Screening for mutations by enzyme mismatch cleavage with T4 endonuclease VII.

Each of four possible sets of mismatches (G.A/C.T, C.C/G.G, A.A/T.T, and C.A/G.T) containing the 8 possible single-base-pair mismatches derived from isolated mutations were examined to test the ability of T4 endonuclease VII to consistently detect mismatches in heteroduplexes. At least two examples of each set of mismatches were studied for cleavage in the complementary pairs of heteroduplexes formed between normal and mutant DNA. Four deletion mutations were also included in this study. The various PCR-derived products used in the formation of heteroduplexes ranged from 133 to 1502 bp. At least one example of each set showed cleavage of at least one strand containing a mismatch. Cleavage of at least one strand of the pairs of heteroduplexes occurred in 17 of the 18 known single-base-pair mutations tested, with an A.A/T.T set not being cleaved in any mismatched strand. We propose that this method may be effective in detecting and positioning almost all mutational changes when DNA is screened for mutations.     

Partial purification of an enzyme from Saccharomyces cerevisiae that cleaves Holliday junctions.

An enzyme from Saccharomyces cerevisiae that cleaves Holliday junctions was partially purified approximately 500- to 1000-fold by DEAE-cellulose chromatography, gel filtration on Sephacryl S300, and chromatography on single-stranded DNA-cellulose. The partially purified enzyme did not have any detectable nuclease activity when tested with single-stranded or double-stranded bacteriophage T7 substrate DNA and did not have detectable endonuclease activity when tested with bacteriophage M13 viral DNA or plasmid pBR322 covalently closed circular DNA. Analysis of the products of the cruciform cleavage reaction by electrophoresis on polyacrylamide gels under denaturing conditions revealed that the cruciform structure was cleaved at either of two sites present in the stem of the cruciform and was not cleaved at the end of the stem. The cruciform cleavage enzyme was able to cleave the Holliday junction present in bacteriophage G4 figure-8 molecules. Eighty percent of these Holliday junctions were cleaved in the proper orientation to generate intact chromosomes during genetic recombination.     

RuvC protein resolves Holliday junctions via cleavage of the continuous (noncrossover) strands.

The RuvC protein of Escherichia coli resolves Holliday junctions during genetic recombination and the postreplicational repair of DNA damage. Using synthetic Holliday junctions that are constrained to adopt defined isomeric configurations, we show that resolution occurs by symmetric cleavage of the continuous (noncrossing) pair of DNA strands. This result contrasts with that observed with phage T4 endonuclease VII, which cleaves the pair of crossing strands. In the presence of RuvC, the pair of continuous strands (i.e., the target strands for cleavage) exhibit a hypersensitivity to hydroxyl radicals. These results indicate that the continuous strands are distorted within the RuvC/Holliday junction complex and that RuvC-mediated resolution events require protein-directed structural changes to the four-way junction.     

Retrotransposon R1Bm endonuclease cleaves the target sequence

The R1Bm element, found in the silkworm Bombyx mori, is a member of a group of widely distributed retrotransposons that lack long terminal repeats. Some of these elements are highly sequence-specific and others, like the human L1 sequence, are less so. The majority of R1Bm elements are associated with ribosomal DNA (rDNA). R1Bm inserts into 28S rDNA at a specific sequence; after insertion it is flanked by a specific 14-bp target site duplication of the 28S rDNA. The basis for this sequence specificity is unknown. We show that R1Bm encodes an enzyme related to the endonuclease found in the human L1 retrotransposon and also to the apurinic/apyrimidinic endonucleases. We expressed and purified the enzyme from bacteria and showed that it cleaves in vitro precisely at the positions in rDNA corresponding to the boundaries of the 14-bp target site duplication. We conclude that the function of the retrotransposon endonucleases is to define and cleave target site DNA.     

Identification of four acidic amino acids that constitute the catalytic center of the RuvC Holliday junction resolvase.

