Involvement of single-stranded tails in homologous recombination of DNA injected into Xenopus laevis oocyte nuclei.

Homologous recombination of DNA molecules injected into Xenopus laevis oocyte nuclei is extremely efficient when those molecules are linear and have overlapping homologous ends. It was previously shown that a 5'----3' exonuclease activity in oocytes attacks injected linear DNAs and leaves them with single-stranded 3' tails. We tested the hypothesis that such tailed molecules are early intermediates on the pathway to recombination products. Substrates with 3' tails were made in vitro and injected into oocytes, where they recombined rapidly and efficiently. In experiments with mixed substrates, molecules with 3' tails entered recombination intermediates and products more rapidly than did molecules with flush ends. Molecules endowed in vitro with 5' tails also recombined efficiently in oocytes, but their rate was not faster than for flush-ended substrates. In most cases, the 5' tails served as templates for resynthesis of the 3' strands, regenerating duplex ends which then entered the normal recombination pathway. In oocytes from one animal, some of the 5' tails were removed, and this was exacerbated when resynthesis was partially blocked. Analysis by two-dimensional gel electrophoresis of recombination intermediates from 5'-tailed substrates confirmed that they had acquired 3' tails as a result of the action of the 5'----3' exonuclease. These results demonstrate that homologous recombination in oocytes proceeds via a pathway that involves single-stranded 3' tails. Molecular models incorporating this feature are discussed.     

Structure of chromatin at deoxyribonucleic acid replication forks: location of the first nucleosomes on newly synthesized simian virus 40 deoxyribonucleic acid

Exonucleases specific for either 3' ends (Escherichia coli exonuclease III) or 5' ends (bacteriophage T7 gene 6 exonuclease) of nascent DNA chains have been used to determine the number of nucleotides from the actual sites of DNA synthesis to the first nucleosome on each arm of replication forks in simian virus 40 (SV40) chromosomes labeled with [3H]thymidine in whole cells. Whereas each enzyme excised all of the nascent [3H]DNA from purified replicating SV40 DNA, only a fraction of the [3H]DNA was excised from purified replicating SV40 chromosomes. The latter result was attributable to the inability of either exonuclease to digest nucleosomal DNA in native replicating SV40 chromosomes, as demonstrated by the following observations: (i) digestion with either exonuclease did not reduce the amount of newly synthesized nucleosomal DNA released by micrococcal nuclease during a subsequent digestion period; (ii) in briefly labeled molecules, as much as 40% of the [3H]DNA was excised from long nascent DNA chains; (iii) the fraction of [3H]DNA excised by exonuclease III was reduced in proportion to the actual length of the radiolabeled DNA; (iv) the effects of the two exonucleases were additive, consistent with each enzyme trimming only the 3' or 5' ends of nascent DNA chains without continued excision through to the opposite end. When the fraction of nascent [3H]DNA excised from replicating SV40 DNA by exonuclease III was compared with the fraction of [32P]DNA simultaneously excised from an SV40 DNA restriction fragment, the actual length of nascent [3H]DNA was calculated. From this number, the fraction of [3H]DNA excised from replicating SV40 chromosomes was converted into the number of nucleotides. Accordingly, the average distance from either 3' or 5' ends of long nascent DNA chains to the first nucleosome on either arm of replication forks was found to be 125 nucleotides. Furthermore, each exonuclease excised about 80% of the radiolabel in Okazaki fragments, suggesting that less than one-fifth of the Okazaki fragments were contained in nucleosomes. On the basis of these and other results, a model for eukaryotic replication forks is presented in which nucleosomes appear rapidly on both the forward and retrograde arms, about 125 and 300 nucleotides, respectively, from the actual site of DNA synthesis. In addition, it is proposed that Okazaki fragments are initiated on nonnucleosomal DNA and then assembled into nucleosomes, generally after ligation to the 5' ends of long nascent DNA chains is completed.     

