Structure of discontinuities in kinetoplast DNA-associated minicircles during S phase in Crithidia fasciculata

Kinetoplast DNA (kDNA) is a novel form of mitochondrial DNA consisting of thousands of interlocked minicircles and 20–30 maxicircles. The minicircles replicate free of the kDNA network but nicks and gaps in the newly synthesized strands remain at the time of reattachment to the kDNA network. We show here that the steady-state population of replicated, network-associated minicircles only becomes repaired to the point of having nicks with a 3′OH and 5′deoxyribonucleoside monophosphate during S phase. These nicks represent the origin/terminus of the strand and occur within the replication origins (oriA and oriB) located 180° apart on the minicircle. Minicircles containing a new L strand have a single nick within either oriA or oriB but not in both origins in the same molecule. The discontinuously synthesized H strand contains single nicks within both oriA and oriB in the same molecule implying that discontinuities between the H-strand Okazaki fragments become repaired except for the fragments initiated within the two origins. Nicks in L and H strands at the origins persist throughout S phase and only become ligated as a prelude to network division. The failure to ligate these nicks until just prior to network division is not due to inappropriate termini for ligation.     

Direct detection of RNA in vitro and in situ by target-primed RCA: The impact of E. coli RNase III on the detection efficiency of RNA sequences distanced far from the 3′-end

We improved the target RNA-primed RCA technique for direct detection and analysis of RNA in vitro and in situ. Previously we showed that the 3′ → 5′ single-stranded RNA exonucleolytic activity of Phi29 DNA polymerase converts the target RNA into a primer and uses it for RCA initiation. However, in some cases, the single-stranded RNA exoribonucleolytic activity of the polymerase is hindered by strong double-stranded structures at the 3′-end of target RNAs. We demonstrate that in such hampered cases, the double-stranded RNA-specific Escherichia coli RNase III efficiently assists Phi29 DNA polymerase in converting the target RNA into a primer. These observations extend the target RNA-primed RCA possibilities to test RNA sequences distanced far from the 3′-end and customize this technique for the inner RNA sequence analysis.     

DNA strand transfer catalyzed by vaccinia topoisomerase: ligation of DNAs containing a 3' mononucleotide overhang

The specificity of vaccinia topoisomerase for transesterification to DNA at the sequence 5′-CCCTT and its versatility in strand transfer have illuminated the recombinogenic properties of type IB topoisomerases and spawned topoisomerase-based strategies for DNA cloning. Here we characterize a pathway of topoisomerase-mediated DNA ligation in which enzyme bound covalently to a CCCTT end with an unpaired +1T nucleotide rapidly and efficiently joins the CCCTT strand to a duplex DNA containing a 3′ A overhang. The joining reaction occurs with high efficiency, albeit slowly, to duplex DNAs containing 3′ G, T or C overhangs. Strand transfer can be restricted to the correctly paired 3′ A overhang by including 0.5 M NaCl in the ligation reaction mixture. The effects of base mismatches and increased ionic strength on the rates of 3′ overhang ligation provide a quantitative picture of the relative contributions of +1 T:A base pairing and electrostatic interactions downstream of the scissile phosphate to the productive binding of an unlinked acceptor DNA to the active site. The results clarify the biochemistry underlying topoisomerase-cloning of PCR products with non-templated 3′ overhangs.     

Detection of single-nucleotide polymorphisms in the <Emphasis Type="Italic">p53</Emphasis> gene by LDR/RCA in hydrogel microarrays

To find single-nucleotide polymorphisms (SNPs) in the human genome, three modern technologies of molecular genetic analysis were combined: the ligase detection reaction (LDR), rolling circle amplification (RCA), and immobilized microarray of gel elements (IMAGE). SNPs were detected in target DNA by selective ligation of allele-specific nucleotides in microarrays. The ligation product was assayed in microarray gel pads by RCA. Two variants of microarray analysis were compared. One included selective ligation of short oligonu-cleotides immobilized in a microarray with subsequent amplification with a preformed circular probe (a common circle). The probe was especially designed for human genome research. The other variant employed immobilized allele-specific padlock probes, which could be circularized as a result of selective ligation. Codon 72 SNP of the human p53 gene was used as a model. RCA in microarrays proved to be a quantitative assay and, in combination with LDR, allowed efficient discrimination of alleles. The principles and prospects of LDR/RCA in microarrays are discussed.Translated from Molekulyarnaya Biologiya, Vol. 39, No. 1, 2005, pp. 30–39.Original Russian Text Copyright © 2005 by Kashkin, Strizhkov, Gryadunov, Surzhikov, Grechishnikova, Kreindlin, Chupeeva, Evseev, Turygin, Mirzabekov.     