Escherichia coli RuvC protein is a specific endonuclease that resolves Holliday junctions during homologous recombination. Since the endonucleolytic activity of RuvC requires a divalent cation and since 3 or 4 acidic residues constitute the catalytic centers of several nucleases that require a divalent cation for the catalytic activity, we examined whether any of the acidic residues of RuvC were required for the nucleolytic activity. By site-directed mutagenesis, we constructed a series of ruvC mutant genes with similar amino acid replacements in 1 of the 13 acidic residues. Among them, the mutant genes with an alteration at Asp-7, Glu-66, Asp-138, or Asp-141 could not complement UV sensitivity of a ruvC deletion strain, and the multicopy mutant genes showed a dominant negative phenotype when introduced into a wild-type strain. The products of these mutant genes were purified and their biochemical properties were studied. All of them retained the ability to form a dimer and to bind specifically to a synthetic Holliday junction. However, they showed no, or extremely reduced, endonuclease activity specific for the junction. These 4 acidic residues, which are dispersed in the primary sequence, are located in close proximity at the bottom of the putative DNA binding cleft in the three-dimensional structure. From these results, we propose that these 4 acidic residues constitute the catalytic center for the Holliday junction resolvase and that some of them play a role in coordinating a divalent metal ion in the active center.     

Structure of Hjc, a Holliday junction resolvase, from Sulfolobus solfataricus

The 2.15-A structure of Hjc, a Holliday junction-resolving enzyme from the archaeon Sulfolobus solfataricus, reveals extensive structural homology with a superfamily of nucleases that includes type II restriction enzymes. Hjc is a dimer with a large DNA-binding surface consisting of numerous basic residues surrounding the metal-binding residues of the active sites. Residues critical for catalysis, identified on the basis of sequence comparisons and site-directed mutagenesis studies, are clustered to produce two active sites in the dimer, about 29 A apart, consistent with the requirement for the introduction of paired nicks in opposing strands of the four-way DNA junction substrate. Hjc displays similarity to the restriction endonucleases in the way its specific DNA-cutting pattern is determined but uses a different arrangement of nuclease subunits. Further structural similarity to a broad group of metal/phosphate-binding proteins, including conservation of active-site location, is observed. A high degree of conservation of surface electrostatic character is observed between Hjc and T4-phage endonuclease VII despite a complete lack of structural homology. A model of the Hjc-Holliday junction complex is proposed, based on the available functional and structural data.     

The heteromeric Nanoarchaeum equitans splicing endonuclease cleaves noncanonical bulge-helix-bulge motifs of joined tRNA halves

Among the tRNA population of the archaeal parasite Nanoarchaeum equitans are five species assembled from separate 5' and 3' tRNA halves and four species derived from tRNA precursors containing introns. In both groups an intervening sequence element must be removed during tRNA maturation. A bulge-helix-bulge (BHB) motif is the hallmark structure required by the archaeal splicing endonuclease for recognition and excision of all introns. BHB motifs are recognizable at the joining sites of all five noncontinuous tRNA species, although deviations from the canonical BHB motif are clearly present in at least two of them. Here, we show that the N. equitans splicing endonuclease cleaves tRNA precursors containing normal introns, as well as all five noncontinuous precursor tRNAs, at the predicted splice sites, indicating the enzyme's dual role in the removal of tRNA introns and processing of tRNA halves to be joined in trans. The cleavage activity on a set of synthetic canonical and noncanonical BHB constructs showed that the N. equitans splicing endonuclease accepts a broader range of substrates than the homodimeric Archaeoglobus fulgidus enzyme. In contrast to the A. fulgidus endonuclease, the N. equitans splicing enzyme possesses two different subunits. This heteromeric endonuclease type, found in N. equitans, in all Crenarchaeota, and in Methanopyrus kandleri, is able to act on the noncanonical tRNA introns present only in these organisms, which suggests coevolution of enzyme and substrate.     

Crystal structure of the Holliday junction migration motor protein RuvB from Thermus thermophilus HB8

We report here the crystal structure of the RuvB motor protein from Thermus thermophilus HB8, which drives branch migration of the Holliday junction during homologous recombination. RuvB has a crescent-like architecture consisting of three consecutive domains, the first two of which are involved in ATP binding and hydrolysis. DNA is likely to interact with a large basic cleft, which encompasses the ATP-binding pocket and domain boundaries, whereas the junction-recognition protein RuvA may bind a flexible beta-hairpin protruding from the N-terminal domain. The structures of two subunits, related by a noncrystallographic pseudo-2-fold axis, imply that conformational changes of motor protein coupled with ATP hydrolysis may reflect motility essential for its translocation around double-stranded DNA.     

