Single-stranded DNA-binding Domain Research Articles

PhieDBEs: a DBD-containing, PAM-flexible, high-efficiency dual base editor toolbox with wide targeting scope for use in plants.

Dual base editors (DBEs) enable simultaneous A-to-G and C-to-T conversions, expanding mutation types. However, low editing efficiency and narrow targeting range limit the widespread use of DBEs in plants. The single-strand DNA binding domain of RAD51 DBD can be fused to base editors to improve their editing efficiency. However, it remains unclear how the DBD affects dual base editing performance in plants. In this study, we generated a series of novel plant DBE-SpGn tools consisting of nine constructs using the high-activity cytidine deaminase evoFERNY, adenosine deaminase TadA8e and DBD in various fusion modes with the PAM-flexible Streptococcus pyogenes Cas9 (SpCas9) nickase variant SpGn (with NG-PAM). By analysing their editing performance on 48 targets in rice, we found that DBE-SpGn constructs containing a single DBD and deaminases located at the N-terminus of SpGn exhibited the highest editing efficiencies. Meanwhile, constructs with deaminases located at the C-terminus and/or multiple DBDs failed to function normally and exhibited inhibited editing activity. We identified three particularly high-efficiency dual base editors (C-A-SpGn, C-A-D-SpGn and A-C-D-SpGn), named PhieDBEs (Plant high-efficiency dual base editors), capable of producing efficient dual base conversions within a narrow editing window (M5 ~ M9, M = A/C). The editing efficiency of C-A-D-SpGn was as high as 95.2% at certain target sites, with frequencies of simultaneous C-to-T and A-to-G conversions as high as 81.0%. In summary, PhieDBEs (especially C-A-D-SpGn) can produce diverse mutants and may prove useful in a wide variety of applications, including plant functional genomics, precise mutagenesis, directed evolution and crop genetic improvement, among others.

Development of plant cytosine base editors with the Cas12a system

Base editors of the Cas9 system have been widely used for precise nucleotide substitution in crops. In this study, Cas12a was applied to construct plant cytosine base editors (CBEs). The main elements of Cas12a-CBEs were engineered and their efficiency was evaluated in stably transformed rice cells. An optimized ttCas12a-hyA3Bctd editor, consisting of a LbCas12a variant carrying catalytic inactive D832A and temperature-tolerance D156R double mutations, a truncated human APOBEC3B deaminase, a human RAD51 single-stranded DNA-binding domain, and double copies of UGI, outperformed other Cas12a-CBEs in base editing efficiency. In T0 transgenic rice plants, ttCas12a-hyA3Bctd edited an average of 42.01% and a maximum of 68.75% of lines at six genomic targets. A-to-G conversions were generated in rice by an adenine base editor with a similar architecture to the optimized CBE. Our results provide preliminary evidence for the feasibility of robust and efficient plant Cas12a base editing systems, which could be useful for precise crop breeding.

Expanding the Editing Window of Cytidine Base Editors With the Rad51 DNA-Binding Domain in Rice.

Recently developed base editors provide a powerful tool for plant research and crop improvement. Although a number of different deaminases and Cas proteins have been used to improve base editors the editing efficiency, and editing window are still not optimal. Fusion of a non-sequence-specific single-stranded DNA-binding domain (DBD) from the human Rad51 protein between Cas9 nickase and the deaminase has been reported to dramatically increase the editing efficiency and expand the editing window of base editors in the mammalian cell lines and mouse embryos. We report the use of this strategy in rice, by fusing a rice codon-optimized human Rad51 DBD to the cytidine base editors AncBE4max, AncBE4max-NG, and evoFERNY. Our results show that the addition of Rad51 DBD did not increase editing efficiency in the major editing window but the editing range was expanded in all the three systems. Replacing the human Rad51 DBD with the rice Rad51 DBD homolog also expanded the editing window effectively.

PhieABEs: a PAM-less/free high-efficiency adenine base editor toolbox with wide target scope in plants.

