of the exposed, mobile regions. In turn, the MD fluctuations correspond with the crystallographic B-factors, with the fluctuations derived from a Gaussian network model, and with the experimental H/D exchange rates. An Essential Dynamics Analysis identified the dynamic aspects of the toxin as a crab-claw-like mechanism of two topological domains, along with coupled deformations of exposed motifs. The “crab-claw” movement resembles the motion of C3-like toxins and emerges as a property of the central β scaffold of catalytic single domain toxins. The exposure and high mobility of the cis side motifs in the Scabin β-core suggest involvement in DNA substrate binding. A ternary Scabin·NAD+·DNA model was produced via an independent docking methodology, where the intermolecular interactions correspond to the region of high mobility identified by dynamics analyses and agree with binding and kinetic data reported for wild-type and Scabin variants. Based on data for the Pierisin-like toxin group, the sequence motif Rβ1–RLa–NLc–STTβ2–WPN–WARTT–(QxE)ARTT emerges as a catalytic signature involved in the enzymatic activity of these DNA-acting toxins. However, these results also show that Scabin possesses a unique DNA-binding motif within the Pierisin-like toxin group.

Molecular Interactions of a DNA Modifying Enzyme APOBEC3F Catalytic Domain with a Single-Stranded DNA

The single-stranded DNA (ssDNA) cytidine deaminase APOBEC3F (A3F) deaminates cytosine (C) to uracil (U) and is a known restriction factor of HIV-1. Its C-terminal catalytic domain (CD2) alone is capable of binding single-stranded nucleic acids and is important for deamination. However, little is known about how the CD2 interacts with ssDNA. Here we report a crystal structure of A3F-CD2 in complex with a 10-nucleotide ssDNA composed of poly-thymine, which reveals a novel positively charged nucleic acid binding site distal to the active center that plays a key role in substrate DNA binding and catalytic activity. Lysine and tyrosine residues within this binding site interact with the ssDNA, and mutating these residues dramatically impairs both ssDNA binding and catalytic activity. This binding site is not conserved in APOBEC3G (A3G), which may explain differences in ssDNA-binding characteristics between A3F-CD2 and A3G-CD2. In addition, we observed an alternative Zn-coordination conformation around the active center. These findings reveal the structural relationships between nucleic acid interactions and catalytic activity of A3F.

Analysis of DNA binding by human factor xeroderma pigmentosum complementation group A (XPA) provides insight into its interactions with nucleotide excision repair substrates

Xeroderma pigmentosum (XP) complementation group A (XPA) is an essential scaffolding protein in the multiprotein nucleotide excision repair (NER) machinery. The interaction of XPA with DNA is a core function of this protein; a number of mutations in the DNA-binding domain (DBD) are associated with XP disease. Although structures of the central globular domain of human XPA and data on binding of DNA substrates have been reported, the structural basis for XPA's DNA-binding activity remains unknown. X-ray crystal structures of the central globular domain of yeast XPA (Rad14) with lesion-containing DNA duplexes have provided valuable insights, but the DNA substrates used for this study do not correspond to the substrates of XPA as it functions within the NER machinery. To better understand the DNA-binding activity of human XPA in NER, we used NMR to investigate the interaction of its DBD with a range of DNA substrates. We found that XPA binds different single-stranded/double-stranded junction DNA substrates with a common surface. Comparisons of our NMR-based mapping of binding residues with the previously reported Rad14-DNA crystal structures revealed similarities and differences in substrate binding between XPA and Rad14. This includes direct evidence for DNA contacts to the residues extending C-terminally from the globular core, which are lacking in the Rad14 construct. Moreover, mutation of the XPA residue corresponding to Phe-262 in Rad14, previously reported as being critical for DNA binding, had only a moderate effect on the DNA-binding activity of XPA. The DNA-binding properties of several disease-associated mutations in the DBD were investigated. These results suggest that for XPA mutants exhibiting altered DNA-binding properties, a correlation exists between the extent of reduction in DNA-binding affinity and the severity of symptoms in XP patients.

Characterization of DNA Substrate Binding to the Phosphatase Domain of the DNA Repair Enzyme Polynucleotide Kinase/Phosphatase.

