Linear Duplex Research Articles

Novel zinc finger nuclease created by combining the Cys 2His 2- and His 4-type zinc finger domains

To improve the DNA hydrolytic activity of the zinc finger nuclease, we have created a new artificial zinc finger nuclease (ZWH4) by connecting two distinct zinc finger domains possessing different types of Zn(II) binding sites (Cys 2His 2- and His 4-types). The overall fold of ZWH4 is similar to that of the wild-type Sp1 zinc finger (Sp1(zf123)) as revealed by circular dichroism spectroscopy. The gel mobility shift assay demonstrated that ZWH4 binds to the GC box DNA, although the DNA-binding affinity is lower than that of Sp1(zf123). Evidently, ZWH4 hydrolyzes the covalently closed circular plasmid DNA (form I) containing the GC box (pBSGC) to the linear duplex DNA (form III) in the presence of a higher concentration (50 times) of the protein than DNA for a 24-h reaction. Of special interest is the fact that the novel mixed zinc finger protein containing the Cys 2His 2- and His 4-type domains was first created. The present results provide the useful information for the redesign strategy of an artificial nuclease based on the zinc finger motif.

Optimization of linear double-stranded RNA for the production of multiple siRNAs targeting hepatitis C virus.

RNA interference (RNAi)-based gene silencing possesses great therapeutic potential for inhibiting replication of human viruses such as hepatitis C virus (HCV). However, one of the putative limitations for its use as a therapy is the rapid emergence of escape variants. These contain deletions or mutations within the viral genome sequences complementary to the small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) being used for treatment. As a potential solution to this problem, we constructed an expression system for duplex RNAs harboring two siRNA units using convergent H1 and U6 Pol III promoters. Here, the length and orientation of the transcript, tandem siRNA (tsiRNA), were optimized to be processed by the intracellular ribonuclease Dicer into functional siRNAs targeting different sequences. Assessment in transfected cells indicates that the length of the tsiRNA duplex (40-42 base pairs) is more critical for both siRNA-producing capacity and gene silencing activity than the orientation of each siRNA unit. In Huh7 cells replicating full-length HCV RNA, expression of length-optimized tsiRNA inhibited viral protein levels as efficiently as a single 21-nucleotide siRNA-expression construct, without affecting miRNA maturation or induction of an interferon response. We verified that the anti-viral activity of tsiRNA was achieved by precise cleavage of two target sites. A distinct advantage of this strategy is that each side of the optimized linear duplex RNA could enter into the Dicer-mediated processing machinery, thus likely providing more equal and efficient production of multiple siRNAs required for reducing the chance of viral escape.

Sequence Specificity of BAL 31 Nuclease for ssDNA Revealed by Synthetic Oligomer Substrates Containing Homopolymeric Guanine Tracts

BackgroundThe extracellular nuclease from Alteromonas espejiana, BAL 31 catalyzes the degradation of single-stranded and linear duplex DNA to 5′-mononucleotides, cleaves negatively supercoiled DNA to the linear duplex form, and cleaves duplex DNA in response to the presence of apurinic sites.Principal FindingsIn this work we demonstrate that BAL 31 activity is affected by the presence of guanine in single-stranded DNA oligomers. Specifically, nuclease activity is shown to be affected by guanine's presence in minimal homopolymeric tracts in the middle of short oligomer substrates and also by its presence at the 3′ end of ten and twenty base oligomers. G•C rich regions in dsDNA are known to cause a decrease in the enzyme's nuclease activity which has been attributed to the increased thermal stability of these regions, thus making it more difficult to unwind the strands required for enzyme access. Our results indicate that an additional phenomenon could be wholly or partly responsible for the loss of activity in these G•C rich regions. Thus the presence of a guanine tract per se impairs the enzyme's functionality, possibly due to the tract's bulky nature and preventing efficient progression through the active site.ConclusionsThis study has revealed that the general purpose BAL 31 nuclease commonly used in molecular genetics exhibits a hithertofore non-characterized degree of substrate specificity with respect to single-stranded DNA (ssDNA) oligomers. Specifically, BAL 31 nuclease activity was found to be affected by the presence of guanine in ssDNA oligomers.