Specific action of T4 endonuclease V on damaged DNA in xeroderma pigmentosum cells in vivo.

The specific action of T4 endonuclease V on damaged DNA in xeroderma pigmentosum cells was examined using an in vivo assay system with hemagglutinating virus of Japan (Sendai virus) inactivated by UV light. A clear dose response was observed between the level of UV-induce unscheduled DNA synthesis of xeroderma pigmentosum cells and the amount of T4 endonuclease V activity added. The T4 enzyme was unstable in human cells, and its half-life was 3 hr. Fractions derived from an extract of Escherichia coli infected with T4V1, a mutant defective in the endonuclease V gene, showed no ability to restore the UV-induced unscheduled DNA synthesis of xeroderma pigmentosum cells. However, fractions derived from an extract of T4D-infected E. coli with endonuclease V activity were effective. The T4 enzyme was effective in xeroderma pigmentosum cells on DNA damaged by UV light but not in cells damaged by 4-nitroquinoline 1-oxide. The results of these experiments show that the T4 enzyme has a specific action on human cell DNA in vivo. Treatment with the T4 enzyme increased the survival of group A xeroderma pigmentosum cells after UV irradiation.     

Claudin multigene family encoding four-transmembrane domain protein components of tight junction strands

Two related integral membrane proteins, claudin-1 and -2, recently were identified as novel components of tight junction (TJ) strands. Here, we report six more claudin-like proteins, indicating the existence of a claudin gene family. Three of these were reported previously as RVP1, Clostridium perfringens enterotoxin receptor, and TMVCF, but their physiological functions were not determined. Through similarity searches followed by PCR, we isolated full length cDNAs of mouse RVP1, Clostridium perfringens enterotoxin receptor, and TMVCF as well as three mouse claudin-like proteins and designated them as claudin-3 to -8, respectively. All of these claudin family members showed similar patterns on hydrophilicity plots, which predicted four transmembrane domains in each molecule. Northern blotting showed that the tissue distribution pattern varied significantly, depending on claudin species. Similarly to claudin-1 and -2, when these claudins were HA-tagged and introduced into cultured Madin–Darby canine kidney cells, all showed a tendency to concentrate at TJs. Immunofluorescence and immunoelectron microscopy with polyclonal antibodies specific for claudin-3, -4, or -8 revealed that these molecules were exclusively concentrated at TJs in the liver and/or kidney. These findings indicated that multiple claudin family members are involved in the formation of TJ strands in various tissues.     

Correction of proliferation and drug sensitivity defects in the progeroid Werner's Syndrome by Holliday junction resolution

The progeroid Werner's syndrome (WS) represents the best current model of human aging. It is caused by loss of the WRN helicase/exonuclease, resulting in high levels of replication fork stalling and genomic instability. Current models suggest that characteristic WS phenotypes of poor S phase progression, low proliferative capacity, and drug hypersensitivity are the result of accumulation of alternative DNA structures at stalled or collapsed forks during DNA replication, and Holliday junction resolution has been shown to enhance survival of cis-platin-treated WS cells. Here, we present a direct test of the hypothesis that the replication/repair defect in unstressed WS cells is the result of an inability to resolve recombination intermediates. We have created isogenic WS cell lines expressing a nuclear-targeted bacterial Holliday junction endonuclease, RusA, and show that Holliday junction resolution by RusA restores DNA replication capacity in primary WS fibroblasts and enhances their proliferation. Furthermore, RusA expression rescues WS fibroblast hypersensitivity to replication fork blocking agents camptothecin and 4NQO, suggesting that the hypersensitivity is caused by inappropriate recombination at DNA structures formed when the replication fork arrests or collapses at 4NQO- or camptothecin-induced lesions. This work is the first to demonstrate that Holliday junction accumulation in primary Werner syndrome fibroblasts results in their poor proliferative capacity, and to rescue WS hypersensitivity to camptothecin and 4NQO by Holliday junction resolution.     