Polynucleotide kinase/phosphatase (PNKP) is a DNA strand break repair enzyme that uses separate 5' kinase and 3' phosphatase active sites to convert damaged 5'-hydroxyl/3'-phosphate strand termini to ligatable 5'-phosphate/3'-hydroxyl ends. The phosphatase active site has received particular attention as a target of inhibition in cancer therapy development. The phosphatase domain dephosphorylates a range of single- and double-stranded substrates; however, structural studies have shown that the phosphatase catalytic cleft can bind only single-stranded substrates. Here we use a catalytically inactive but structurally intact phosphatase mutant to probe interactions between PNKP and a variety of single- and double-stranded DNA substrates using an electrophoretic mobility shift assay. This work indicates that the phosphatase domain binds 3'-phosphorylated single-stranded DNAs in a manner that is highly dependent on the presence of the 3'-phosphate. Double-stranded substrate binding, in contrast, is not as dependent on the 3'-phosphate. Experiments comparing blunt-end, 3'-overhanging, and frayed-end substrates indicate that the predicted loss of energy due to base pair disruption upon binding of the phosphatase active site is likely balanced by favorable interactions between the liberated complementary strand and PNKP. Comparison of the effects on substrate binding of mutations within the phosphatase active site cleft with mutations in surrounding positively charged surfaces suggests that the surrounding surfaces are important for binding to double-stranded substrates. We further show that while fluorescence polarization methods can detect specific binding of single-stranded DNAs with the phosphatase domain, this method does not detect specific interactions between the PNKP phosphatase and double-stranded substrates.

The kinetic analysis of recognition of the damaged nucleotides by mutant forms of the 8-oxoguanine DNA glycosylase hOGG1

We have investigated the role of Tyr-203, His-270, and Lys-249 amino acid residues from the 8-oxoguanine glycosylase (hOGG1) active site in the process of recognition of 7,8-dihydro-8-oxoguanine (oxoG) damaged nucleotide and in the catalytic stages of enzymatic reaction. The pre-steady state kinetic analysis of conformational transitions of mutant forms of the enzyme and model DNA substrates during the enzymatic process revealed that the studied amino acid residues are involved in the specific binding of DNA substrates. The Tyr-203 is responsible for recognition of the damaged nucleotide; interaction between His-270 and DNA is necessary for the formation of the catalytically active complex with the oxoG-containing DNA. The Lys-249 acts not only as one of the catalytically important amino acids of the active site of the enzyme, but also plays a significant role in the formation of specific enzyme–substrate complex. The present study significantly complements the molecular-kinetic model of the enzymatic reaction and helps to clarify the origin of the high specificity of hOGG1 to oxidized bases in DNA.

Thermodynamic Analysis of Fast Stages of Specific Lesion Recognition by DNA Repair Enzymes.

The methodology of determination of the thermodynamic parameters of fast stages of recognition and cleavage of DNA substrates is described for the enzymatic processes catalyzed by DNA glycosylases Fpg and hOGG1 and AP endonuclease APE1 during base excision repair (BER) pathway. For this purpose, stopped-flow pre-steady-state kinetic analysis of tryptophan fluorescence intensity changes in proteins and fluorophores in DNA substrates was performed at various temperatures. This approach made it possible to determine the changes of standard Gibbs free energy, enthalpy, and entropy of sequential steps of DNA-substrate binding, as well as activation enthalpy and entropy for the transition complex formation of the catalytic stage. The unified features of mechanism for search and recognition of damaged DNA sites by various enzymes of the BER pathway were discovered.

Thermodynamics of Damaged DNA Binding and Catalysis by Human AP Endonuclease 1

Apurinic/apyrimidinic (AP) endonucleases play an important role in DNA repair and initiation of AP site elimination. One of the most topical problems in the field of DNA repair is to understand the mechanism of the enzymatic process involving the human enzyme APE1 that provides recognition of AP sites and efficient cleavage of the 5'-phosphodiester bond. In this study, a thermodynamic analysis of the interaction between APE1 and a DNA substrate containing a stable AP site analog lacking the C1' hydroxyl group (F site) was performed. Based on stopped-flow kinetic data at different temperatures, the steps of DNA binding, catalysis, and DNA product release were characterized. The changes in the standard Gibbs energy, enthalpy, and entropy of sequential specific steps of the repair process were determined. The thermodynamic analysis of the data suggests that the initial step of the DNA substrate binding includes formation of non-specific contacts between the enzyme binding surface and DNA, as well as insertion of the amino acid residues Arg177 and Met270 into the duplex, which results in the removal of "crystalline" water molecules from DNA grooves. The second binding step involves the F site flipping-out process and formation of specific contacts between the enzyme active site and the everted 5'-phosphate-2'-deoxyribose residue. It was shown that non-specific interactions between the binding surfaces of the enzyme and DNA provide the main contribution into the thermodynamic parameters of the DNA product release step.

An intact putative mouse telomerase essential N-terminal domain is necessary for proper telomere maintenance.