Oligonucleotide Analogues with Integrated Bases and Backbone

AbstractThe thiomethylene‐linked U*[s]U(*) dimers 9–14 were synthesized by substitution of the 6‐[(mesyloxy)methyl]uridine 6 by the thiolate derived from the uridine‐5′‐thioacetates 7 and 8 followed by O‐deprotection. Similarly, the thiomethylene‐linked A*[s]A(*) dimers 9–14 were obtained from the 8‐(bromomethyl)adenosine 15 and the adenosine‐5′‐thioacetates 16 and 17. The concentration dependence of both HN(3) of the U*[s]U(*) dimers 9–14 evidences the formation of linear and cyclic duplexes, and of linear higher associates, C(8 or 6)CH2OH and/or C(5′/II)OH groups favouring the formation of cyclic duplexes. The concentration dependence of the chemical shift for both H2NC(6) of the A*[s]A(*) dimers 18–23 evidences the formation of mainly linear associates. The heteroassociation of U*[s]U(*) to A*[s]A(*) dimers is stronger than the homoassociation of U*[s]U(*) dimers, as evidenced by diluting equimolar mixtures of 11/20 and 13/22. A 1 : 1 stoichiometry of the heteroassociation is evidenced by a Job's plot for 11/20, and by mole ratio plots for 9/18, 10/19, 12/21, 13/22, and 14/23.

Defective Dissociation of a “Slow” RecA Mutant Protein Imparts an Escherichia coli Growth Defect

The RecA and some related proteins possess a simple motif, called (KR)X(KR), that (in RecA) consists of two lysine residues at positions 248 and 250 at the subunit-subunit interface. This study and previous work implicate this RecA motif in the following: (a) catalyzing ATP hydrolysis in trans,(b) coordinating the ATP hydrolytic cycles of adjacent subunits, (c) governing the rate of ATP hydrolysis, and (d) coupling the ATP hydrolysis to work (in this case DNA strand exchange). The conservative K250R mutation leaves RecA nucleoprotein filament formation largely intact. However, ATP hydrolysis is slowed to less than 15% of the wild-type rate. DNA strand exchange is also slowed commensurate with the rate of ATP hydrolysis. The results reinforce the idea of a tight coupling between ATP hydrolysis and DNA strand exchange. When a plasmid-borne RecA K250R protein is expressed in a cell otherwise lacking RecA protein, the growth of the cells is severely curtailed. The slow growth defect is alleviated in cells lacking RecFOR function, suggesting that the defect reflects loading of RecA at stalled replication forks. Suppressors occur as recA gene alterations, and their properties indicate that limited dissociation by RecA K250R confers the slow growth phenotype. Overall, the results suggest that recombinational DNA repair is a common occurrence in cells. RecA protein plays a sufficiently intimate role in the bacterial cell cycle that its properties can limit the growth rate of a bacterial culture.

A Biochemically Defined System for Coding Joint Formation in V(D)J Recombination

V(D)J recombination is one of the most complex DNA transactions in biology. The RAG complex makes double-stranded breaks adjacent to signal sequences and creates hairpin coding ends. Here, we find that the kinase activity of the Artemis:DNA-PKcs complex can be activated by hairpin DNA ends in cis, thereby allowing the hairpins to be nicked and then to undergo processing and joining by nonhomologous DNA end joining. Based on these insights, we have reconstituted many aspects of the antigen receptor diversification of V(D)J recombination by using 13 highly purified polypeptides, thereby permitting variable domain exon assembly by using this fully defined system in accord with the 12/23 rule for this process. The features of the recombination sites created by this system include all of the features observed in vivo (nucleolytic resection, P nucleotides, and N nucleotide addition), indicating that most, if not all, of the end modification enzymes have been identified.

DNA Binding Mode Transitions of Escherichia coli HU αβ: Evidence for Formation of a Bent DNA — Protein Complex on Intact, Linear Duplex DNA

