pDUAL: a transposon-based cosmid cloning vector for generating nested deletions and DNA sequencing templates in vivo

We describe a transposon gamma delta-containing cosmid cloning vector, pDUAL (previously called pJANUS), and demonstrate an efficient strategy for isolating nested deletions in both large-scale and small-scale DNA sequencing efforts. This "deletion factory" strategy takes advantage of the ability of gamma delta (Tn1000) to generate deletions that extend from an end of the transposon into adjacent DNA when gamma delta transposes to new sites in the same DNA molecule. pDUAL contains the contraselectable (conditional lethal) sacB+ (sucrose sensitivity) and strA+ (streptomycin sensitivity) genes just outside each end of an engineered gamma delta and selectable kan+ (Kanr) and tet+ (Tetr) genes between the cloning site and sacB and strA, respectively. Selection on sucrose tetracycline medium yields deletions that extend from one gamma delta end for various distances into the cloned DNA, while selection on streptomycin kanamycin medium yields comparable deletions in the other direction. Both types of deletions are recoverable because the essential plasmid replication origin is embedded in the gamma delta component and is thereby retained in each deletion product. Pilot experiments with pDUAL clones showed that deletion end points can be mapped or selected by plasmid size and that both DNA strands of any single clone can be accessed for sequencing by using a pair of universal primers specific for sequences that are just interior to the gamma delta ends.     

Functional characterization of the T4 DNA ligase: a new insight into the mechanism of action

ATP-dependent DNA ligases are essential enzymes in both DNA replication and DNA repair processes. Here we report a functional characterization of the T4 DNA ligase. One N-terminal and two C-terminal deletion mutants were expressed in Escherichia coli as histidine- tagged proteins. An additional mutant bore a substitution of Lys159 in the active site that abolished ATP binding. All the proteins were tested in biochemical assays for ATP-dependent self-adenylation, DNA binding, nick joining, blunt-end ligation and AMP- dependent DNA relaxation. From this analysis we conclude that binding to DNA is mediated by sequences at both protein ends and plays a key role in the reaction. The enzyme establishes two different complexes with DNA: (i) a transient complex (T.complex) involving the adenylated enzyme; (ii) a stable complex (S.complex) requiring the deadenylated T4 DNA ligase. The formation of an S. complex seems to be relevant during both blunt-end ligation and DNA relaxation. Moreover the inactive His-K159L substitution mutant, although unable to self-adenylate, still possesses AMP-dependent DNA nicking activity.     

Cell cycle and genetic requirements of two pathways of nonhomologous end-joining repair of double-strand breaks in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, an HO endonuclease-induced double-strand break can be repaired by at least two pathways of nonhomologous end joining (NHEJ) that closely resemble events in mammalian cells. In one pathway the chromosome ends are degraded to yield deletions with different sizes whose endpoints have 1 to 6 bp of homology. Alternatively, the 4-bp overhanging 3' ends of HO-cut DNA (5'-AACA-3') are not degraded but can be base paired in misalignment to produce +CA and +ACA insertions. When HO was expressed throughout the cell cycle, the efficiency of NHEJ repair was 30 times higher than when HO was expressed only in G1. The types of repair events were also very different when HO was expressed throughout the cell cycle; 78% of survivors had small insertions, while almost none had large deletions. When HO expression was confined to the G1 phase, only 21% were insertions and 38% had large deletions. These results suggest that there are distinct mechanisms of NHEJ repair producing either insertions or deletions and that these two pathways are differently affected by the time in the cell cycle when HO is expressed. The frequency of NHEJ is unaltered in strains from which RAD1, RAD2, RAD51, RAD52, RAD54, or RAD57 is deleted; however, deletions of RAD50, XRS2, or MRE11 reduced NHEJ by more than 70-fold when HO was not cell cycle regulated. Moreover, mutations in these three genes markedly reduced +CA insertions, while significantly increasing the proportion of both small (-ACA) and larger deletion events. In contrast, the rad5O mutation had little effect on the viability of G1-induced cells but significantly reduced the frequency of both +CA insertions and -ACA deletions in favor of larger deletions. Thus, RAD50 (and by extension XRS2 and MRE11) exerts a much more important role in the insertion-producing pathway of NHEJ repair found in S and/or G2 than in the less frequent deletion events that predominate when HO is expressed only in G1.     