The excision of AP sites by the 3'-5' exonuclease activity of the Klenow fragment of Escherichia coli DNA polymerase I

The 3' AP endonucleases (class I) are said to hydrolyze the phosphodiester bond 3' to AP sites yielding 3'-OH and 5'-phosphate ends; on the other hand, the resulting 3' terminal AP site is not removed by the 3'-5' exonuclease activity of the Klenow fragment [1]. We show that AP sites in DNA are easily removed by the 3'-5' exonuclease activity of the Klenow fragment and that they are excised as deoxyribose-5-phosphate. It is suggested that the 3' AP endonucleases are perhaps not the hydrolases they are supposed to be.     

Characterization of an endonuclease IV 3'-5' exonuclease activity

Previous characterization of Escherichia coli endonuclease IV has shown that the enzyme specifically cleaves the DNA backbone at apurinic/apyrimidinic sites and removes 3' DNA blocking groups. By contrast, and unlike the major apurinic/apyrimidinic endonuclease exonuclease III, negligible exonuclease activity has been associated with endonuclease IV. Here we report that endonuclease IV does possess an intrinsic 3'-5' exonuclease activity. The activity was detected in purified preparations of the endonuclease IV protein from E. coli and from the distantly related thermophile Thermotoga maritima; it co-eluted with both enzymes under different chromatographic conditions. Induction of either endonuclease IV in an E. coli overexpression system resulted in induction of the exonuclease activity, and the E. coli exonuclease activity had similar heat stability to the endonuclease IV AP endonuclease activity. Characterization of the exonuclease activity showed that its progression on substrate is sensitive to ionic strength, metal ions, EDTA, and reducing conditions. Substrates with 3' recessed ends were preferred substrates for the 3'-5' exonuclease activity. Comparison of the relative apurinic/apyrimidinic endonuclease and exonuclease activity of endonuclease IV shows that the relative exonuclease activity is high and is likely to be significant in vivo.     

Complete enzymatic synthesis of DNA containing the SV40 origin of replication

The replication of simian virus 40 origin-containing DNA has been reconstituted in vitro with SV40 large T antigen and purified proteins isolated from HeLa cells. Covalently closed circular DNA (RF I') daughter molecules are formed in the presence of T antigen, a single-stranded DNA binding protein and DNA polymerase alpha-primase complex, together with ribonuclease H, DNA ligase, topoisomerase II, and a double-stranded specific exonuclease that has been purified to homogeneity. The 44-kDa exonuclease-digested oligo(rA) annealed to poly(dT) in the 5'----3' direction. DNA ligase and the 5'----3' exonuclease were essential for RF I' formation. Covalently closed circular duplex DNA and full length linear single-stranded DNA were detected by alkaline gel electrophoresis as products of the complete system. DNA replication in the absence of either DNA ligase or the 5'----3' exonuclease yielded DNA products that were half length (approximately 1500 nucleotides) and smaller Okazaki-like fragments (approximately 200 nucleotides). Hybridization experiments showed that the longer chains were synthesized from the leading strand template, while the small products were synthesized from the lagging strand template. These results suggest that the RNA primers attached to 5' ends of replicated DNA are completely removed by the 5'----3' exonuclease, with the assistance of RNase H.     

Characterization of a 3'-5' exonuclease associated with VDJP.

VDJP (V(D)J RSS Dependent DNA Joining Protein) was cloned based on binding to the nonamer portion of the V(D)J recombinational signal sequence (RSS), and genetic analysis revealed that VDJP is encoded by the same gene as the large subunit of Replication Factor C (RF-C). Recombinant VDJP has a site directed DNA joining activity and is capable of forming a covalent bond between DNA fragments containing an RSS element near their ends and exhibits 3' to 5' exonuclease activity. In this report, we examine the biochemical properties of the VDJP exonuclease activity such as directionality of nuclease action (3' to 5' or 5' to 3'), single-strand substrate preference, cleavage products, dependence on cofactors and metal cations, and optimal reaction conditions. From this analysis, we conclude that VDJP has an intrinsic 3'-5' exonuclease activity that produces mononucleotide products.     