Role of the basic amino acid cluster and Glu-23 in pyrimidine dimer glycosylase activity of T4 endonuclease V.

T4 endonuclease V [endodeoxyribonuclease (pyrimidine dimer); deoxyribonuclease (pyrimidine dimer), EC 3.1.25.1] initiates repair of damaged DNA by hydrolysis of the N-glycosyl bond at the 5' side of a pyrimidine photodimer in double-stranded DNA. To study one of the active sites of T4 endonuclease V, systematic site-directed mutagenesis was performed on the synthetic T4 endonuclease V gene, in parallel with three-dimensional structure analysis by x-ray crystallography. The mutant proteins were evaluated for DNA glycosylase activity using an oligonucleotide duplex (14-mer) containing a single thymidine dimer as a substrate. Replacement of either Glu-23 with glutamine or asparatic acid or Arg-3 with glutamine completely abolished DNA glycosylase activity. Mutation of Arg-3 to lysine or of Arg-26 to glutamine or lysine in a basic amino acid cluster caused serious defects in DNA glycosylase activity, which are reflected in the increases in Km and decreases in kcat of DNA glycosylase activity. On the other hand, substitutions of lysine for Arg-22 or of glutamine for Arg-117 or Lys-121 resulted in increases in the Km value. The completely inactive mutant proteins, E23Q and R3Q, in which glutamine was substituted for Glu-23 and Arg-3, respectively, were further investigated by CD spectroscopy for their ability to bind the oligonucleotide substrate. It was found that the E23Q protein retained specific substrate-binding ability, whereas the R3Q protein did not. These results indicate that Glu-23 plays an important role in catalysis of the DNA glycosylase reaction, and that Arg-3 is a crucial residue for substrate binding. In addition, Arg-22, Arg-26, Arg-117, and Lys-121 in the basic amino acid cluster also participate in substrate binding. We conclude that the basic amino acid cluster in T4 endonuclease V is an essential structure for DNA glycosylase activity.     

Mechanism of Bloom syndrome complex assembly required for double Holliday junction dissolution and genome stability

The RecQ-like helicase BLM cooperates with topoisomerase IIIα, RMI1, and RMI2 in a heterotetrameric complex (the “Bloom syndrome complex”) for dissolution of double Holliday junctions, key intermediates in homologous recombination. Mutations in any component of the Bloom syndrome complex can cause genome instability and a highly cancer-prone disorder called Bloom syndrome. Some heterozygous carriers are also predisposed to breast cancer. To understand how the activities of BLM helicase and topoisomerase IIIα are coupled, we purified the active four-subunit complex. Chemical cross-linking and mass spectrometry revealed a unique architecture that links the helicase and topoisomerase domains. Using biochemical experiments, we demonstrated dimerization mediated by the N terminus of BLM with a 2:2:2:2 stoichiometry within the Bloom syndrome complex. We identified mutations that independently abrogate dimerization or association of BLM with RMI1, and we show that both are dysfunctional for dissolution using in vitro assays and cause genome instability and synthetic lethal interactions with GEN1/MUS81 in cells. Truncated BLM can also inhibit the activity of full-length BLM in mixed dimers, suggesting a putative mechanism of dominant-negative action in carriers of BLM truncation alleles. Our results identify critical molecular determinants of Bloom syndrome complex assembly required for double Holliday junction dissolution and maintenance of genome stability.