Escherichia coli HU αβ, a major nucleoid-associated protein, organizes chromosomal DNA and facilitates numerous DNA transactions. Using isothermal titration calorimetry, fluorescence resonance energy transfer and a series of DNA lengths (8 bp, 15 bp, 34 bp, 38 bp and 160 bp) we established that HU αβ interacts with duplex DNA using three different nonspecific binding modes. Both the HU to DNA molar ratio ([HU]/[DNA]) and DNA length dictate the dominant HU binding mode. On sufficiently long DNA (≥ 34 bp), at low [HU]/[DNA], HU populates a noncooperative 34 bp binding mode with a binding constant of 2.1 ± 0.4 × 10 6 M − 1 , and a binding enthalpy of + 7.7 ± 0.6 kcal/mol at 15 °C and 0.15 M Na +. With increasing [HU]/[DNA], HU bound in the noncooperative 34 bp mode progressively converts to two cooperative (ω∼20) modes with site sizes of 10 bp and 6 bp. These latter modes exhibit smaller binding constants (1.1 ± 0.2 × 10 5 M − 1 for the 10 bp mode, 3.5 ± 1.4 × 10 4 M − 1 for the 6 bp mode) and binding enthalpies (4.2 ± 0.3 kcal/mol for the 10 bp mode, − 1.6 ± 0.3 kcal/mol for the 6 bp mode). As DNA length increases to 34 bp or more at low [HU]/[DNA], the small modes are replaced by the 34 bp binding mode. Fluorescence resonance energy transfer data demonstrate that the 34 bp mode bends DNA by 143 ± 6° whereas the 6 bp and 10 bp modes do not. The model proposed in this study provides a novel quantitative and comprehensive framework for reconciling previous structural and solution studies of HU, including single molecule (force extension measurement), fluorescence, and electrophoretic gel mobility-shift assays. In particular, it explains how HU condenses or extends DNA depending on the relative concentrations of HU and DNA.

Method to eliminate linear DNA from mixture containing nicked circular, supercoiled, and linear plasmid DNA

Preparations of circular plasmid DNA in either supercoiled or nicked circular form often are contaminated with undesired linear DNA fragments arising from shearing/degradation of chromosomal DNA or linearization of plasmid DNA itself. We report a simple enzymatic method, using a combination of λ exonuclease and RecJ f, for the selective removal of linear DNA from such mixtures. λ exonuclease digests one strand of linear duplex DNA in the 5′ to 3′ direction, whereas RecJ f, a single-strand-specific exonuclease, digests the remaining complementary single strand into mononucleotides. This combination of exonucleases can remove linear DNA from a mixture of linear and supercoiled DNA, leaving the supercoiled form intact. Furthermore, the inability of λ exonuclease to initiate digestion at nicks or gaps enables the removal of undesired linear DNA when nicked circular DNA has been enzymatically prepared from supercoiled DNA. This method can be useful in the preparation of homogeneous circular plasmid DNA required for therapeutic applications and biophysical studies.

Analysis of the DNA Binding Activity of BRCA1 and Its Modulation by the Tumour Suppressor p53

BackgroundThe breast cancer susceptibility protein, BRCA1 functions to maintain the integrity of the genome. The exact mechanisms by which it does so, however, remain unclear. The ability of BRCA1 to bind directly to DNA suggests a more direct role. However, little research has been conducted to understand the functional relevance of this characteristic of BRCA1. In this study we examine the DNA substrate specificity of BRCA1 and how this may be controlled by one of its interacting partners, p53.Methodology/Principal FindingsUsing competition gel retardation assays we have examined the ability of residues 230-534 of BRCA1 to discriminate between different synthetic DNA substrates that mimic those recognised by the DNA damage response i.e. four-way junction DNA, mismatch containing DNA, bulge containing DNA and linear DNA. Of those tested the highest affinity observed was for four-way junction DNA, with a 20 fold excess of each of the other synthetic DNA's unable to compete for any of the bound BRCA1 230-534. We also observed a higher affinity for C∶C and bulge containing DNA compared to linear duplex and G∶T containing DNA. BRCA1 230-534 also has interaction sites for the tumour suppressor p53 and we show that titration of this complex into the DNA binding assays significantly reduces the affinity of BRCA1 for DNA.Conclusions/SignificanceIn this paper we show that BRCA1 can discriminate between different types of DNA damage and we discuss the implications of this with respect to its function in DNA repair. We also show that the DNA binding activity can be inhibited by the tumour suppressor p53 and suggest that this may prevent genome destabilizing events such as HR between non-homologous sequences.