Nonsense-mediated mRNA decay in mammalian cells involves decapping, deadenylating, and exonucleolytic activities

Nonsense-mediated mRNA decay (NMD) is a mechanism by which cells recognize and degrade mRNAs that prematurely terminate translation. To date, the polarity and enzymology of NMD in mammalian cells is unknown. We show here that downregulating the Dcp2 decapping protein or the PM/Scl100 component of the exosome (1) significantly increases the abundance of steady-state nonsense-containing but not nonsense-free mRNAs, and (2) significantly slows the decay rate of transiently induced nonsense-containing but not nonsense-free mRNA. Downregulating poly(A) ribonuclease (PARN) also increases the abundance of nonsense-containing mRNAs. Furthermore, NMD factors Upf1, Upf2, and Upf3X coimmunopurify with the decapping enzyme Dcp2, the putative 5'-->3' exonuclease Rat1, the proven 5'-->3' exonuclease Xrn1, exosomal components PM/Scl100, Rrp4, and Rrp41, and PARN. From these and other data, we conclude that NMD in mammalian cells degrades mRNAs from both 5' and 3' ends by recruiting decapping and 5'-->3' exonuclease activities as well as deadenylating and 3'-->5' exonuclease activities.     

DNase I footprinting and enhanced exonuclease function of the bipartite Werner syndrome protein (WRN) bound to partially melted duplex DNA.

Werner syndrome is a premature aging and cancer-prone hereditary disorder caused by deficiency of the WRN protein that harbors 3' -->5' exonuclease and RecQ-type 3' --> 5' helicase activities. To assess the possibility that WRN acts on partially melted DNA intermediates, we constructed a substrate containing a 21-nucleotide noncomplementary region asymmetrically positioned within a duplex DNA fragment. Purified WRN shows an extremely efficient exonuclease activity directed at both blunt ends of this substrate, whereas no activity is observed on a fully duplex substrate. High affinity binding of full-length WRN protects an area surrounding the melted region of the substrate from DNase I digestion. ATP binding stimulates but is not required for WRN binding to this region. Thus, binding of WRN to the melted region underlies the efficient exonuclease activity directed at the nearby ends. In contrast, a WRN deletion mutant containing only the functional exonuclease domain does not detectably bind or degrade this substrate. These experiments indicate a bipartite structure and function for WRN, and we propose a model by which its DNA binding, helicase, and exonuclease activities function coordinately in DNA metabolism. These studies also suggest that partially unwound or noncomplementary regions of DNA could be physiological targets for WRN.     

Degradation of linear DNA by a strand-specific exonuclease activity in Xenopus laevis oocytes.

Linear DNA injected into Xenopus laevis oocyte nuclei recombines with high efficiency if homologous sequences are present at overlapping molecular ends. We found that injected linear DNA was degraded by a 5'----3' strand-specific exonuclease activity during incubation in the oocyte nucleus to leave a heterogeneous population of 3'-tailed molecules. Decreasing the concentration of DNA injected increased the heterogeneity and the average rate of degradation. The 3' tails created were relatively stable; among molecules persisting after overnight incubation, many had 3' tails intact to within 10 bases of the original ends. DNA molecules that were efficient substrates for homologous recombination in oocytes were also partially degraded, leaving 3' tails. We found no evidence for other potent nuclease activities. If molecules with recessed 3'-OH ends were injected, endogenous polymerase efficiently resynthesized complementary strands before degradation of the 5' tails occurred. 3'-tailed molecules are plausible intermediates in the initiation of homologous recombination events in Xenopus oocyte nuclei.     

