The core and carboxyl-terminal domains of the integrase protein of human immunodeficiency virus type 1 each contribute to nonspecific DNA binding.

The integrase protein of human immunodeficiency virus type 1 removes two nucleotides from the 3' ends of reverse-transcribed human immunodeficiency virus type 1 DNA (3' processing) and covalently inserts the processed ends into a target DNA (DNA strand transfer). Mutant integrase proteins that lack the amino-and/or carboxyl-terminal domains are incapable of catalyzing 3' processing and DNA strand transfer but are competent for an apparent reversal of the DNA strand transfer reaction (disintegration) in vitro. Here, we investigate the binding of integrase to DNA by UV cross-linking. Cross-linked complexes form with a variety of DNA substrates independent of the presence of divalent metal ion. Analysis with amino- and carboxyl-terminal deletion mutant proteins shows that residues 213 to 266 of the 288-residue protein are required for efficient cross-linking in the absence of divalent metal ion. Carboxyl-terminal deletion mutants that lack this region efficiently cross-link only to the branched disintegration DNA substrate, and this reaction is dependent on the presence of metal ion. Both the core and C-terminal domains of integrase therefore contribute to nonspecific DNA binding.     

Construction and evaluation of a kinetic scheme for RecA-mediated DNA strand exchange

The Escherichia coli RecA protein is the prototype of a class of proteins playing a central role in genomic repair and recombination in all organisms. The unresolved mechanistic strategy by which RecA aligns a single strand of DNA with a duplex DNA and mediates a DNA strand switch is central to understanding its recombinational activities. Toward a molecular-level understanding of RecA-mediated DNA strand exchange, we explored its mechanism using oligonucleotide substrates and the intrinsic fluorescence of 6-methylisoxanthopterin (6MI). Steady- and presteady-state spectrofluorometric data demonstrate that the reaction proceeds via a sequential four-step mechanism comprising a rapid, bimolecular association step followed by three slower unimolecular steps. Previous authors have proposed multistep mechanisms involving two or three steps. Careful analysis of the differences among the experimental systems revealed a previously undiscovered intermediate (N1) whose formation may be crucial in the kinetic discrimination of homologous and heterologous sequences. This observation has important implications for probing the fastest events in DNA strand exchange using 6MI to further elucidate the molecular mechanisms of recombination and recombinational repair.     

Sequential excision of internal eliminated DNA sequences in the differentiating macronucleus of the hypotrichous ciliate Stylonychia lemnae.

Elimination of internal eliminated sequences (IES) during macronuclear development of the hypotrichous ciliate Stylonychia lemnae was analyzed in one cluster of macronuclear precursor DNA sequences. The results indicate that IES elimination is a highly ordered process, it starts very early during macronuclear development and has only finished immediately before DNA fragmentation takes place. It occurs in distinct steps and the IES are eliminated in a specific order, where a defined IES is only removed after complete elimination of other IES. Transfection experiments clearly demonstrate that the structure of the IES itself is not sufficient for its correct excision but other cis-acting sequences or additional structural requirements are needed for IES elimination.     

DNase I cleavage of branched DNA molecules

We report here a potentially useful signature of branched DNA structures. The base 5' to the branch and the five bases flanking the 3' side of the branch site are protected from cleavage by DNase I in both three- and four-arm branched DNA molecules. Our procedure is to measure the cleavage profile for each 5' -labeled strand in a control duplex and compare this with that of the same strand in a branched structure under conditions yielding less than one cut per strand. The resulting cleavage pattern in an immobile four-arm junction is roughly 2-fold symmetric, consistent with the pattern of Fe(II).EDTA-induced cleavage that has been observed previously. In the three-arm junction, the DNase I cleavage pattern is asymmetric, indicating lack of 3-fold symmetry. A variable pattern of protection occurs to the 5' side of the branch in some strands only for both three- and four-arm junctions, extending 2-4 residues 5' to the branch.     