The Human RecQ Helicases, BLM and RECQ1, Display Distinct DNA Substrate Specificities

RecQ helicases maintain chromosome stability by resolving a number of highly specific DNA structures that would otherwise impede the correct transmission of genetic information. Previous studies have shown that two human RecQ helicases, BLM and WRN, have very similar substrate specificities and preferentially unwind noncanonical DNA structures, such as synthetic Holliday junctions and G-quadruplex DNA. Here, we extend this analysis of BLM to include new substrates and have compared the substrate specificity of BLM with that of another human RecQ helicase, RECQ1. Our findings show that RECQ1 has a distinct substrate specificity compared with BLM. In particular, RECQ1 cannot unwind G-quadruplexes or RNA-DNA hybrid structures, even in the presence of the single-stranded binding protein, human replication protein A, that stimulates its DNA helicase activity. Moreover, RECQ1 cannot substitute for BLM in the regression of a model replication fork and is very inefficient in displacing plasmid D-loops lacking a 3'-tail. Conversely, RECQ1, but not BLM, is able to resolve immobile Holliday junction structures lacking an homologous core, even in the absence of human replication protein A. Mutagenesis studies show that the N-terminal region (residues 1-56) of RECQ1 is necessary both for protein oligomerization and for this Holliday junction disruption activity. These results suggest that the N-terminal domain or the higher order oligomer formation promoted by the N terminus is essential for the ability of RECQ1 to disrupt Holliday junctions. Collectively, our findings highlight several differences between the substrate specificities of RECQ1 and BLM (and by inference WRN) and suggest that these enzymes play nonoverlapping functions in cells.

End Modification of a Linear DNA Duplex Enhances NER-Mediated Excision of an Internal Pt(II)-Lesion

The study of DNA repair has been facilitated by the development of extract-based in vitro assay systems and the use of synthetic DNA duplexes that contain site-specific lesions as repair substrates. Unfortunately, exposed DNA termini can be a liability when working in crude cell extracts because they are targets for DNA end-modifying enzymes and binding sites for proteins that recognize DNA termini. In particular, the double-strand break repair protein Ku is an abundant DNA end-binding protein that has been shown to interfere with nucleotide excision repair (NER) in vitro. To facilitate the investigation of NER in whole-cell extracts, we explored ways of modifying the exposed ends of synthetic repair substrates to prevent Ku binding and improve in vitro NER efficiency. Replacement of six contiguous phosphodiester linkages at the 3'-ends of the duplex repair substrate with nuclease-resistant nonionic methylphosphonate linkages resulted in a 280-fold decrease in binding affinity between Ku and the modified duplex. These results are consistent with the published crystal structure of a Ku/DNA complex [Walker et al. (2001) Nature 412, 607-614] and show that the 3'-terminal phosphodiester linkages of linear DNA duplexes are important determinants in DNA end-binding by Ku. Using HeLa whole-cell extracts and a 149-base pair DNA duplex repair substrate, we tested the effects of modification of exposed DNA termini on NER-mediated in vitro excision of a 1,3-GTG-Pt(II) intrastrand cross-link. Methylphosphonate modification at the 3'-ends of the repair substrate resulted in a 1.6-fold increase in excision. Derivatization of the 5'-ends of the duplex with biotin and subsequent conjugation with streptavidin to block Ku binding resulted in a 2.3-fold increase excision. By combining these modifications, we were able to effectively reduce Ku-derived interference of NER excision in vitro and observed a 4.4-fold increase in platinum lesion excision. These modifications are easy to incorporate into synthetic oligonucleotides and may find general utility whenever synthetic linear duplex DNAs are used as substrates to investigate DNA repair in whole-cell extracts.

Multiscale modeling of nucleic acids: Insights into DNA flexibility

The elastic rod theory is used together with all-atom normal mode analysis in implicit solvent to characterize the mechanical flexibility of duplex DNA. The bending, twisting, stretching rigidities extracted from all-atom simulations (on linear duplexes from 60 to 150 base pairs in length and from 94-bp minicircles) are in reasonable agreement with experimental results. We focus on salt concentration and sequence effects on the overall flexibility. Bending persistence lengths are about 20% higher than most experimental estimates, but the transition from low-salt to high-salt behavior is reproduced well, as is the dependence of the stretching modulus on salt (which is opposite to that of bending). CTG and CGG trinucleotide repeats, responsible for several degenerative disorders, are found to be more flexible than random DNA, in agreement with several recent studies, whereas poly(dA).poly(dT) is the stiffest sequence we have encountered. The results suggest that current all-atom potentials, which were parameterized on small molecules and short oligonucleotides, also provide a useful description of duplex DNA at much longer length scales.