Protein-free parallel triple-stranded DNA complex formation

A 14 nt DNA sequence 5′-AGAATGTGGCAAAG-3′ from the zinc finger repeat of the human KRAB zinc finger protein gene ZNF91 bearing the intercalator 2-methoxy,6-chloro,9-amino acridine (Acr) attached to the sugar–phosphate backbone in various positions has been shown to form a specific triple helix (triplex) with a 16 bp hairpin (intramolecular) or a two-stranded (intermolecular) duplex having the identical sequence in the same (parallel) orientation. Intramolecular targets with the identical sequence in the antiparallel orientation and a non-specific target sequence were tested as controls. Apparent binding constants for formation of the triplex were determined by quantitating electrophoretic band shifts. Binding of the single-stranded oligonucleotide probe sequence to the target led to an increase in the fluorescence anisotropy of acridine. The parallel orientation of the two identical sequence segments was confirmed by measurement of fluorescence resonance energy transfer between the acridine on the 5′-end of the probe strand as donor and BODIPY-Texas Red on the 3′-amino group of either strand of the target duplex as acceptor. There was full protection from OsO₄-bipyridine modification of thymines in the probe strand of the triplex, in accordance with the presumed triplex formation, which excluded displacement of the homologous duplex strand by the probe–intercalator conjugate. The implications of these results for the existence of protein-independent parallel triplexes are discussed.     

The phosphatase activity of mammalian polynucleotide kinase takes precedence over its kinase activity in repair of single strand breaks

The dual function mammalian DNA repair enzyme, polynucleotide kinase (PNK), facilitates strand break repair through catalysis of 5′-hydroxyl phosphorylation and 3′-phosphate dephosphorylation. We have examined the relative activities of the kinase and phosphatase functions of PNK using a novel assay, which allows the simultaneous characterization of both activities in processing nicks and gaps containing both 3′-phosphate and 5′-hydroxyl. Under multiple turnover conditions the phosphatase activity of the purified enzyme is significantly more active than its kinase activity. Consistent with this result, phosphorylation of the 5′-hydroxyl is rate limiting in cell extract mediated-repair of a nicked substrate. On characterizing the effects of individually mutating the two active sites of PNK we find that while site-directed mutagenesis of the kinase domain of PNK does not affect its phosphatase activity, disruption of the phosphatase domain also abrogates kinase function. This loss of kinase function requires the presence of a 3′-phosphate, but it need not be present in the same strand break as the 5′-hydroxyl. PNK preferentially binds 3′-phosphorylated substrates and DNA binding to the phosphatase domain blocks further DNA binding by the kinase domain.     

Novel application of Phi29 DNA polymerase: RNA detection and analysis in vitro and in situ by target RNA-primed RCA

We present a novel Phi29 DNA polymerase application in RCA-based target RNA detection and analysis. The 3′→5′ RNase activity of Phi29 DNA polymerase converts target RNA into a primer and the polymerase uses this newly generated primer for RCA initiation. Therefore, using target RNA-primed RCA, padlock probes may be targeted to inner RNA sequences and their peculiarities can be analyzed directly. We demonstrate that the exoribonucleolytic activity of Phi29 DNA polymerase can be successfully applied in vitro and in situ. These findings expand the potential for detection and analysis of RNA sequences distanced from 3′-end.     

Single molecule linear analysis of DNA in nano-channel labeled with sequence specific fluorescent probes

An array of nano-channels was fabricated from silicon based semiconductor materials to stretch long, native dsDNA. Here we present a labeling scheme in which it is possible to identify the location of specific sequences along the stretched DNA molecules. The scheme proceeds by first using the strand displacement activity of the Vent (exo-) polymerase to generate single strand flaps on nicked dsDNA. These single strand flaps are hybridized with sequence specific fluorophore-labeled probes. Subsequent imaging of the DNA molecules inside a nano-channel array device allows for quantitative identification of the location of probes. The highly efficient DNA hybridization on the ss-DNA flaps is an excellent method to identify the sequence motifs of dsDNA as it gives us unique ability to control the length of the probe sequence and thus the frequency of hybridization sites on the DNA. We have also shown that this technique can be extended to a multi color labeling scheme by using different dye labeled probes or by combining with a DNA- polymerase-mediated incorporation of fluorophore-labeled nucleotides on nicking sites. Thus this labeling chemistry in conjunction with the nano-channel platform can be a powerful tool to solve complex structural variations in DNA which is of importance for both research and clinical diagnostics of genetic diseases.     