Replication strand preference for deletions associated with DNA palindromes

We have isolated and sequenced a set of deletions stimulated by DNA palindromes in Escherichia coli . All of the deletions are asymmetric with respect to the parental sequence and have occurred at short direct repeats. This is consistent with deletion by strand slippage during DNA replication. The orientation of the asymmetry in such deletion products is diagnostic of the direction of the strand slippage event. It is therefore also diagnostic of its occurrence on the leading or lagging strand of the replication fork when the direction of replication is known. In all cases in which the orientation of the asymmetry could be determined with respect to DNA replication, the products were consistent with a preference for deletion on the lagging strand of the fork. The data include replication slippage in three situations: on the chromosome of E . coli , in bacteriophage λ and in high-copy-number pUC-based plasmids.     

Asymmetric structure of a three-arm DNA junction

We present here experimental evidence that three-arm branched DNA molecules form an asymmetric structure in the presence of Mg2+. Electrophoretic mobility and chemical and enzymatic footprinting experiments on a three-arm branched DNA molecule formed from three 16-mer strands are described. The electrophoretic mobilities of three species of a three-arm junction in which pairs of arms are extended are found to differ in the presence of Mg2+: one combination of elongated arms migrates significantly faster than the other two. This effect is eliminated in the absence of Mg2+, leading us to suggest that the three-arm DNA junction forms an asymmetric structure due to preferential stacking of two of the arms at the junction in the presence of Mg2+. The pattern of self-protection of each 16-mer strand of the core complex exposed to Fe(II).EDTA and DNase I scission is unique, consistent with formation of an asymmetric structure in the presence of Mg2+. We conclude that three-arm junctions resemble four-arm junctions in showing preferential stacking effects at the branch site. Comparison of the scission patterns of linear duplexes and the branched trimer by the reactive probes methidiumpropyl-EDTA.Fe(II) [MPE.Fe(II)] and Cu(I)-[o-phenanthroline]2 [(OP)2CuI] further indicates that the branch point represents a site of enhanced binding for drugs, as it does in the four-arm case. Reaction with diethyl pyrocarbonate (DEPC), a purine-specific probe sensitive to conformation, is enhanced at the branch site, consistent with loosening of base pairing or unpairing at this point.(ABSTRACT TRUNCATED AT 250 WORDS)     

Drosophila melanogaster strand transferase. A protein that forms heteroduplex DNA in the absence of both ATP and single-strand DNA binding protein

The purification of a Drosophila strand transfer protein is described, which involves Bio-Rex 70, Superose 6, Mono S, and single-stranded DNA-agarose chromatography. A 105,000-dalton polypeptide copurifies with the strand transfer activity on the last two column steps. The strand transferase carries out strand transfer at an unusually low protein:single-stranded DNA ratio and requires neither a nucleotide cofactor nor exogenous single-strand DNA binding protein to form heteroduplex DNA. Biochemical analysis of the reaction products has established that one strand of the DNA duplex is displaced during the reaction. Several properties, including the kinetics and stoichiometry of strand transfer, differentiate this activity from previously characterized strand transferases.     