Oligonucleotide Analogues with Integrated Bases and Backbone. Part 17

AbstractThe formation of cyclic duplexes (pairing) of known oxymethylene‐linked self‐complementary U*[o]A(*) dinucleosides contrasts with the absence of pairing of the ethylene‐linked U*[ca]A(*) analogues. The origin of this difference, and the expected association of U*[x]A(*) and A*[x]U(*) dinucleosides with x=CH2, O, or S was analysed. According to this analysis, pairing occurs via constitutionally isomeric Watson–Crick, reverse Watson–Crick, Hoogsteen, or reverse Hoogsteen H‐bonded linear duplexes. Each one of them may give rise to three diastereoisomeric cyclic duplexes, and each one of them can adopt three main conformations. The relative stability of all conformers with x=CH2, O, or S were analysed. U*[x]A(*) dinucleosides with x=CH2 do not form stable cyclic duplexes, dinucleosides with x=O may form cyclic duplexes with a gg‐conformation about the C(4′)C(5′) bond, and dinucleosides with x=S may form cyclic duplexes with a gt‐conformation about this bond.The temperature dependence of the chemical shift of HN(3) of the self‐complementary, oxymethylene‐linked U*[o]A(*) dinucleosides 1–6 in CDCl3 in the concentration range of 0.4–50 mM evidences equilibria between the monoplex, mainly linear duplexes, and higher associates for 3, between the monoplex and cyclic duplexes for 6, and between the monoplex, linear, and cyclic duplexes as well as higher associates for 1, 2, 4, and 5.The self‐complementary, thiomethylene‐linked U*[s]A(*) dinucleosides 27–32 and the sequence isomeric A*[s]U(*) analogues 33–38 were prepared by S‐alkylation of the 6‐(mesyloxymethyl)uridine 12 and the 8‐(bromomethyl)adenosine 22. The required thiolates were prepared in situ from the C(5′)‐acetylthio derivatives 9, 15, 19, and 25. The association in CHCl3 of the thiomethylene‐linked dinucleoside analogues was studied by 1H‐NMR and CD spectroscopy, and by vapour‐pressure osmometric determination of the apparent molecular mass. The U*[s]A(*) alcohols 28, 30, and 31 form cyclic duplexes connected by Watson–Crick H‐bonds, while the fully protected dimers 27 and 29 form mainly linear duplexes and higher associates. The diol 32 forms mainly cyclic duplexes in solution and corrugated ribbons in the solid state. The nucleobases of crystalline 32 form reverse Hoogsteen H‐bonds, and the resulting ribbons are cross‐linked by H‐bonds between HOCH2C(8/I) and N(3/I). Among the A*[s]U(*) dimers, only the C(8/I)‐hydroxymethylated 37 forms (mainly) a cyclic duplex, characterized by reverse Hoogsteen base pairing. The dimers 34–36 form mainly linear duplexes and higher associates. Dimers 34 and particularly 38 gelate CHCl3. Temperature‐dependent CD spectra of 28, 30, 31, and 37 evidence π‐stacking in the cyclic duplexes. Base stacking in the particularly strongly associating diol 32 in CHCl3 solution is evidenced by a melting temperature of ca. 2°.

The Phage T4 Protein UvsW Drives Holliday Junction Branch Migration

The phage T4 UvsW protein has been shown to play a crucial role in the switch from origin-dependent to recombination-dependent replication in T4 infections through the unwinding of origin R-loop initiation intermediates. UvsW also functions with UvsX and UvsY to repair damaged DNA through homologous recombination, and, based on genetic evidence, has been proposed to act as a Holliday junction branch migration enzyme. Here we report the purification and characterization of UvsW. Using oligonucleotide-based substrates, we confirm that UvsW unwinds branched DNA substrates, including X and Y structures, but shows little activity in unwinding linear duplex substrates with blunt or single-strand ends. Using a novel Holliday junction-containing substrate, we also demonstrate that UvsW promotes the branch migration of Holliday junctions efficiently through more than 1000 bp of DNA. The ATP hydrolysis-deficient mutant protein, UvsW-K141R, is unable to promote Holliday junction branch migration. However, both UvsW and UvsW-K141R are capable of stabilizing Holliday junctions against spontaneous branch migration when ATP is not present. Using two-dimensional agarose gel electrophoresis we also show that UvsW acts on T4-generated replication intermediates, including Holliday junction-containing X-shaped intermediates and replication fork-shaped intermediates. Taken together, these results strongly support a role for UvsW in the branch migration of Holliday junctions that form during T4 recombination, replication, and repair.