Naturally occurring telomerase reverse transcriptase (TERT) isoforms may regulate telomerase activity, and possibly function independently of telomeres to modulate embryonic stem (ES) cell self-renewal and differentiation. We report the characterisation of two novel mouse TERT (mTERT) splice variants, Ins-i1[1-102] (Insi1 for short) and Del-e12[1-40] (Dele12 for short) that have not been previously described. Insi1 represents an in-frame insertion of nucleotides 1-102 from intron 1, encoding a 34 amino acid insertion at amino acid 73. Based on known functions of this region in human and Tetrahymena TERTs, the insertion interrupts the RNA interaction domain 1 implicated in low-affinity RNA binding and the telomerase essential N-terminal domain implicated in DNA substrate interactions. Dele12 contains a 40 nucleotide deletion of exon 12 which generates a premature stop codon, and possible protein lacking the C-terminus. We found Insi1 expressed in adult mouse brain and kidney and Dele12 expressed in adult mouse ovary. Dele12 was inactive in vitro and in mTERT(-/-) ES cells and Insi1 retained 26-48% of telomerase activity reconstituted by wild-type mTERT in vitro and in mTERT(-/-) ES cells. The Insi1 variant exhibited reduced DNA substrate binding in vitro and both variants exhibited a reduction in binding the telomerase RNA, mTR, when expressed in mTERT(-/-) ES cells. Stable expression of Dele12 in the mouse fibroblast CB17 cell line inhibited telomerase activity and slowed cell growth, suggesting a potential dominant-negative effect. Levels of signal-free ends, representing short telomeres, and end-to-end fusions were higher in mTERT(-/-) ES cells expressing mTERT-Insi1 and mTERT-Dele12, compared with levels observed in mTERT(-/-) ES cells expressing wild-type mTERT. In addition, in mTERT(-/-) cells expressing mTERT-Insi1, we observed chromosomes that were products of repeated breakage-bridge-fusion cycles and other telomere dysfunction-related aberrations. An intact mTERT N-terminus which contributes to mTR binding, DNA binding and telomerase activity is necessary for elongation of short telomeres and the maintenance of functional telomeres. It is reasonable to speculate that relative levels of mTERT-Insi1 may regulate telomere function in specific tissues.

The All-Alpha Domains of Coupling Proteins from the Agrobacterium tumefaciens VirB/VirD4 and Enterococcus faecalis pCF10-Encoded Type IV Secretion Systems Confer Specificity to Binding of Cognate DNA Substrates.

Bacterial type IV coupling proteins (T4CPs) bind and mediate the delivery of DNA substrates through associated type IV secretion systems (T4SSs). T4CPs consist of a transmembrane domain, a conserved nucleotide-binding domain (NBD), and a sequence-variable helical bundle called the all-alpha domain (AAD). In the T4CP structural prototype, plasmid R388-encoded TrwB, the NBD assembles as a homohexamer resembling RecA and DNA ring helicases, and the AAD, which sits at the channel entrance of the homohexamer, is structurally similar to N-terminal domain 1 of recombinase XerD. Here, we defined the contributions of AADs from the Agrobacterium tumefaciens VirD4 and Enterococcus faecalis PcfC T4CPs to DNA substrate binding. AAD deletions abolished DNA transfer, whereas production of the AAD in otherwise wild-type donor strains diminished the transfer of cognate but not heterologous substrates. Reciprocal swaps of AADs between PcfC and VirD4 abolished the transfer of cognate DNA substrates, although strikingly, the VirD4-AADPcfC chimera (VirD4 with the PcfC AAD) supported the transfer of a mobilizable plasmid. Purified AADs from both T4CPs bound DNA substrates without sequence preference but specifically bound cognate processing proteins required for cleavage at origin-of-transfer sequences. The soluble domains of VirD4 and PcfC lacking their AADs neither exerted negative dominance in vivo nor specifically bound cognate processing proteins in vitro. Our findings support a model in which the T4CP AADs contribute to DNA substrate selection through binding of associated processing proteins. Furthermore, MOBQ plasmids have evolved a docking mechanism that bypasses the AAD substrate discrimination checkpoint, which might account for their capacity to promiscuously transfer through many different T4SSs. For conjugative transfer of mobile DNA elements, members of the VirD4/TraG/TrwB receptor superfamily bind cognate DNA substrates through mechanisms that are largely undefined. Here, we supply genetic and biochemical evidence that a helical bundle, designated the all-alpha domain (AAD), of T4SS receptors functions as a substrate specificity determinant. We show that AADs from two substrate receptors, Agrobacterium tumefaciens VirD4 and Enterococcus faecalis PcfC, bind DNA without sequence or strand preference but specifically bind the cognate relaxases responsible for nicking and piloting the transferred strand through the T4SS. We propose that interactions of receptor AADs with DNA-processing factors constitute a basis for selective coupling of mobile DNA elements with type IV secretion channels.