Mechanism of exonuclease action of BAL 31 nuclease

Two kinetically and molecularly distinct forms ('fast' (F) and 'slow' (S] of nuclease BAL 31 from Alteromonas espejiana effect the length reduction of linear duplex DNAs through a 3'----5'-directed exonuclease activity in conjunction with an endonuclease activity against the 5'-terminated single-stranded tails generated by the exonuclease activity. No evidence for a 5'----3' mode of exonuclease action was seen. Single-stranded DNA is degraded predominantly by the 3'----5' exonuclease action. There is a pronounced decrease, to roughly constant values, of the average lengths of the tails in partially digested duplexes at a constant extent of digestion with increasing nuclease concentration. This decrease correlates with an increasing extent of ligatability, in the absence of repair, under conditions favoring the joining of fully base-paired ends. The exonuclease action, at least against duplex substrates, is quasi-processive and removes approx. 18 and 28 nucleotides per productive enzyme-substrate encounter for the S and F species, respectively. The dependence on Ca2+ and Mg2+ concentrations of the activities has been determined.     

A single-stranded DNA exonuclease from Schizosaccharomyces pombe.

We have purified to near homogeneity a DNA exonuclease from meiotic cells of Schizosaccharomyces pombe. The enzyme, designated exonuclease II (ExoII), had an apparent molecular weight of 134,000 and was abundant in the cell. It specifically degraded single-stranded DNA in the 5'----3' direction with an apparent Km for 5' DNA ends of 3.6 x 10(-11) M and produced 5' deoxynucleoside monophosphates. Its mode of degradation is similar to that of the RecJ protein from Escherichia coli; ExoII may, therefore, be involved in genetic recombination and DNA damage repair.     

RNA-linked nascent DNA pieces in phage T7-infected Escherichia coli. III. Detection of intact primer RNA

RNA-linked DNA fragments of T7-infected Escherichiacoli were labeled with [(32)P]orthophosphate invivo. The RNA segments of the labeled fragments were isolated by degrading the DNA portion with the 3'--> 5' exonuclease intrinsic to bacteriophage T4 DNA polymerase and fractionated according to net charge by a DEAE-Sephadex A-25 column chromatography in the presence of 7 M urea. Tri-, tetra- and pentanucleotides were obtained which have ATP residues at their 5' ends. Most of the pentanucleotides had a single deoxynucleotide at the 3' end but a minor portion was totally an oligoribonucleotide. In the light of prior results, the former is a cooligomer of an intact tetraribonucleotide primer and a monodeoxynucleotide and the latter is an intact pentaribonucleotide primer. Tri- and tetraribonucleotides with ATP at the 5' ends had no deoxynucleotide at the 3' ends, therefore it is not clear if intact triribonucleotide primers are present. The 5'-terminal dinucleotides of the tetra- and pentanucleotides were mostly pppApC and a trace amount of pppApA was present.Images     

Adenovirus terminal protein protects single stranded DNA from digestion by a cellular exonuclease.

Adenovirus 5 DNA-protein complex is isolated from virions as a duplex DNA molecule covalently attached by the 5' termini of each strand to virion protein of unknown function. The DNA-protein complex can be digested with E. coli exonuclease III to generate molecules analogous to DNA replication intermediates in that they contain long single stranded regions ending in 5' termini bound to terminal protein. The infectivity of pronase digested Adenovirus 5 DNA is greatly diminished by exonuclease III digestion. However, the infectivity of the DNA-protein complex is not significantly altered when up to at least 2400 nucleotides are removed from the 3' ends of each strand. This indicates that the terminal protein protects 5' terminated single stranded regions from digestion by a cellular exonuclease. DNA-protein complex prepared from a host range mutant with a mutation mapping in the left 4% of the genome was digested with exonuclease III, hybridized to a wild type restriction fragment comprising the left 8% of the genome, and transfected into HeLa cells. Virus with wild type phenotype was recovered at high frequency.     