Improved deoxyribozymes for synthesis of covalently branched DNA and RNA

A covalently branched nucleic acid can be synthesized by joining the 2′-hydroxyl of the branch-site ribonucleotide of a DNA or RNA strand to the activated 5′-phosphorus of a separate DNA or RNA strand. We have previously used deoxyribozymes to synthesize several types of branched nucleic acids for experiments in biotechnology and biochemistry. Here, we report in vitro selection experiments to identify improved deoxyribozymes for synthesis of branched DNA and RNA. Each of the new deoxyribozymes requires Mn²⁺ as a cofactor, rather than Mg²⁺ as used by our previous branch-forming deoxyribozymes, and each has an initially random region of 40 rather than 22 or fewer combined nucleotides. The deoxyribozymes all function by forming a three-helix-junction (3HJ) complex with their two oligonucleotide substrates. For synthesis of branched DNA, the best new deoxyribozyme, 8LV13, has k_obs on the order of 0.1 min⁻¹, which is about two orders of magnitude faster than our previously identified 15HA9 deoxyribozyme. 8LV13 also functions at closer-to-neutral pH than does 15HA9 (pH 7.5 versus 9.0) and has useful tolerance for many DNA substrate sequences. For synthesis of branched RNA, two new deoxyribozymes, 8LX1 and 8LX6, were identified with broad sequence tolerances and substantial activity at pH 7.5, versus pH 9.0 for many of our previous deoxyribozymes that form branched RNA. These experiments provide new, and in key aspects improved, practical catalysts for preparation of synthetic branched DNA and RNA.     

Deletion loop mutagenesis: a novel method for the construction of point mutations using deletion mutants

Deletion loop mutagenesis is a new, general method for site-directed mutagenesis that allows point mutations to the introduced within a sequence of DNA defined by a previously isolated deletion mutant. Wild type and deletion mutant DNA are cloned into a bacterial plasmid and each is cleaved with a different single cut restriction enzyme. Heteroduplexes are formed between the two DNAs to produce circular molecules containing a nick in each strand and a single-stranded deletion loop. The deletion loops are mutagenised using sodium bisulphite and the DNA transfected directly into a uracil repair deficient strain of Escherichia coli. Up to half of the resultant clones contain DNA produced by replication of the wild-type length strand and bear mutations exclusively within the target area. An example is given in which a deletion mutant lacking 21 nucleotides from the region coding for SV40 large-T was used. Eight of the possible nine target cytosine residues were mutagenised. The method described is specific, efficient and simple.     

Effects of deletion and site-directed mutations on ligation steps of NAD+-dependent DNA ligase: a biochemical analysis of BRCA1 C-terminal domain

DNA strand joining entails three consecutive steps: enzyme adenylation to form AMP-ligase, substrate adenylation to form AMP-DNA, and nick closure. In this study, we investigate the effects on ligation steps by deletion and site-directed mutagenesis of the BRCA1 C-terminal (BRCT) domain using NAD(+)-dependent DNA ligase from Thermus species AK16D. Deletion of the BRCT domain resulted in substantial loss of ligation activity, but the mutant was still able to form an AMP-ligase intermediate, suggesting that the defects caused by deletion of the entire BRCT domain occur primarily at steps after enzyme adenylation. The lack of AMP-DNA accumulation by the domain deletion mutant as compared to the wild-type ligase indicates that the BRCT domain plays a role in the substrate adenylation step. Gel mobility shift analysis suggests that the BRCT domain and helix-hairpin-helix subdomain play a role in DNA binding. Similar to the BRCT domain deletion mutant, the G617I mutant showed a low ligation activity and lack of accumulation of AMP-DNA intermediate. However, the G617I mutant was only weakly adenylated, suggesting that a point mutation in the BRCT domain could also affect the enzyme adenylation step. The significant reduction of ligation activity by G634I appears to be attributable to a defect at the substrate adenylation step. The greater ligation of mismatched substrates by G638I is accountable by accelerated conversion of the AMP-DNA intermediate to a ligation product at the final nick closure step. The mutational effects of the BRCT domain on ligation steps in relation to protein-DNA and potential protein-protein interactions are discussed.     