The differential extension in dsDNA bound to Rad51 filaments may play important roles in homology recognition and strand exchange

RecA and Rad51 proteins play an important role in DNA repair and homologous recombination. For RecA, X-ray structure information and single molecule force experiments have indicated that the differential extension between the complementary strand and its Watson–Crick pairing partners promotes the rapid unbinding of non-homologous dsDNA and drives strand exchange forward for homologous dsDNA. In this work we find that both effects are also present in Rad51 protein. In particular, pulling on the opposite termini (3′ and 5′) of one of the two DNA strands in a dsDNA molecule allows dsDNA to extend along non-homologous Rad51-ssDNA filaments and remain stably bound in the extended state, but pulling on the 3′5′ ends of the complementary strand reduces the strand-exchange rate for homologous filaments. Thus, the results suggest that differential extension is also present in dsDNA bound to Rad51. The differential extension promotes rapid recognition by driving the swift unbinding of dsDNA from non-homologous Rad51-ssDNA filaments, while at the same time, reducing base pair tension due to the transfer of the Watson–Crick pairing of the complementary strand bases from the highly extended outgoing strand to the slightly less extended incoming strand, which drives strand exchange forward.     

Coordinate 5' and 3' endonucleolytic trimming of terminally blocked blunt DNA double-strand break ends by Artemis nuclease and DNA-dependent protein kinase

Previous work showed that, in the presence of DNA-dependent protein kinase (DNA-PK), Artemis slowly trims 3′-phosphoglycolate-terminated blunt ends. To examine the trimming reaction in more detail, long internally labeled DNA substrates were treated with Artemis. In the absence of DNA-PK, Artemis catalyzed extensive 5′→3′ exonucleolytic resection of double-stranded DNA. This resection required a 5′-phosphate, but did not require ATP, and was accompanied by endonucleolytic cleavage of the resulting 3′ overhang. In the presence of DNA-PK, Artemis-mediated trimming was more limited, was ATP-dependent and did not require a 5′-phosphate. For a blunt end with either a 3′-phosphoglycolate or 3′-hydroxyl terminus, endonucleolytic trimming of 2–4 nucleotides from the 3′-terminal strand was accompanied by trimming of 6 nt from the 5′-terminal strand. The results suggest that autophosphorylated DNA-PK suppresses the exonuclease activity of Artemis toward blunt-ended DNA, and promotes slow and limited endonucleolytic trimming of the 5′-terminal strand, resulting in short 3′ overhangs that are trimmed endonucleolytically. Thus, Artemis and DNA-PK can convert terminally blocked DNA ends of diverse geometry and chemical structure to a form suitable for polymerase-mediated patching and ligation, with minimal loss of terminal sequence. Such processing could account for the very small deletions often found at DNA double-strand break repair sites.     

Characterization of Biochemical Properties of Bacillus subtilis RecQ Helicase

RecQ family helicases function as safeguards of the genome. Unlike Escherichia coli, the Gram-positive Bacillus subtilis bacterium possesses two RecQ-like homologues, RecQ[Bs] and RecS, which are required for the repair of DNA double-strand breaks. RecQ[Bs] also binds to the forked DNA to ensure a smooth progression of the cell cycle. Here we present the first biochemical analysis of recombinant RecQ[Bs]. RecQ[Bs] binds weakly to single-stranded DNA (ssDNA) and blunt-ended double-stranded DNA (dsDNA) but strongly to forked dsDNA. The protein exhibits a DNA-stimulated ATPase activity and ATP- and Mg²⁺-dependent DNA helicase activity with a 3′→5′ polarity. Molecular modeling shows that RecQ[Bs] shares high sequence and structure similarity with E. coli RecQ. Surprisingly, RecQ[Bs] resembles the truncated Saccharomyces cerevisiae Sgs1 and human RecQ helicases more than RecQ[Ec] with regard to its enzymatic activities. Specifically, RecQ[Bs] unwinds forked dsDNA and DNA duplexes with a 3′-overhang but is inactive on blunt-ended dsDNA and 5′-overhung duplexes. Interestingly, RecQ[Bs] unwinds blunt-ended DNA with structural features, including nicks, gaps, 5′-flaps, Kappa joints, synthetic replication forks, and Holliday junctions. We discuss these findings in the context of RecQ[Bs]''s possible functions in preserving genomic stability.     