Selective affinity chromatography of DNA polymerases with associated 3' to 5' exonuclease activities

The use of 5'-AMP as a ligand for the affinity chromatography of DNA polymerases with intrinsic 3' to 5' exonuclease activities was investigated. The basis for this is that 5'-AMP would be expected to act as a ligand for the associated 3' to 5' exonuclease. The requirements for binding of Escherichia coli DNA polymerase I, T4 DNA polymerase, and calf thymus DNA polymerase delta, all of which have associated 3' to 5' exonuclease activities, to several commercially available 5'-AMP supports with different linkages of 5'-AMP to either agarose or cellulose were examined. The DNA polymerases which possessed 3' to 5' exonuclease activities were bound to agarose types in which the 5'-phosphoryl group and the 3'-hydroxyl group of the AMP were unsubstituted. Bound enzyme could be eluted by either an increase in ionic strength or competitive binding of nucleoside 5'-monophosphates. Magnesium was found to reinforce the binding of the enzyme to these affinity supports. DNA polymerase alpha, which does not have an associated 3' to 5' exonuclease activity, did not bind to any of these columns. These differences can be used to advantage for the purification of DNA polymerases that have associated 3' to 5' exonuclease activities, as well as a means for establishing the association of 3' to 5' exonuclease activities with DNA polymerases.     

Exonuclease associated with bacteriophage T5-Induced DNA polymerase.

T-5-induced DNA polymerase has been shown to possess a 3' leads to 5'-exonucleolytic activity. The exonuclease acts on both native and denatured DNA, but the apparent rate of degradation of denatured DNA is about five times faster than that for native DNA. The enzyme appears to act only on 3'-OH ends and produces mainly 5'-dNMP's. Like polymerase activity, exonuclease activity shows a pH optimum around 8.6. Mg2+, dithiothreitol, and N-ethylmaleimide had identical effects on both the activities. Nicked DNA was almost totally protected from exonuclease action under synthetic conditions, i.e., in the presence of 4dNTP's. Denatured DNA was partly degraded in the early phase of incubation with 4dNTP's, presumably due to unhybridized tails at the 3'-OH primer ends. However, the exonuclease activity was operative in both cases under synthetic conditions, as evidenced by template-dependent conversion of [3H]dTTP to [3H]dTMP.     

Characteristics of the nascent and non-nascent small DNA molecules found in Escherichia coli

We have purified nascent DNA molecules from Escherichia coli pulse-labeled with 5-bromo[6-3H]deoxyuridine by repeated chromatography on nitrocellulose and isopycnic centrifugation in CsCl. The nascent molecules were labeled with 32P either at their 5' ends using polynucleotide kinase or at their 3' ends using terminal transferase. Compared to the non-nascent DNA of normal density, the nascent dense DNA contained a higher proportion of molecules terminated at their 5' ends with ribonucleotides. Exposure of the dense DNA to alkali generated 5' OH termini quantitatively equivalent to the number of molecules bearing 5' ribonucleotides. Experiments designed (1) to detect structures at the 5' ends of phosphatase-treated nascent DNA molecules that caused them to be resistant to hydrolysis by spleen exonuclease or (2) to detect polypeptides that were associated covalently with small DNA molecules and could be iodinated with the Bolton-Hunter reagent did not yield positive results. We conclude that many, if not all, of the intermediates in E. coli DNA replication are initiated with one or more ribonucleotides. The nascent molecules are outnumbered by small non-nascent DNA molecules in the cell, many of which appear to become slightly longer when cells are pulsed with thymidine. Many of the non-nascent DNA molecules behave as if they were self-complementary or crosslinked.     

Pyrimidine dimer excision in Escherichia coli strains deficient in exonucleases V and VII and in the 5'' leads to 3'' exonuclease of DNA polymerase I.