Methyl-directed repair of mismatched small heterologous sequences in cell extracts from Escherichia coli

The methyl-directed DNA repair efficiency of a set of M13mp18 heteroduplexes containing 1-8 or 22 unpaired bases was determined by using an in vitro DNA mismatch repair assay. The unpaired bases of each heteroduplex residing at overlapping recognition sites of two restriction endonucleases allow independent assay of repair on either DNA strand. Our results showed that the repair of small nucleotide heterologies in Escherichia coli extracts was very similar to base-base mismatch repair, being strand-specific and highly biased to the unmethylated strand. The in vitro activity was also dependent on products of mutH, mutL, mutS, and uvrD loci and was equally efficient on nucleotide insertions and deletions. The repair levels of small heterologies were affected by base composition of the heterologies. However, the extent of repair of heteroduplexes containing small heterologous sequences was found to decrease with an increase in the number of unpaired bases. Heteroduplexes containing an extra nucleotide of 22 bases provoked very low level of methyl-directed repair.     

Sequence specificity of illegitimate plasmid recombination in Bacillus subtilis: possible recognition sites for DNA topoisomerase I

Previous work in our group indicated that structural plasmid instability in Bacillus subtilis is often caused by illegitimate recombination between non-repeated sequences, characterized by a relatively high AT content. Recently we developed a positive selection vector for analysis of plasmid recombination events in B. subtilis which enables measurement of recombination frequencies without interference of selective growth differences of cells carrying wild-type or deleted plasmids. Here we have used this system to further analyse the sequence specificity of illegitimate plasmid recombination events and to assess the role of the host-encoded DNA topoisomerase I enzyme in this process. Several lines of evidence suggest that single-strand DNA nicks introduced by DNA topoisomerase I are a major source of plasmid deletions in pGP100. First, strains overproducing DNA topoisomerase I showed increased levels of plasmid deletion. Second, these deletions occurred predominantly (>90% of the recombinants) between non-repeated DNA sequences, the majority of which resemble potential DNA topoisomerase I target sites. Sequence alignment of 66 deletion end-points confirmed the previously reported high AT content and, most importantly, revealed a highly conserved C residue at position -4 relative to the site of cleavage at both deletion termini. Based on these genetic data we propose the following putative consensus cleavage site for DNA topoisomerase I of B.subtilis: 5'-A/TCATA/TTAA/TA/TA-3'.     

DNA nick processing by exonuclease and polymerase activities of bacteriophage T4 DNA polymerase accounts for acridine-induced mutation specificities in T4

Acridine-induced frameshift mutagenesis in bacteriophage T4 has been shown to be dependent on T4 topoisomerase. In the absence of a functional T4 topoisomerase, in vivo acridine-induced mutagenesis is reduced to background levels. Further, the in vivo sites of acridine-induced deletions and duplications correlate precisely with in vitro sites of acridine-induced T4 topoisomerase cleavage. These correlations suggest that acridine-induced discontinuities introduced by topoisomerase could be processed into frameshift mutations. The induced mutations at these sites have a specific arrangement about the cleavage site. Deletions occur adjacent to the 3' end and duplications occur adjacent to the 5' end of the cleaved bond. It was proposed that at the nick, deletions could be produced by the 3'-->5' removal of bases by DNA polymerase-associated exonuclease and duplications could be produced by the 5'-->3' templated addition of bases. We have tested in vivo for T4 DNA polymerase involvement in nick processing, using T4 phage having DNA polymerases with altered ratios of exonuclease to polymerase activities. We predicted that the ratios of the deletion to duplication mutations induced by acridines in these polymerase mutant strains would reflect the altered exonuclease/polymerase ratios of the mutant T4 DNA polymerases. The results support this prediction, confirming that the two activities of the T4 DNA polymerase contribute to mutagenesis. The experiments show that the influence of T4 DNA polymerase in acridine-induced mutation specificities is due to its processing of acridine-induced 3'-hydroxyl ends to generate deletions and duplications by a mechanism that does not involve DNA slippage.     