SummaryAdenine base editors (ABEs), which are generally engineered adenosine deaminases and Cas variants, introduce site‐specific A‐to‐G mutations for agronomic trait improvement. However, notably varying editing efficiencies, restrictive requirements for protospacer‐adjacent motifs (PAMs) and a narrow editing window greatly limit their application. Here, we developed a robust high‐efficiency ABE (PhieABE) toolbox for plants by fusing an evolved, highly active form of the adenosine deaminase TadA8e and a single‐stranded DNA‐binding domain (DBD), based on PAM‐less/free Streptococcus pyogenes Cas9 (SpCas9) nickase variants that recognize the PAM NGN (for SpCas9n‐NG and SpGn) or NNN (for SpRYn). By targeting 29 representative targets in rice and assessing the results, we demonstrate that PhieABEs have significantly improved base‐editing activity, expanded target range and broader editing windows compared to the ABE7.10 and general ABE8e systems. Among these PhieABEs, hyper ABE8e‐DBD‐SpRYn (hyABE8e‐SpRY) showed nearly 100% editing efficiency at some tested sites, with a high proportion of homozygous base substitutions in the editing windows and no single guide RNA (sgRNA)‐dependent off‐target changes. The original sgRNA was more compatible with PhieABEs than the evolved sgRNA. In conclusion, the DBD fusion effectively promotes base‐editing efficiency, and this novel PhieABE toolbox should have wide applications in plant functional genomics and crop improvement.

NgAgo possesses guided DNA nicking activity.

Prokaryotic Argonautes (pAgos) have been proposed as more flexible tools for gene-editing as they do not require sequence motifs adjacent to their targets for function, unlike popular CRISPR/Cas systems. One promising pAgo candidate, from the halophilic archaeon Natronobacterium gregoryi (NgAgo), has been the subject of debate regarding its potential in eukaryotic systems. Here, we revisit this enzyme and characterize its function in prokaryotes. NgAgo expresses poorly in non-halophilic hosts with most of the protein being insoluble and inactive even after refolding. However, we report that the soluble fraction does indeed act as a nicking DNA endonuclease. NgAgo shares canonical domains with other catalytically active pAgos but also contains a previously unrecognized single-stranded DNA binding domain (repA). Both repA and the canonical PIWI domains participate in DNA cleavage activities of NgAgo. NgAgo can be programmed with guides to nick targeted DNA in Escherichia coli and in vitro 1 nt outside the 3′ end of the guide sequence. We also found that these endonuclease activities are essential for enhanced NgAgo-guided homologous recombination, or gene-editing, in E. coli. Collectively, our results demonstrate the potential of NgAgo for gene-editing and provide new insight into seemingly contradictory reports.

Purine-rich element binding protein B attenuates the coactivator function of myocardin by a novel molecular mechanism of smooth muscle gene repression.

Myocardin is a potent transcriptional coactivator protein, which functions as the master regulator of vascular smooth muscle cell differentiation. The cofactor activity of myocardin is mediated by its physical interaction with serum response factor, a ubiquitously expressed transactivator that binds to CArG boxes in genes encoding smooth muscle-restricted proteins. Purine-rich element binding protein B (Purβ) represses the transcription of the smooth muscle α-actin gene (Acta2) in fibroblasts and smooth muscle cells by interacting with single-stranded DNA sequences flanking two 5' CArG boxes in the Acta2 promoter. In this study, the ability of Purβ to modulate the cofactor activity of myocardin was investigated using a combination of cellular and biochemical approaches. Results of smooth muscle gene promoter-reporter assays indicated that Purβ specifically inhibits the coactivator function of myocardin in a manner requiring the presence of all three single-stranded DNA binding domains in the Purβ homodimer. DNA binding analyses demonstrated that Purβ interacts with CArG-containing DNA elements with a much lower affinity compared to other purine-rich target sequences present in the Acta2 promoter. Co-immunoprecipitation and DNA pull-down assays revealed that Purβ associates with myocardin and serum response factor when free or bound to duplex DNA containing one or more CArG boxes. Functional analysis of engineered Purβ point mutants identified several amino acid residues essential for suppression of myocardin activity. Collectively, these findings suggest an inhibitory mechanism involving direct protein-protein interaction between the homodimeric Purβ repressor and the myocardin-serum response factor-CArG complex.

Increasing the efficiency and targeting range of cytidine base editors through fusion of a single-stranded DNA-binding protein domain.

Cytidine base editors are powerful genetic tools that catalyse cytidine to thymidine conversion at specific genomic loci, and further improvement of the editing range and efficiency is critical for their broader applications. Through insertion of a non-sequence-specific single-stranded DNA-binding domain from Rad51 protein between Cas9 nickase and the deaminases, serial hyper cytidine base editors were generated with substantially increased activity and an expanded editing window towards the protospacer adjacent motif in both cell lines and mouse embryos. Additionally, hyeA3A-BE4max selectively catalysed cytidine conversion in TC motifs with a broader editing range and much higher activity (up to 257-fold) compared with eA3A-BE4max. Moreover, hyeA3A-BE4max specifically generated a C-to-T conversion without inducing bystander mutations in the haemoglobin gamma gene promoter to mimic a naturally occurring genetic variant for amelioration of β-haemoglobinopathy, suggesting the therapeutic potential of the improved base editors.