Optical Antisense Imaging of Tumor with Fluorescent DNA Duplexes

Antisense targeting of tumor with fluorescent conjugated DNA oligomers has the potential of improving tumor/normal tissue ratios over that achievable by nuclear antisense imaging. When administered as a linear duplex of two fluorophore-conjugated oligomers arranged in a manner that inhibits fluorescence as the duplex and designed to dissociate only in the presence of the target mRNA, the fluorescence signal should in principle be inhibited everywhere except in the target cell. Optical imaging by fluorescence quenching using linear fluorophore-conjugated oligomers has not been extensively investigated and may not have been previously considered for antisense targeting. We evaluated in cell culture and in KB-G2 tumor bearing nude mice a 25-mer phosphorothioate (PS) anti- mdr1 antisense DNA conjugated with the Cy5.5 emitter on its 3' equivalent end and hybridized as a linear duplex with a shorter 18-mer phosphodiester (PO) complementary DNA (cDNA) with the Black Hole inhibitor BHQ3 on its 5' end. In serum environments, 90% of the DNA25-Cy5.5 fluorescence was inhibited immediately following addition of the cDNA18-BHQ3 and showed only slight loss of inhibition over 24 h at 37 degrees C. As evidence of antisense specific binding, when incubated with the DNA25-Cy5.5/cDNA18-BHQ3 duplex, the fluorescence was lower in KB-31 (Pgp +/-) cells compared to KB-G2 (Pgp++) cells, but when incubated with the control cDNA18-Cy5.5/DNA25-BHQ3 duplex in which the fluorophores were reversed, the fluorescence of both cell types was low. As further evidence of specific binding, the fluorescent intensity of total RNA from KB-G2 cells incubated with the study duplex showed evidence of dissociation and hybridization with the target mRNA. Furthermore, the fluorescence microscopy images of KB-G2 cells incubated with DNA25-Cy5.5 as the singlet or study duplex show that migration in both cases is to the nucleus. The animal studies were performed in mice bearing KB-G2 tumor in one thigh and receiving iv the study or control duplexes. The tumor/normal thigh fluorescence ratio was clearly positive as early as 30 min postinjection in the study mice and reached a maximum at 5 h. By contrast, much lower fluorescence was observed in mice receiving the control duplex at the same dosage. Fluorescence microscope imaging showed that the Cy5.5 fluorescence was much higher in tumor sections from the animal that had received the study rather than control duplex. Thus combining a fluorophore-conjugated antisense DNA with an inhibitor-conjugated shorter complementary cDNA inhibited fluorescence both in cell culture and in tumored animals except in the presence of the target mRNA. This proof of concept investigation of optical antisense targeting therefore suggests that further studies including optimization of this approach are appropriate.

Saccharomyces cerevisiaeHop1 Protein Zinc Finger Motif Binds to the Holliday Junction and Distorts the DNA Structure: Implications for Holliday Junction Migration

Saccharomyces cerevisiae HOP1, which encodes a component of synaptonemal complex, plays an important role in crossing over between homologues. Hop1p contains a zinc finger motif, and substitution of a conserved Cys371 by Ser rendered the hop1 mutant allele defective in sporulation and meiosis. However, the molecular mechanism underlying the function of Hop1 zinc finger motif (ZnF) remains obscure. Here we show that wild-type Hop1 ZnF binds significantly better to the Holliday junction compared with other recombination intermediates. Consequently, the salt titration midpoint for dissociation of the Holliday junction-ZnF complex was higher than the complexes containing flush-ended linear or tailed duplex DNA. Although DNase I footprinting showed that Hop1 ZnF binds to each of the four arms of the junction, KMnO4 probing and 2-aminopurine fluorescence emission data disclosed that it distorts the DNA structure along a pair of symmetrical arms. Molecular modeling studies show that Hop1 ZnF forms a unique zinc-binding fold, reminiscent of the basic helix-loop-helix motif. In the presence of Zn2+, docking studies show that alpha helix 1, which is replete with basic amino acid residues, makes stabilizing contacts with the sugar-phosphate backbone. Structural comparison revealed a striking similarity between RecG wedge domain and Hop1 ZnF motif. We propose that Hop1 ZnF motif plays a key role in the physical monitoring of recombination intermediates and branch migration of the Holliday junction.