Role of micronucleus-limited DNA in programmed deletion of mse2.9 during macronuclear development of Tetrahymena thermophila

Extensive programmed DNA rearrangements occur during the development of the somatic macronucleus from the germ line micronucleus in the sexual cycle of the ciliated protozoan Tetrahymena thermophila. Using an in vivo processing assay, we analyzed the role of micronucleus-limited DNA during the programmed deletion of mse2.9, an internal eliminated sequence (IES). We identified a 200-bp region within mse2.9 that contains an important cis-acting element which is required for the targeting of efficient programmed deletion. Our results, obtained with a series of mse2.9-based chimeric IESs, led us to suggest that the cis-acting elements in both micronucleus-limited and macronucleus-retained flanking DNAs stimulate programmed deletion to different degrees depending on the particular eliminated sequence. The mse2.9 IES is situated within the second intron of the micronuclear locus of the ARP1 gene. We show that the expression of ARP1 is not essential for the growth of Tetrahymena. Our results also suggest that mse2.9 is not subject to epigenetic regulation of DNA deletion, placing possible constraints on the scan RNA model of IES excision.     

Opposing roles for DNA structure-specific proteins Rad1, Msh2, Msh3, and Sgs1 in yeast gene targeting

Targeted gene replacement (TGR) in yeast and mammalian cells is initiated by the two free ends of the linear targeting molecule, which invade their respective homologous sequences in the chromosome, leading to replacement of the targeted locus with a selectable gene from the targeting DNA. To study the postinvasion steps in recombination, we examined the effects of DNA structure-specific proteins on TGR frequency and heteroduplex DNA formation. In strains deleted of RAD1, MSH2, or MSH3, we find that the frequency of TGR is reduced and the mechanism of TGR is altered while the reverse is true for deletion of SGS1, suggesting that Rad1 and Msh2:Msh3 facilitate TGR while Sgs1 opposes it. The altered mechanism of TGR in the absence of Msh2:Msh3 and Rad1 reveals a separate role for these proteins in suppressing an alternate gene replacement pathway in which incorporation of both homology regions from a single strand of targeting DNA into heteroduplex with the targeted locus creates a mismatch between the selectable gene on the targeting DNA and the targeted gene in the chromosome.     

Mechanism of DNA binding and localized strand separation by Pur alpha and comparison with Pur family member, Pur beta

Pur alpha is a single-stranded (ss) DNA- and RNA-binding protein with three conserved signature repeats that have a specific affinity for guanosine-rich motifs. Pur alpha unwinds a double-stranded oligonucleotide containing purine-rich repeats by maintaining contact with the purine-rich strand and displacing the pyrimidine-rich strand. Mutational analysis indicates that arginine and aromatic residues in the repeat region of Pur alpha are essential for both ss- and duplex DNA binding. Pur alpha binds either linearized or supercoiled plasmid DNA, generating a series of regularly spaced bands in agarose gels. This series is likely due to localized unwinding by quanta of Pur alpha since removal of Pur alpha in the gel eliminates the series and since Pur alpha binding increases the sensitivity of plasmids to reaction with potassium permanganate, a reaction specific for unwound regions. Pur alpha binding to linear duplex DNA creates binding sites for the phage T4 gp32 protein, an ss-DNA binding protein that does not itself bind linearized DNA. In contrast, Pur beta lacking the Pur alpha C-terminal region binds supercoiled DNA but not linearized DNA. Similarly, a C-terminal deletion of Pur alpha can bind supercoiled pMYC7 plasmid, but cannot bind the same linear duplex DNA segment. Therefore, access to linear DNA initially requires C-terminal sequences of Pur alpha.     

C-terminal deletions of the Escherichia coli RecA protein. Characterization of in vivo and in vitro effects

A set of C-terminal deletion mutants of the RecA protein of Escherichia coli, progressively removing 6, 13, 17, and 25 amino acid residues, has been generated, expressed, and purified. In vivo, the deletion of 13 to 17 C-terminal residues results in increased sensitivity to mitomycin C. In vitro, the deletions enhance binding to duplex DNA as previously observed. We demonstrate that much of this enhancement involves the deletion of residues between positions 339 and 346. In addition, the C-terminal deletions cause a substantial upward shift in the pH-reaction profile of DNA strand exchange reactions. The C-terminal deletions of more than 13 amino acid residues result in strong inhibition of DNA strand exchange below pH 7, where the wild-type protein promotes a proficient reaction. However, at the same time, the deletion of 13-17 C-terminal residues eliminates the reduction in DNA strand exchange seen with the wild-type protein at pH values between 7.5 and 9. The results suggest the existence of extensive interactions, possibly involving multiple salt bridges, between the C terminus and other parts of the protein. These interactions affect the pK(a) of key groups involved in DNA strand exchange as well as the direct binding of RecA protein to duplex DNA.     