Unexpected Evolution of Lesion-Recognition Modules in Eukaryotic NER and Kinetoplast DNA Dynamics Proteins from Bacterial Mobile Elements

SummaryThe provenance of several components of major uniquely eukaryotic molecular machines are increasingly being traced back to prokaryotic biological conflict systems. Here, we demonstrate that the N-terminal single-stranded DNA-binding domain from the anti-restriction protein ArdC, deployed by bacterial mobile elements against their host, was independently acquired twice by eukaryotes, giving rise to the DNA-binding domains of XPC/Rad4 and the Tc-38-like proteins in the stem kinetoplastid. In both instances, the ArdC-N domain tandemly duplicated forming an extensive DNA-binding interface. In XPC/Rad4, the ArdC-N domains (BHDs) also fused to the inactive transglutaminase domain of a peptide-N-glycanase ultimately derived from an archaeal conflict system. Alongside, we delineate several parallel acquisitions from conjugative elements/bacteriophages that gave rise to key components of the kinetoplast DNA (kDNA) replication apparatus. These findings resolve two outstanding questions in eukaryote biology: (1) the origin of the unique DNA lesion-recognition component of NER and (2) origin of the unusual, plasmid-like features of kDNA.

Characterization of single-stranded DNA-binding proteins from the psychrophilic bacteria Desulfotalea psychrophila, Flavobacterium psychrophilum, Psychrobacter arcticus, Psychrobacter cryohalolentis, Psychromonas ingrahamii, Psychroflexus torquis, and Photobacterium profundum.

BackgroundSingle-stranded DNA-binding proteins (SSBs) play essential roles in DNA replication, recombination and repair in Bacteria, Archaea and Eukarya. In recent years, there has been an increasing interest in SSBs, since they find numerous applications in diverse molecular biology and analytical methods.ResultsWe report the characterization of single-stranded DNA-binding proteins from the psychrophilic bacteria Desulfotalea psychrophila (DpsSSB), Flavobacterium psychrophilum (FpsSSB), Psychrobacter arcticus (ParSSB), Psychrobacter cryohalolentis (PcrSSB), Psychromonas ingrahamii (PinSSB), Photobacterium profundum (PprSSB), and Psychroflexus torquis (PtoSSB). The proteins show a high differential within the molecular mass of their monomers and the length of their amino acid sequences. The high level of identity and similarity in respect to the EcoSSB is related to the OB-fold and some of the last amino acid residues. They are functional as homotetramers, with each monomer encoding one single stranded DNA binding domain (OB-fold). The fluorescence titrations indicated that the length of the ssDNA-binding site size is approximately 30 ± 2 nucleotides for the PinSSB, 31 ± 2 nucleotides for the DpsSSB, and 32 ± 2 nucleotides for the ParSSB, PcrSSB, PprSSB and PtoSSB. They also demonstrated that it is salt independent. However, when the ionic strength was changed from low salt to high, binding-mode transition was observed for the FpsSSB, at 31 ± 2 nucleotides and 45 ± 2 nucleotides, respectively. As expected, the SSB proteins under study cause duplex DNA destabilization. The greatest decrease in duplex DNA melting temperature was observed in the presence of the PtoSSB 17°C. The SSBs in question possess relatively high thermostability for proteins derived from cold-adapted bacteria.ConclusionThe results showed that SSB proteins from psychrophilic microorganisms are typical bacterial SSBs and possess relatively high thermostability, offering an attractive alternative to other thermostable SSBs in molecular biology applications.

Structure analysis of FAAP24 reveals single-stranded DNA-binding activity and domain functions in DNA damage response

The FANCM/FAAP24 heterodimer has distinct functions in protecting cells from complex DNA lesions such as interstrand crosslinks. These functions rely on the biochemical activity of FANCM/FAAP24 to recognize and bind to damaged DNA or stalled replication forks. However, the DNA-binding activity of this complex was not clearly defined. We investigated how FAAP24 contributes to the DNA-interacting functions of the FANCM/FAAP24 complex by acquiring the N-terminal and C-terminal solution structures of human FAAP24. Modeling of the FAAP24 structure indicates that FAAP24 may possess a high affinity toward single-stranded DNA (ssDNA). Testing of various FAAP24 mutations in vitro and in vivo validated this prediction derived from structural analyses. We found that the DNA-binding and FANCM-interacting functions of FAAP24, although both require the C-terminal (HhH)2 domain, can be distinguished by segregation-of-function mutations. These results demonstrate dual roles of FAAP24 in DNA damage response against crosslinking lesions, one through the formation of FANCM/FAAP24 heterodimer and the other via its ssDNA-binding activity required in optimized checkpoint activation.