Signal amplification of padlock probes by rolling circle replication.

Circularizing oligonucleotide probes (padlock probes) have the potential to detect sets of gene sequences with high specificity and excellent selectivity for sequence variants, but sensitivity of detection has been limiting. By using a rolling circle replication (RCR) mechanism, circularized but not unreacted probes can yield a powerful signal amplification. We demonstrate here that in order for the reaction to proceed efficiently, the probes must be released from the topological link that forms with target molecules upon hybridization and ligation. If the target strand has a nearby free 3' end, then the probe-target hybrids can be displaced by the polymerase used for replication. The displaced probe can then slip off the targetstrand and a rolling circle amplification is initiated. Alternatively, the target sequence itself can prime an RCR after its non-base paired 3' end has been removed by exonucleolytic activity. We found the Phi29 DNA polymerase to be superior to the Klenow fragment in displacing the target DNA strand, and it maintained the polymerization reaction for at least 12 h, yielding an extension product that represents several thousand-fold the length of the padlock probe.     

Fluorescence-based detection of short DNA sequences under non-denaturing conditions

The ability of peptide nucleic acid (PNA) to open up duplex DNA in a highly sequence-specific manner makes it possible to detect short DNA sequences on the background of or within genomic DNA under non-denaturing conditions. To do so, chosen marker sites in double-stranded DNA are locally opened by a pair of PNA openers, thus transforming one strand within the target region (20-30 bp) into the single-stranded form. Onto this accessible DNA sequence a circular oligonucleotide probe is assembled, which serves as a template for rolling circle amplification (RCA). Both homogeneous and heterogeneous assay formats are investigated, as are different formats for fluorescence-based amplicon detection. Our recent data with immobilized analytes suggest that marker sequences in plasmid and bacterial chromosomal DNA can be successfully detected.     

Functional dissection of siRNA sequence by systematic DNA substitution: modified siRNA with a DNA seed arm is a powerful tool for mammalian gene silencing with significantly reduced off-target effect

Short interfering RNA (siRNA)-based RNA interference (RNAi) is widely used for target gene knockdown in mammalian cells. To clarify the position-dependent functions of ribonucleotides in siRNA, siRNAs with various DNA substitutions were constructed. The following could be simultaneously replaced with DNA without substantial loss of gene-silencing activity: the seed arm, which occupies positions 2–8 from the 5′end of the guide strand; its complementary sequence; the 5′end of the guide strand and the 3′overhang of the passenger strand. However, most part of the 3′ two-thirds of the guide strand could not be replaced with DNA, possibly due to binding of RNA-recognition proteins such as TRBP2 and Ago2. The passenger strand with DNA in the 3′end proximal region was incapable of inducing off-target effect. Owing to lesser stability of DNA–RNA hybrid than RNA duplex, modified siRNAs with DNA substitution in the seed region were, in most cases, incapable to exert unintended gene silencing due to seed sequence homology. Thus, it may be possible to design DNA–RNA chimeras which effectively silence mammalian target genes without silencing unintended genes.     

Rolling-circle amplification under topological constraints

We have performed rolling-circle amplification (RCA) reactions on three DNA templates that differ distinctly in their topology: an unlinked DNA circle, a linked DNA circle within a pseudorotaxane-type structure and a linked DNA circle within a catenane. In the linked templates, the single-stranded circle (dubbed earring probe) is threaded, with the aid of two peptide nucleic acid openers, between the two strands of double-stranded DNA (dsDNA). We have found that the RCA efficiency of amplification was essentially unaffected when the linked templates were employed. By showing that the DNA catenane remains intact after RCA reactions, we prove that certain DNA polymerases can carry out the replicative synthesis under topological constraints allowing detection of several hundred copies of a dsDNA marker without DNA denaturation. Our finding may have practical implications in the area of DNA diagnostics.     