Reassociation rate limited displacement of DNA strands by branch migration.

Large branched DNA structures are constructed by two-step reassociation of separated complementary strands from restriction fragments of different lengths. The displacement of DNA strands initially annealed to longer complementary DNA sequences, a process mediated by branch migration, is very rapid and has thus far been followed only under conditions which are second order, DNA reassociation rate limiting. The average lifetime of branched DNA leading to displacement of 1.6 Kb strands is estimated to be less than 10 seconds under conditions of DNA reassociation, Tm-25 degrees C. Several DNA-binding drugs, including intercalating dyes, have been tested to determine their influence, if any, on the kinetics of DNA strand displacements by branch migration. Only actinomycin D was found to have significant effect under the conditions we have described. The kinetics of the strand displacement in the presence of low concentrations of actinomycin D remain second order and slower rate of strand displacement must be attributed to decreased rate of reassociation of DNA strands to form the branched intermediates. Consideration is given to the potential manipulation of DNA structures at site-directed branches and the limitations due to rapid strand displacements. The feasibility of constructing sufficiently large branched DNA regions to approach first order, branch migration rate limiting kinetics is also discussed.     

Mechanism of DNA binding and localized strand separation by Purα and comparison with Pur family member,Purβ

Purα is a single-stranded (ss) DNA- and RNA-binding protein with three conserved signature repeats that have a specific affinity for guanosine-rich motifs. Purα unwinds a double-stranded oligonucleotide containing purine-rich repeats by maintaining contact with the purine-rich strand and displacing the pyrimidine-rich strand. Mutational analysis indicates that arginine and aromatic residues in the repeat region of Purα are essential for both ss- and duplex DNA binding. Purα binds either linearized or supercoiled plasmid DNA, generating a series of regularly spaced bands in agarose gels. This series is likely due to localized unwinding by quanta of Purα since removal of Purα in the gel eliminates the series and since Purα binding increases the sensitivity of plasmids to reaction with potassium permanganate, a reaction specific for unwound regions. Purα binding to linear duplex DNA creates binding sites for the phage T4 gp32 protein, an ss-DNA binding protein that does not itself bind linearized DNA. In contrast, Purβ lacking the Purα C-terminal region binds supercoiled DNA but not linearized DNA. Similarly, a C-terminal deletion of Purα can bind supercoiled pMYC7 plasmid, but cannot bind the same linear duplex DNA segment. Therefore, access to linear DNA initially requires C-terminal sequences of Purα.     

Mismatch repair factor MSH2-MSH3 binds and alters the conformation of branched DNA structures predicted to form during genetic recombination

Genetic studies in Saccharomyces cerevisiae predict that the mismatch repair (MMR) factor MSH2-MSH3 binds and stabilizes branched recombination intermediates that form during single strand annealing and gene conversion. To test this model, we constructed a series of DNA substrates that are predicted to form during these recombination events. We show in an electrophoretic mobility shift assay that S. cerevisiae MSH2-MSH3 specifically binds branched DNA substrates containing 3' single-stranded DNA and that ATP stimulates its release from these substrates. Chemical footprinting analyses indicate that MSH2-MSH3 specifically binds at the double-strand/single-strand junction of branched substrates, alters its conformation and opens up the junction. Therefore, MSH2-MSH3 binding to its substrates creates a unique nucleoprotein structure that may signal downstream steps in repair that include interactions with MMR and nucleotide excision repair factors.