Human single-stranded DNA binding proteins are essential for maintaining genomic stability

The double-stranded conformation of cellular DNA is a central aspect of DNA stabilisation and protection. The helix preserves the genetic code against chemical and enzymatic degradation, metabolic activation, and formation of secondary structures. However, there are various instances where single-stranded DNA is exposed, such as during replication or transcription, in the synthesis of chromosome ends, and following DNA damage. In these instances, single-stranded DNA binding proteins are essential for the sequestration and processing of single-stranded DNA. In order to bind single-stranded DNA, these proteins utilise a characteristic and evolutionary conserved single-stranded DNA-binding domain, the oligonucleotide/oligosaccharide-binding (OB)-fold. In the current review we discuss a subset of these proteins involved in the direct maintenance of genomic stability, an important cellular process in the conservation of cellular viability and prevention of malignant transformation. We discuss the central roles of single-stranded DNA binding proteins from the OB-fold domain family in DNA replication, the restart of stalled replication forks, DNA damage repair, cell cycle-checkpoint activation, and telomere maintenance.

Structures of apo- and ssDNA-bound YdbC from Lactococcus lactis uncover the function of protein domain family DUF2128 and expand the single-stranded DNA-binding domain proteome

Single-stranded DNA (ssDNA) binding proteins are important in basal metabolic pathways for gene transcription, recombination, DNA repair and replication in all domains of life. Their main cellular role is to stabilize melted duplex DNA and protect genomic DNA from degradation. We have uncovered the molecular function of protein domain family domain of unknown function DUF2128 (PF09901) as a novel ssDNA binding domain. This bacterial domain strongly associates into a dimer and presents a highly positively charged surface that is consistent with its function in non-specific ssDNA binding. Lactococcus lactis YdbC is a representative of DUF2128. The solution NMR structures of the 20 kDa apo-YdbC dimer and YdbC:dT19G1 complex were determined. The ssDNA-binding energetics to YdbC were characterized by isothermal titration calorimetry. YdbC shows comparable nanomolar affinities for pyrimidine and mixed oligonucleotides, and the affinity is sufficiently strong to disrupt duplex DNA. In addition, YdbC binds with lower affinity to ssRNA, making it a versatile nucleic acid-binding domain. The DUF2128 family is related to the eukaryotic nuclear protein positive cofactor 4 (PC4) family and to the PUR family both by fold similarity and molecular function.

Whole-Exome Sequencing Studies of Nonhereditary (Sporadic) Parathyroid Adenomas

Genetic abnormalities, such as those of multiple endocrine neoplasia type 1 (MEN1) and Cyclin D1 (CCND1) genes, occur in <50% of nonhereditary (sporadic) parathyroid adenomas. To identify genetic abnormalities in nonhereditary parathyroid adenomas by whole-exome sequence analysis. Whole-exome sequence analysis was performed on parathyroid adenomas and leukocyte DNA samples from 16 postmenopausal women without a family history of parathyroid tumors or MEN1 and in whom primary hyperparathyroidism due to single-gland disease was cured by surgery. Somatic variants confirmed in this discovery set were assessed in 24 other parathyroid adenomas. Over 90% of targeted exons were captured and represented by more than 10 base reads. Analysis identified 212 somatic variants (median eight per tumor; range, 2-110), with the majority being heterozygous nonsynonymous single-nucleotide variants that predicted missense amino acid substitutions. Somatic MEN1 mutations occurred in six of 16 (∼35%) parathyroid adenomas, in association with loss of heterozygosity on chromosome 11. However, no other gene was mutated in more than one tumor. Mutations in several genes that may represent low-frequency driver mutations were identified, including a protection of telomeres 1 (POT1) mutation that resulted in exon skipping and disruption to the single-stranded DNA-binding domain, which may contribute to increased genomic instability and the observed high mutation rate in one tumor. Parathyroid adenomas typically harbor few somatic variants, consistent with their low proliferation rates. MEN1 mutation represents the major driver in sporadic parathyroid tumorigenesis although multiple low-frequency driver mutations likely account for tumors not harboring somatic MEN1 mutations.