Homologous recombination (HR) is an essential and highly regulated cellular process required for maintenance of genome stability during mitosis and sexual reproduction. A key intermediate in HR and recombinational DNA repair is a four-way DNA junction known as a Holliday junction (HJ). During meiosis, double Holliday junctions (dHJs) are cut by endonucleases (resolution) in order to create the cross-overs necessary for proper chromosome segregation (1, 2). In somatic cells, however, cross-overs are potentially deleterious, and their formation is suppressed by the BLM protein (3). Failure to suppress cross-over formation leads to an increased frequency of sister chromatid exchanges and has the potential to result in translocations and/or somatic cell loss of heterozygosity (4), key drivers of genome instability and cancer. As a likely consequence, individuals with homozygous BLM mutations exhibit Bloom syndrome and are highly cancer prone (5). Heterozygous BLM mutation carriers are also significantly overrepresented in breast cancer families (6, 7).The 1,417-amino acid BLM protein suppresses DNA cross-overs by promoting dHJ “dissolution,” a process that unlinks the recombination intermediates back to their prerecombination state (8). Critical to dHJ dissolution by BLM is a RecQ-type superfamily II helicase domain. The ATPase function of this domain is required for DNA unwinding, translocation, and strand exchange within the HJ (9, 10). Dissolution also requires the concerted action of three additional factors, topoisomerase IIIα (TopoIIIα), RMI1, and RMI2. Together, these four proteins form the Bloom syndrome complex (BS complex; also known as the “dissolvasome” or “BTRR [BLM-TopoIIIα-RMI1-RMI2]”) (11). TopoIIIα belongs to the type IA class of topoisomerases, padlock-shaped enzymes that effect changes in DNA topology through a “strand-passage” mechanism (12). When coupled with TopoIIIα, BLM promotes DNA decatenation under physiological conditions by catalyzing DNA unlinking in an ATPase-dependent manner.The ancillary factors RMI1 (625 amino acids) and RMI2 (147 amino acids) are Replication Protein A–like, OB (oligonucleotide binding) fold–containing proteins that stimulate the dissolution reaction and have been implicated in either nucleic acid engagement (13–15) or protein–protein interactions (16–19). In particular, in the yeast dissolvasome complex, RMI1 stabilizes the covalently bound open form of the TopoIIIα DNA gate in a configuration that favors DNA strand passage (10). The significance of these components to BLM function is underscored by recent discoveries of homozygous mutant TOP3Α, RMI1, or RMI2 individuals who have Bloom syndrome–like disorders (20, 21).X-ray crystal structures of the RecQ helicase domain of BLM [encompassing residues 636 to 1298, Protein Data Bank (PDB) ID codes 4CGZ, 4CDG, and 403M (22, 23)] revealed a unique position of the DNA duplex relative to the helicase core and visualization of both pre- and posttranslocation states that flip out at least three bases of single-strand DNA (ssDNA). An additional structure of TopoIIIα (residues 21 to 639) in complex with RMI1 [residues 2 to 216; PDB ID code 4CHT (12)] also revealed the characteristic toroidal structure of type I topoisomerases, with a gate mechanism that is regulated by an insertion loop within the first OB fold of RMI1. While these structures informed on key residues and the biophysical action of each protein, they did not explain how the BS complex assembles or point to an overall mechanism of dHJ dissolution. Key to understanding this mechanism is a molecular characterization of the number and relative position of helicases and topoisomerases within the complex. However, there are contrasting reports in the literature on the oligomeric state of BLM. Size exclusion chromatography and atomic force microscopy support monomeric, dimeric, and larger oligomeric states, while EM data reveal five- or six-lobed structures reminiscent of ring-shaped multimeric ATPases (24–27). The oligomeric state of BLM within the BS complex has never been investigated, although RMI1:RMI2 purified in isolation forms a 1:1 heterodimer in published reports (16, 18, 19).The primary goal of this study was to establish how the BS complex assembles and how this assembly promotes dHJ dissolution and maintenance of genome stability. To achieve this, we purified an active recombinant BS complex. Using a combination of biochemical, biophysical, and cross-linking mass spectrometry (XL-MS) experiments, we determined that recombinant BS complex exists predominantly in a 2:2:2:2 stoichiometry. We found that dimerization of the BS complex and interaction with TopoIIIα/RMI1 are mediated by the N terminus of BLM, and we define critical residues required for these interactions and dHJ dissolution in vitro. To validate our in vitro experiments, we used cell complementation experiments with mutant BLM proteins and showed that these interactions are also required for genome stability in cells.     