Protection of DNA Ends by Telomeric 3′ G-Tail Sequences

The extreme ends of eukaryotic chromosomes contain 3' extensions in the form of single-stranded G-rich repeats, referred to as telomeric 3' G-tails or overhangs. Increasing evidence has suggested that telomeric 3' G-tails can adopt a G-quadruplex conformation both in vitro and in vivo. However, the role of G-quadruplexes on the structure and function of telomeric 3' G-tails remains unclear. In the current study, we showed that the human telomeric 3' G-tail sequence protected the duplex DNA ends in cis from being recognized as double strand breaks. This protection is dependent on the G-quadruplex conformation of the 3' G-tail sequence. These results suggest that the ability of telomeric 3' G-tails to adopt the endprotecting G-quadruplex conformation may be one of the reasons for the existence of the evolutionarily conserved G-stretch motifs in telomeric DNA sequences.

Oligonucleotide Analogues with Integrated Bases and Backbone. Part 15

AbstractThe self‐complementary (Z)‐configured U*[ce]A(*) dinucleotide analogues 6, 8, 10, 12, 14, and 16, and the A*[ce]U(*) dimers 19, 21, 23, 25, 27, and 29 were prepared by partial hydrogenation of the corresponding ethynylene linked dimers. Photolysis of 14 led to the (E)‐alkene 17. These dinucleotide analogues associate in CDCl3 solution, as evidenced by NMR and CD spectroscopy. The thermodynamic parameters of the duplexation were determined by van't Hoff analysis. The (Z)‐configured U*[ce]A(*) dimers 14 and 16 form cyclic duplexes connected by Watson–Crick H‐bonds, the (E)‐configured U*[ce]A dimer 17 forms linear duplexes, and the U*[ce]A(*) allyl alcohols 6, 8, 10, and 12 form mixtures of linear and cyclic duplexes. The C(6/I)‐unsubstituted A*[ce]U allyl alcohols 19 and 23 form linear duplexes, whereas the C(6/I)‐substituted A*[ce]U* allyl alcohols 21 and 25, and the C(5′/I)‐deoxy A*[ce]U(*) dimers 27 and 29 also form minor amounts of cyclic duplexes. The influence of intra‐ and intermolecular H‐bonding of the allyl alcohols and the influence of the base sequence upon the formation of cyclic duplexes are discussed.

Oligonucleotide Analogues with Integrated Bases and Backbone. Part 16

AbstractThe self‐complementary, ethylene‐linked U*[ca]A(*) dinucleotide analogues 8, 10, 12, 14, 16, and 18, and the sequence‐isomeric A*[ca]U(*) analogues 20, 22, 24, 26, 28, and 30 were obtained by Pd/C‐catalyzed hydrogenation of the corresponding, known ethynylene‐linked dimers. The association of the ethylene‐linked dimers was investigated by NMR and CD spectroscopy. The U*[ca]A(*) dimers form linear duplexes and higher associates (K between 29 and 114M−1). The A*[ca]U(*) dimers, while associating more strongly (K between 88 and 345M−1), lead mostly to linear duplexes and higher associates; they form only minor amounts of cyclic duplexes. The enthalpy–entropy compensation characterizing the association of the U*[cx]A(*) and A*[cx]U(*) dimers (x=y, e, and a) is discussed.

SSB Protein Limits RecOR Binding onto Single-stranded DNA

The RecO and RecR proteins form a complex that promotes the nucleation of RecA protein filaments onto SSB protein-coated single-stranded DNA (ssDNA). However, even when RecO and RecR proteins are provided at optimal concentrations, the loading of RecA protein is surprisingly slow, typically proceeding with a lag of 10 min or more. The rate-limiting step in RecOR-promoted RecA nucleation is the binding of RecOR protein to ssDNA, which is inhibited by SSB protein despite the documented interaction between RecO and SSB. Full activity of RecOR is seen only when RecOR is preincubated with ssDNA prior to the addition of SSB. The slow binding of RecOR to SSB-coated ssDNA involves the C terminus of SSB. When an SSB variant that lacks the C-terminal 8 amino acids is used, the capacity of RecOR to facilitate RecA loading onto the ssDNA is largely abolished. The results are used in an expanded model for RecOR action.