Identification of a soybean chloroplast DNA replication origin-binding protein

Replication of chloroplast DNA (ctDNA) in several plants and in Chlamydomonas reinhardii has been shown to occur by a double displacement loop (D-loop) mechanism and potentially also by a rolling circle mechanism. D-loop replication origins have been mapped in several species. Minimal replication origin sequences used as probes identified two potential binding proteins by southwestern blot analysis. A 28 kDa (apparent molecular weight by SDS-PAGE analysis) soybean protein has been isolated by origin sequence-specific DNA affinity chromatography from total chloroplast proteins. Mass spectrometry analysis identified this protein as the product of the soybean C6SY33 gene (accession number ACU14156), which is annotated as encoding a putative uncharacterized protein with a molecular weight of 25,897 Da, very near the observed molecular weight of the purified protein based on gel electrophoresis. Western blot analysis using an antibody against a homologous Arabidopsis protein indicates that this soybean protein is localized specifically in chloroplasts. The soybean protein shares some homology within a single-stranded DNA binding (SSB) domain of E. coli SSB and an Arabidopsis thaliana mitochondrial-localized SSB of about 21 kDa (mtSSB). However, the soybean protein induces a specific electrophoretic mobility shift only when incubated with a double-stranded fragment containing the previously mapped ctDNA replication oriA region. This protein has no electrophoretic mobility shift activity when incubated with single-stranded DNA. In contrast, the Arabidopsis mtSSB causes a mobility shift only with single-stranded DNA but not with the oriA fragment or with control dsDNA of unrelated sequence. These results suggest that the 26 kDa soybean protein is a specific origin binding protein that may be involved in initiation of ctDNA replication.

Isolation and characterization of the core single-stranded DNA-binding domain of purine-rich element binding protein B (Purβ)

Purβ is a single-stranded nucleic acid-binding protein implicated in the injury-induced repression of genes encoding certain muscle-restricted isoforms of actin and myosin expressed in the heart, skeletal muscle, and vasculature. To better understand how the modular arrangement of the primary sequence of Purβ affects the higher order structure and function of the protein, purified recombinant Purβ was subjected to partial proteolysis in an attempt to identify a well-folded truncation protein that retained purine-rich single-stranded DNA-binding activity. Limited tryptic digestion of Purβ liberated a core ∼30 kDa fragment corresponding to residues 29–305 as determined by epitope mapping and mass spectrometry. Size exclusion chromatography indicated that the isolated core fragment retains the ability to self-associate while circular dichroism analysis confirmed that the Purβ core domain is stably folded in the absence of glycine-rich N- and C-terminal sequences. Comparative DNA-binding assays revealed that the isolated core domain interacts with purine-rich cis-elements from the smooth muscle α-actin gene with similar specificity but increased affinity compared to full-length Purβ. These findings suggest that the highly conserved modular repeats of Purβ fold to form a core functional domain, which mediates the specific and high affinity binding of the protein to single-stranded DNA.

Characterization of exceptionally thermostable single-stranded DNA-binding proteins from Thermotoga maritima and Thermotoga neapolitana

BackgroundIn recent years, there has been an increasing interest in SSBs because they find numerous applications in diverse molecular biology and analytical methods.ResultsWe report the characterization of single-stranded DNA binding proteins (SSBs) from the thermophilic bacteria Thermotoga maritima (TmaSSB) and Thermotoga neapolitana (TneSSB). They are the smallest known bacterial SSB proteins, consisting of 141 and 142 amino acid residues with a calculated molecular mass of 16.30 and 16.58 kDa, respectively. The similarity between amino acid sequences of these proteins is very high: 90% identity and 95% similarity. Surprisingly, both TmaSSB and TneSSB possess a quite low sequence similarity to Escherichia coli SSB (36 and 35% identity, 55 and 56% similarity, respectively). They are functional as homotetramers containing one single-stranded DNA binding domain (OB-fold) in each monomer. Agarose mobility assays indicated that the ssDNA-binding site for both proteins is salt independent, and fluorescence spectroscopy resulted in a size of 68 ± 2 nucleotides. The half-lives of TmaSSB and TneSSB were 10 h and 12 h at 100°C, respectively. When analysed by differential scanning microcalorimetry (DSC) the melting temperature (Tm) was 109.3°C and 112.5°C for TmaSSB and TneSSB, respectively.ConclusionThe results showed that TmaSSB and TneSSB are the most thermostable SSB proteins identified to date, offering an attractive alternative to TaqSSB and TthSSB in molecular biology applications, especially with using high temperature e. g. polymerase chain reaction (PCR).