Generation of double Holliday junction DNAs and their dissolution/resolution within a chromatin context

Four-way DNA intermediates, also known as Holliday junctions (HJs), are formed during homologous recombination and DNA repair, and their resolution is necessary for proper chromosome segregation. To facilitate the biochemical analysis of HJ processing, we developed a method involving DNAzyme self-cleavage to generate 1.8-kb DNA molecules containing either single (sHJ) or double Holliday junctions (dHJs). We show that dHJ DNAs (referred to as HoJo DNAs) are dissolved by the human BLM–TopIIIα–RMI1–RMI2 complex to form two noncrossover products. However, structure-selective endonucleases (human GEN1 and SMX complex) resolve DNA containing single or double HJs to yield a mixture of crossover and noncrossover products. Finally, we demonstrate that chromatin inhibits the resolution of the double HJ by GEN or SMX while allowing BTRR-mediated dissolution.

Homologous recombination (HR) provides an important mechanism for the repair of DNA double-stranded breaks and the restoration of broken replication forks (1). Recombination in mitotic cells generally occurs between sister chromatids and can lead to the formation of DNA intermediates in which the sisters are covalently linked by four-way DNA junctions, known as Holliday junctions (HJs) (2). Failure to process these DNA intermediates leads to improper chromosome segregation and cell death (3, 4). Recombination also plays an important role in meiotic cells, when interactions occur between homologous chromosomes, and is responsible for the generation of genetic diversity.In mitotic cells, HJs are primarily processed by “dissolution,” in which two adjacent HJs (double Holliday junctions [dHJs]) converge in an adenosine triphosphate (ATP)-hydrolysis–dependent reaction to form a hemicatenane that is subsequently decatenated by topoisomerase action. In human cells, this two-step process involves the BLM–topoisomerase IIIα–RMI1–RMI2 (BTRR) complex (5–7). In yeast, similar reactions are driven by the Sgs1–Top3–Rmi1 (STR) complex (8, 9). Dissolution yields exclusively noncrossover products, which help to maintain the heterozygous state, as a loss of heterozygosity can cause cancer development (10). Mutations in the BLM gene are linked to a human inherited disorder known as Bloom syndrome, which is characterized by short stature, sensitivity to sunlight, and a greatly increased risk of a broad range of cancers (11, 12). In the clinic, patients with Bloom syndrome are diagnosed by a cytogenetic test that detects elevated levels of sister chromatid exchanges. Pathogenic mutations in the TOP3A and RMI1 genes also cause a Bloom syndrome–like disorder, consistent with the fact that these genes participate in the same molecular pathway (12).Persistent HJs that escape processing by BTRR, and single Holliday junctions (sHJs), are resolved by structure-selective endonucleases (SSEs), which specifically recognize and cleave HJs by mediating a nucleolytic attack on two opposing strands at the junction point (2). In humans, these endonucleases include GEN1 and the SMX trinuclease, comprising SLX1–SLX4–MUS81–EME1–XPF–ERCC1 (3, 13–15). Unlike dissolution, resolution gives rise to both crossover and noncrossover products, thereby elevating the frequency of sister chromatid exchanges and increasing the potential for loss of heterozygosity.In contrast to BTRR-mediated dHJ dissolution, which is active throughout the cell cycle, the actions of SMX and GEN1 are tightly regulated. Firstly, SMX complex formation is restricted to prometaphase, as it is dependent upon the phosphorylation of EME1 by CDK1 and PLK1, which stimulates its association with the SLX4 scaffold (15, 16). Secondly, GEN1 is mainly sequestered from the cell nucleus and gains access to DNA after the breakdown of the nuclear envelope during cell division (17).Despite the importance of the BTRR complex in maintaining genetic stability, a detailed picture of dissolution is lacking. Mechanistic studies, using protein complexes from various organisms, led to a model in which two HJs are converged by the branch migration activity of the BLM helicase (18–20). Convergent migration generates positive supercoiling that is relaxed by topoisomerase IIIα and generates a hemicatenane that is processed by topoisomerase IIIα with the aid of RMI1–RMI2 (6–9, 21).Studies of dHJ dissolution have utilized two model systems: 1) a small dHJ prepared by annealing two synthetic oligos (5–7), and 2) a larger plasmid-sized molecule in which two HJs are separated by 165 bp (20, 22). However, the small size of the synthetic DNA substrate eliminates any possibility for branch migration as the two HJs are separated by only 14 bp, raising concerns as to whether these substrates recapitulate the physiological aspects of dissolution (19). The plasmid-sized substrate has been utilized for the dissolution of dHJs by Saccharomyces cerevisiae STR, Drosophila melanogaster BTR, and more recently human BTRR complex (8, 20, 23). However, there is the significant drawback that generation of this substrate is laborious (taking several weeks), requires purified Cre recombinase and reverse gyrase, and leads to low yields of product.To facilitate mechanistic analysis of dissolution and resolution, we developed a rapid and scalable methodology to prepare a 1.8-kb DNA containing single or double HJs. In the dHJ molecules, the two HJs are separated by a maximum of 746 bp of homologous sequence, allowing the two HJs to migrate within the region of homology. We demonstrate that these dHJ molecules are efficiently dissolved by the human BTRR complex to generate noncrossover products. We also show that GEN1 or SMX resolves the single or double HJs to yield the expected mixture of crossover and noncrossover products. Finally, we find that GEN1/SMX are unable to resolve HJs on chromatinized templates, whereas BTRR-mediated dissolution events are unaffected by nucleosome assembly, potentially indicative of an additional level of regulatory control that favors dissolution over resolution.     