T-linker-specific ligation PCR (T-linker PCR): an advanced PCR technique for chromosome walking or for isolation of tagged DNA ends

Dozens of PCR-based methods are available for chromosome walking from a known sequence to an unknown region. These methods are of three types: inverse PCR, ligation-mediated PCR and randomly primed PCR. However, none of them has been generally applied for this purpose, because they are either difficult or inefficient. Here we describe a simple and efficient PCR strategy—T-linker-specific ligation PCR (T-linker PCR) for gene or chromosome walking. The strategy amplifies the template molecules in three steps. First, genomic DNA is digested with 3′ overhang enzymes. Secondly, primed by a specific primer, a strand of the target molecule is replicated by Taq DNA polymerase and a single A tail is generated on the 3′ unknown end of the target molecule, and then a 3′ overhang-T linker (named T-linker) is specifically ligated onto the target. Thirdly, the target is amplified by two rounds of nested PCR with specific primers and T-linker primers. T-linker PCR significantly improves the existing PCR methods for walking because it uses specific T/A ligation instead of arbitrary ligation or random annealing. To show the feasibility and efficiency of T-linker PCR, we have exploited this method to identify vector DNA or T-DNA insertions in transgenic plants.     

Structure specific DNA recognition by the SLX1–SLX4 endonuclease complex

The SLX1–SLX4 structure-specific endonuclease complex is involved in processing diverse DNA damage intermediates, including resolution of Holliday junctions, collapse of stalled replication forks and removal of DNA flaps. The nuclease subunit SLX1 is inactive on its own, but become activated upon binding to SLX4 via its conserved C-terminal domain (CCD). Yet, how the SLX1–SLX4 complex recognizes specific DNA structure and chooses cleavage sites remains unknown. Here we show, through a combination of structural, biochemical and computational analyses, that the SAP domain of SLX4 is critical for efficient and accurate processing of 5′-flap DNA. It binds the minor groove of DNA about one turn away from the flap junction, and the 5′-flap is implicated in binding the core domain of SLX1. This binding mode accounts for specific recognition of 5′-flap DNA and specification of cleavage site by the SLX1–SLX4 complex.     

Regulation of yeast DNA polymerase δ-mediated strand displacement synthesis by 5′-flaps

The strand displacement activity of DNA polymerase δ is strongly stimulated by its interaction with proliferating cell nuclear antigen (PCNA). However, inactivation of the 3′–5′ exonuclease activity is sufficient to allow the polymerase to carry out strand displacement even in the absence of PCNA. We have examined in vitro the basic biochemical properties that allow Pol δ-exo⁻ to carry out strand displacement synthesis and discovered that it is regulated by the 5′-flaps in the DNA strand to be displaced. Under conditions where Pol δ carries out strand displacement synthesis, the presence of long 5′-flaps or addition in trans of ssDNA suppress this activity. This suggests the presence of a secondary DNA binding site on the enzyme that is responsible for modulation of strand displacement activity. The inhibitory effect of a long 5′-flap can be suppressed by its interaction with single-stranded DNA binding proteins. However, this relief of flap-inhibition does not simply originate from binding of Replication Protein A to the flap and sequestering it. Interaction of Pol δ with PCNA eliminates flap-mediated inhibition of strand displacement synthesis by masking the secondary DNA site on the polymerase. These data suggest that in addition to enhancing the processivity of the polymerase PCNA is an allosteric modulator of other Pol δ activities.     

Genomic Analysis of Pseudomonas putida Phage tf with Localized Single-Strand DNA Interruptions

The complete sequence of the 46,267 bp genome of the lytic bacteriophage tf specific to Pseudomonas putida PpG1 has been determined. The phage genome has two sets of convergently transcribed genes and 186 bp long direct terminal repeats. The overall genomic architecture of the tf phage is similar to that of the previously described Pseudomonas aeruginosa phages PaP3, LUZ24 and phiMR299-2, and 39 out of the 72 products of predicted tf open reading frames have orthologs in these phages. Accordingly, tf was classified as belonging to the LUZ24-like bacteriophage group. However, taking into account very low homology levels between tf DNA and that of the other phages, tf should be considered as an evolutionary divergent member of the group. Two distinguishing features not reported for other members of the group were found in the tf genome. Firstly, a unique end structure – a blunt right end and a 4-nucleotide 3′-protruding left end – was observed. Secondly, 14 single-chain interruptions (nicks) were found in the top strand of the tf DNA. All nicks were mapped within a consensus sequence 5′-TACT/RTGMC-3′. Two nicks were analyzed in detail and were shown to be present in more than 90% of the phage population. Although localized nicks were previously found only in the DNA of T5-like and phiKMV-like phages, it seems increasingly likely that this enigmatic structural feature is common to various other bacteriophages.