Interaction between tetraplex structure of mouse telomeric DNA and telomeric DNA binding domains of mouse telomere binding protein Pot1

Mouse telomeric DNA sequence, Tel3.5: 5'-AGGG(T TAGGG)(3)-3', has the ability to form antiparallel tetraplex structure in the presence of Na(+). We examined the interaction between the antiparallel tetraplex structure of Tel3.5 and each of two single-stranded telomeric DNA-binding domains of mouse telomere binding protein Pot1, mPot1OB1 and mPot1OB2. The antiparallel tetraplex of Tel3.5 became unfolded upon the interaction with mPot1OB1. On the other hand, no significant structural change of the antiparallel tetraplex of Tel3.5 was observed upon the interaction with mPot1OB2. Considering that the antiparallel tetraplex inhibits telomerase-mediated telomere elongation, we conclude that the ability of mPot1OB1 to unfold the antiparallel tetraplex of the mouse telomeric DNA is required for telomerase-mediated telomere elongation.

Two highly thermostable paralogous single-stranded DNA-binding proteins from Thermoanaerobacter tengcongensis

The thermophilic bacterium Thermoanaerobacter tengcongensis has two single-stranded DNA-binding (SSB) proteins, designated TteSSB2 and TteSSB3. In a SSB complementation assay in Escherichia coli, only TteSSB3 took over the in vivo function of EcoSSB. We have cloned the ssb genes obtained by PCR and have developed E. coli overexpression systems. The TteSSB2 and TteSSB3 consist of 153 and 150 amino acids with a calculated molecular mass of 17.29 and 16.96 kDa, respectively. They are the smallest known bacterial SSB proteins. The homology between amino acid sequences of these proteins is 40% identity and 53% similarity. They are functional as homotetramers, with each monomer encoding one single-stranded DNA binding domain (OB-fold). In fluorescence titrations with poly(dT), both proteins bind single-stranded DNA with a binding site size of about 40 nt per homotetramer. Thermostability with half-life of about 30 s at 95 degrees C makes TteSSB3 similar to the known SSB of Thermus aquaticus (TaqSSB). The TteSSB2 was fully active even after 6 h incubation at 100 degrees C. Here, we show for the first time paralogous thermostable homotetrameric SSBs, which could be an attractive alternative for known homodimeric thermostable SSB proteins in their applications for molecular biology methods and analytical purposes.

Fission Yeast Telomeric DNA Binding Protein Pot1 Has The Ability To Unfold Tetraplex Structure of Telomeric DNA

To understand the regulation mechanism of fission yeast telomeric DNA, we analyzed the structural properties of 4Gn: d(GnTTAC)4 (n = 3, 4) and their interaction with the single-stranded telomeric DNA binding domain of telomere-binding protein Pot1 (Pot1DBD). 4G4 adopted only an antiparallel tetraplex in spite of a mixture of parallel and antiparallel tetraplexes of 4G3. The antiparallel tetraplex of 4G4 became unfolded upon the interaction with Pot1DBD. Considering that the antiparallel tetraplex inhibits telomerase-mediated telomere elongation, we conclude that the ability of Pot1 to unfold the antiparallel tetraplex is required for telomerase-mediated telomere regulation.

Unfolding of tetraplex structure of mouse telomeric DNA by the interaction with mouse telomeric DNA binding protein Pot1

We analyzed the structural properties of mouse telomeric DNA sequence, Tel3.5: 5'-AGGG(TTAGG G)3-3', and nontelomeric DNA sequence, T22: 5'-T22-3', and examined the interaction with a single-stranded telomeric DNA-binding domain of mouse telomeric DNA-binding protein Pot1 (mPot1DBD). T22 did not form any higher-order structure, but Tel3.5 formed antiparallel tetraplex structure in the presence of Na(+). The antiparallel tetraplex of Tel3.5 became unfolded upon the interaction with mPot1DBD. Considering that the antiparallel tetraplex is known to inhibit telomerase-mediated telomere elongation, we conclude that the ability of Pot1 to unfold the antiparallel tetraplex of the telomeric DNA is required for regulation of telomerase-mediated telomere elongation.