An isogenic series of Escherichia coli strains deficient in various combinations of three 5' leads to 3' exonucleases (exonuclease V, exonuclease VII, and the 5' leads to 3' exonuclease of DNA polymerase I) was constructed and examined for the ability to excise pyrimidine dimers after UV irradiation. Although the recB and recC mutations (deficient in exonuclease V) proved to be incompatible with the polA(Ex) mutation (deficient in the 5' leads to 3' exonuclease of DNA polymerase I), it was possible to reduce the level of the recB,C exonuclease by the use of temperature-sensitive recB270 recC271 mutants. It was found that, by employing strains deficient in exonuclease V, postirradiation DNA degradation could be reduced and dimer excision measurements could be facilitated. Mutants deficient in exonuclease V were found to excise dimers at a rate comparable to that of the wild type. Mutants deficient in exonuclease V and the 5' leads to 3' exonuclease of DNA polymerase I are slightly slower than the wild type at removing dimers accumulated after doses in excess of 40 J/m2. However, although strains with reduced levels of exonuclease VII excised dimers at the same rate as the wild type, the addition of an exonuclease VII deficiency to a strain with reduced levels of exonuclease V and the 5' leads to 3' exonuclease of DNA polymerase I caused a marked decrease in the rate and extent of dimer excision. These observations support previous indications that the 5' leads to 3' exonuclease of DNA polymerase I is important in dimer removal and also suggest a role for exonuclease VII in the excision repair process.     

Characterization of recombination intermediates from DNA injected into Xenopus laevis oocytes: evidence for a nonconservative mechanism of homologous recombination.

Homologous recombination between DNA molecules injected into Xenopus laevis oocyte nuclei is extremely efficient if injected molecules have overlapping homologous ends. Earlier work demonstrated that ends of linear molecules are degraded by a 5'----3' exonuclease activity, yielding 3' tails that participate in recombination. Here, we have characterized intermediates further advanced along the recombination pathway. The intermediates were identified by their unique electrophoretic and kinetic properties. Two-dimensional gel electrophoresis and hybridization with oligonucleotide probes showed that the intermediates had heteroduplex junctions within their homologous overlaps in which strands ending 3' were full length and those ending 5' were shortened. Additional characterization suggested that these intermediates had formed by the annealing of complementary 3' tails. Annealed junctions made in vitro were rapidly processed to products, indicating that they are on the normal recombination pathway. These results support a nonconservative, single-strand annealing mode of recombination. This recombination mechanism appears to be shared by many organisms, including bacteria, fungi, plants, and mammals.     

Genetic recombination: recA protein promotes homologous pairing between duplex DNA molecules without strand unwinding.

The recA protein of Escherichia coli promotes pairing in vitro between covalent circular duplex DNA and homologous circular duplex DNA containing a single stranded region. We have used a filter binding assay to investigate the frequency of homologous pairing between gapped and intact duplex DNA when unwinding of the free 3' and 5' ends of the gapped molecules was blocked. In order to obtain DNA without free ends, the gapped DNA was treated with trimethylpsoralen and 360 nm light so as to introduce about 6 crosslinks per DNA molecule and the double stranded regions on either side of the gaps were then digested up to the first crosslinks with exonuclease III and lambda exonuclease. This treatment did not diminish the frequency of homologous pairing, an observation which is difficult to reconcile with models for recombination requiring strand unwinding before pairing.     

Exonuclease I of Saccharomyces cerevisiae functions in mitotic recombination in vivo and in vitro.

We previously described a 5'-3' exonuclease required for recombination in vitro between linear DNA molecules with overlapping homologous ends. This exonuclease, referred to as exonuclease I (Exo I), has been purified more than 300-fold from vegetatively grown cells and copurifies with a 42-kDa polypeptide. The activity is nonprocessive and acts preferentially on double-stranded DNA. The biochemical properties are quite similar to those of Schizosaccharomyces pombe Exo I. Extracts prepared from cells containing a mutation of the Saccharomyces cerevisiae EXO1 gene, a homolog of S. pombe exo1, had decreased in vitro recombination activity and when fractionated were found to lack the peak of activity corresponding to the 5'-3' exonuclease. The role of EXO1 on recombination in vivo was determined by measuring the rate of recombination in an exo1 strain containing a direct duplication of mutant ade2 genes and was reduced sixfold. These results indicate that EXO1 is required for recombination in vivo and in vitro in addition to its previously identified role in mismatch repair.