The Holliday junction in an inverted repeat DNA sequence: sequence effects on the structure of four-way junctions

Holliday junctions are important structural intermediates in recombination, viral integration, and DNA repair. We present here the single-crystal structure of the inverted repeat sequence d(CCGGTACCGG) as a Holliday junction at the nominal resolution of 2. 1 A. Unlike the previous crystal structures, this DNA junction has B-DNA arms with all standard Watson-Crick base pairs; it therefore represents the intermediate proposed by Holliday as being involved in homologous recombination. The junction is in the stacked-X conformation, with two interconnected duplexes formed by coaxially stacked arms, and is crossed at an angle of 41.4 degrees as a right-handed X. A sequence comparison with previous B-DNA and junction crystal structures shows that an ACC trinucleotide forms the core of a stable junction in this system. The 3'-C x G base pair of this ACC core forms direct and water-mediated hydrogen bonds to the phosphates at the crossover strands. Interactions within this core define the conformation of the Holliday junction, including the angle relating the stacked duplexes and how the base pairs are stacked in the stable form of the junction.     

Use of T4 RNA ligase to construct model substrates for a ribosomal RNA maturation endonuclease

RNase M5 of Bacillus subtilis specifically cleaves a 179-nucleotide precursor 5S rRNA to yield mature 5S rRNA (116 nucleotides) and two fragments derived from the termini. Possible recognition elements for RNase M5 within the precursor structure include nucleotide sequences arranged with 2-fold rotational and translational symmetry about the substrate bonds. We have used bacteriophage T4 RNA ligase to construct, from synthetic oligonucleotides and mature or precursor 5S rRNA fragments, test substrates lacking these symmetry elements. The susceptibilities of the artificial substrates to RNase M5 demonstrate that the symmetrically arranged sequences are not used in the RNase M5 interaction with the precursor. Additionally, the synthetic protocols permitted the invention of an acid-soluble assay for RNase M5 and, potentially, other specific endoribonucleases.     

Template disruptions and failure of double Holliday junction dissolution during double-strand break repair in Drosophila BLM mutants

Previous biochemical studies of the BLM gene product have shown its ability in conjunction with topoisomerase IIIalpha to resolve double Holliday structures through a process called "dissolution." This process could prevent crossing over during repair of double-strand breaks. We report an analysis of the Drosophila BLM gene, DmBlm, in the repair of double-strand breaks in the premeiotic germ line of Drosophila males. With a repair reporter construct, Rr3, and other genetic tools, we show that DmBlm mutants are defective for homologous repair but show a compensating increase in single-strand annealing. Increases of 40- to 50-fold in crossing over and flanking deletions also were seen. Perhaps most significantly, the template used for homologous repair in DmBlm mutants is itself subject to deletions and complex rearrangements. These template disruptions are indicative of failure to resolve double Holliday junctions. These findings, along with the demonstration that a weak allele of topoisomerase IIIalpha has some of the same defects as DmBlm, support the dissolution model. Finally, an analysis of DmBlm mutants in conjunction with mus81 or spnA (Rad51) reveals a second function of BLM distinct from the repair of induced double-strand breaks and possibly related to maintenance of replication forks.