Clinical economics review: the health-care economic implications of minimal access gastrointestinal surgery

Sporadic progressive external ophthalmoplegia and Kearns-Sayre syndrome are usually associated with single large-scale mitochondrial DNA deletions in muscle. In progressive external ophthalmoplegia with autosomal dominant inheritance, multiple mitochondrial DNA deletions have been reported. We studied several members of a Swedish family with autosomal dominant progressive external ophthalmoplegia and multiple mitochondrial DNA deletions by polymerase chain reaction analysis of single muscle fibers and by in situ hybridization, combined with enzyme histochemical analysis. Muscle fiber segments with deficiency of cytochrome c oxidase, which is partially encoded by mitochondrial DNA, had accumulated mitochondrial DNA with deletions and showed reduced levels of wild-type mitochondrial DNA. The deletions varied between individual muscle fibers. There was one predominant deletion in each cytochrome c oxidase-deficient muscle fiber segment. Sequencing of the deletion breakpoints showed that most but not all of the deletions were flanked by direct repeats. Young, clinically affected individuals of this family without limb muscle symptoms did not show mitochondrial DNA deletions or cytochrome c oxidase-deficient muscle fibers. Our results indicate that a nuclear factor predisposes to the development of somatic multiple mitochondrial DNA deletions. Mitochondrial DNA with multiple different deletions shows clonal expansion, which leads to mitochondrial myopathy with ragged-red fibers and muscle weakness.     

Modulation of protein kinase C by taurolithocholic acid in isolated rat hepatocytes

Misalignment of repeated sequences during DNA replication can lead to deletions or duplications in genomic DNA. In Escherichia coli, such genetic rearrangements can occur at high frequencies, independent of the RecA-homologous recombination protein, and are sometimes associated with sister chromosome exchange (SCE). Two mechanisms for RecA-independent genetic rearrangements have been proposed: simple replication misalignment of the nascent strand and its template and SCE-associated misalignment involving both nascent strands. We examined the influence of the 3' exonuclease of DNA polymerase III and exonuclease I on deletion via these mechanisms in vivo. Because mutations in these exonucleases stimulate tandem repeat deletion, we conclude that displaced 3' ends are a common intermediate in both mechanisms of slipped misalignments. Our results also confirm the notion that two distinct mechanisms contribute to slipped misalignments: simple replication misalignment events are sensitive to DNA polymerase III exonuclease, whereas SCE-associated events are sensitive to exonuclease I. If heterologies are present between repeated sequences, the mismatch repair system dependent on MutS and MutH aborts potential deletion events via both mechanisms. Our results suggest that simple slipped misalignment and SCE-associated misalignment intermediates are similarly susceptible to destruction by the mismatch repair system.     

Restriction map of a 35-kb HLA fragment constructed by nested deletion 'drop-out' mapping

An efficient method for generating detailed restriction maps of large cloned DNA segments is demonstrated. The mapping strategy entails comparing restriction fragments from a parent clone and from nested deletion derivatives of that clone. In a set of deletion plasmids of decreasing size, an individual fragment will be lost, or 'drop-out', according to its position in the cloned fragment. In this demonstration, nested deletions were generated in both directions in a 35-kb DNA segment from the human leukocyte antigen (HLA) region by intramolecular transposition of an engineered gamma delta (Tn1000) element present in a special 'deletion factory' cloning vector [Wang et al., Proc. Natl. Acad. Sci. USA 90 (1993) 7874-7878]. Fifteen plasmids with deletions extending in one direction and eleven plasmids with deletions extending in the opposite direction were digested singly by each of four restriction enzymes. A total of 36 cleavage sites were mapped in the 35-kb HLA fragment. This drop-out approach using nested deletions provides a simple and efficient means of mapping restriction sites, genes and other features of interest in cosmid-sized cloned DNA segments or DNAs.     

Exonuclease IX of Escherichia coli removes 3' phosphoglycolate end groups from DNA

The bacteria Escherichia coli contains several exonucleases acting on both double- and single-stranded DNA, and in both a 5'--> 3' and a 3' --> 5' direction. These enzymes are involved in replicative, repair and recombination functions. A new exonuclease recently identified in E. coli, termed exonuclease IX, acts preferentially on single-stranded DNA as a 3'--> 5' exonuclease and also functions as a 3' phosphodiesterase on DNA containing 3' incised apurinic/apyrimidinic (AP) sites to remove the product trans-4-hydroxy-2-pentenal-5-phosphate. We now demonstrate that the enzyme is also able to remove 3' phosphoglycolate end groups from DNA. This activity may have an important role in DNA base excision repair in E. coli.     

Quantitative estimation of sultamicillin p-toluenesulfonate in pharmaceutical preparations by reverse-phase high performance liquid chromatography

Familial growth hormone deficiency type 1A is an autosomal recessive disease, caused by various homogenous deletions of both alleles of growth hormone gene 1 (hGH1). The hGH1 gene deletion is an event occurring between the 5' and the 3' flanking regions by unequal recombination, which causes a deletion in the hGH1 gene, mostly of 6.7 kb and rarely 7.6 or 7.0 kb in size. Two brothers diagnosed with GH deficiency syndrome were sent to our hospital for further evaluation. DNA samples of the probands and controls were amplified by PCR; restriction endonuclease analysis was done with Sma I enzyme and the patterns were evaluated. Gel electrophoresis results showed that the two brothers had a 7.0 kb deletion. These are the third and fourth cases reported with a 7.0 kb deletion. Both patients responded well to replacement therapy and did not develop antibodies against rGH. No other relatives presented with macro deletions in the hGH1 gene.     

Induction of DNA double-strand breaks by restriction enzymes in X-ray-sensitive mutant Chinese hamster ovary cells measured by pulsed-field gel electrophoresis

This investigation was designed to determine whether the cytotoxic effects of different restriction endonucleases are related to the number and type of DNA double-strand breaks (DSBs) they produce. Chinese hamster ovary (CHO) K1 and xrs-5 cells, a radiosensitive mutant of CHO K1, were exposed to restriction endonucleases HaeIII, HinfI, PvuII and BamHI by electroporation. These enzymes represent both blunt and sticky end cutters with differing recognition sequence lengths. The number of DSBs was measured by pulsed-field gel electrophoresis (PFGE). Two forms of PFGE were employed: asymmetric field-inversion gel electrophoresis (AFIGE) for measuring the kinetics of DNA breaks by enzyme digestion and clamped homogeneous gel electrophoresis (CHEF) for examining the size distributions of damaged DNA. The amount of DNA damage induced by exposure to all four restriction enzymes was significantly greater in xrs-5 compared to CHO K1 cells, consistent with the reported DSB repair deficiency in these cells. Since restriction endonucleases produce DSBs alone as opposed to the various types of DNA damage induced by X rays, these results confirm that the repair defect in this mutant involves the rejoining of DSBs. Although the cutting frequency was directly related to the length of the recognition sequence for four restriction enzymes, there was no simple correlation between the cytotoxic effect and the amount of DNA damage produced by each enzyme in either cell line. This finding suggests that the type or nature of the cutting sequence itself may play a role in restriction enzyme-induced cell killing.     

A functional analysis of the Tn5 transposase. Identification of domains required for DNA binding and multimerization

A series of deletions were constructed in the 476 amino acid Tn5 transposase in order to assemble an initial domain structure for this protein. The first four amino acids were found to be important for transposition activity but not for DNA binding to the Tn5 outside end (OE). Larger amino-terminal deletions result in the complete loss of transposition in vivo and the concomitant loss of specific DNA binding. Four point mutants and a six base-pair deletion in the amino terminus between residues 20 and 36 were also found to impair DNA binding to the OE. Analysis of a series of carboxy-terminal deletions has revealed that the carboxy terminus may actually mask the DNA binding domain, since deletions to residues 388 and 370 result in a large increase in DNA binding activity. In addition, the carboxy-terminal deletion to residue 370 results in a significant increase in the mobility of the Tnp-OE complex indicative of a change in the oligomeric state of this complex. Further carboxy-terminal deletions beyond residue 370 also abolished DNA binding activity. These results indicate that the first four amino acids of Tnp are important for transposition but not DNA binding, a region between residues 5 and 36 is critical for DNA binding, the wild-type carboxy terminus acts to inhibit DNA binding, and that a region towards the carboxy terminus, defined by residues 370 to 387, is critical for Tnp multimeric interactions.     

Nick sensing by vaccinia virus DNA ligase requires a 5' phosphate at the nick and occupancy of the adenylate binding site on the enzyme

Vaccinia virus DNA ligase has an intrinsic nick-sensing function. The enzyme discriminates at the substrate binding step between a DNA containing a 5' phosphate and a DNA containing a 5' hydroxyl at the nick. Further insights into nick recognition and catalysis emerge from studies of the active-site mutant K231A, which is unable to form the covalent ligase-adenylate intermediate and hence cannot activate a nicked DNA substrate via formation of the DNA-adenylate intermediate. Nonetheless, K231A does catalyze phosphodiester bond formation at a preadenylated nick. Hence, the active-site lysine of DNA ligase is not required for the strand closure step of the ligation reaction. The K231A mutant binds tightly to nicked DNA-adenylate but has low affinity for a standard DNA nick. The wild-type vaccinia virus ligase, which is predominantly ligase-adenylate, binds tightly to a DNA nick. This result suggests that occupancy of the AMP binding pocket of DNA ligase is essential for stable binding to DNA. Sequestration of an extrahelical nucleotide by DNA-bound ligase is reminiscent of the base-flipping mechanism of target-site recognition and catalysis used by other DNA modification and repair enzymes.     

Camptothecins inhibit the utilization of hydrogen peroxide in the ligation step of topoisomerase I catalysis

The antitumor compounds camptothecin and its derivatives topotecan and irinotecan stabilize topoisomerase I cleavage complexes by inhibiting the religation reaction of the enzyme. Previous studies, using radiolabeled camptothecin or affinity labeling reagents structurally related to camptothecin, suggest that the agent binds at the topoisomerase I-DNA interface of the cleavage complexes, interacting with both the covalently bound enzyme and with the +1 base. In this study, we have investigated the molecular mechanism of camptothecin action further by taking advantage of the ability of topoisomerase I to couple non-DNA nucleophiles to the cleaved strand of the covalent enzyme-DNA complexes. This reaction of topoisomerase I was originally observed at moderate basic pH where active cleavage complexes mediate hydrolysis or alcoholysis by accepting water or polyhydric alcohol compounds as substitutes for a 5'-OH DNA end in the ligation step. Here, we report that a H2O2-derived nucleophile, presumably, the peroxide anion, facilitates the release of topoisomerase I from the cleavage complexes at neutral pH, and we present evidence showing that this reaction is mechanistically analogous to DNA ligation. We find that camptothecin, topotecan, and SN-38 (the active metabolite of irinotecan) inhibit H2O2 ligation mediated by cleavage complexes not containing DNA downstream of the cleavage site, indicating that drug interaction with DNA 3' to the covalently bound enzyme is not strictly required for the inhibition, although the presence of double-stranded DNA in this region enhances the drug effect. The results suggest that camptothecins prevent ligation by blocking the active site of the covalently bound enzyme.     

Deletion between directly repeated DNA sequences measured in extracts of bacteriophage T7-infected Escherichia coli

An in vitro system based upon extracts of bacteriophage T7 infected Escherichia coli was used to study genetic deletions and to examine the importance of DNA replication in the deletion process. When T7 genomes with gene 1.3 inactivated by a 43-bp insert of random sequence DNA bracketed by 11-bp direct repeats were replicated in vitro the inserts were deleted with a frequency of about 10(-5) deletions per genome replication. Under conditions where deletion could take place only by recombination between direct repeats on distinct DNA molecules deletion frequency was at least an order of magnitude lower. These data demonstrate the utility of the in vitro system as a means for examining deletion mechanisms and underscore the importance of DNA replication in deletions.     

Saccharomyces cerevisiae Ku70 potentiates illegitimate DNA double-strand break repair and serves as a barrier to error-prone DNA repair pathways

Ku, a heterodimer of polypeptides of approximately 70 kDa and 80 kDa (Ku70 and Ku80, respectively), binds avidly to DNA double-strand breaks (DSBs). Mammalian cells defective in Ku are hypersensitive to ionizing radiation due to a deficiency in DSB repair. Here, we show that the simple inactivation of the Saccharomyces cerevisiae Ku70 homologue (Yku70p), does not lead to increased radiosensitivity. However, yku70 mutations enhance the radiosensitivity of rad52 strains, which are deficient in homologous recombination. Through establishing a rapid and reproducible in vivo plasmid rejoining assay, we show that Yku70p plays a crucial role in the repair of DSBs bearing cohesive termini. Whereas this damage is repaired accurately in YKU70 backgrounds, in yku70 mutant strains terminal deletions of up to several hundred bp occur before ligation ensues. Interestingly, this error-prone DNA repair pathway utilizes short homologies between the two recombining molecules and is thus highly reminiscent of a predominant form of DSB repair that operates in vertebrates. These data therefore provide evidence for two distinct and evolutionarily conserved illegitimate recombination pathways. One of these is accurate and Yku70p-dependent, whereas the other is error-prone and Yku70-independent. Furthermore, our studies suggest that Yku70 promotes genomic stability both by promoting accurate DNA repair and by serving as a barrier to error-prone repair processes.     

Translesional synthesis on DNA templates containing a single abasic site. A mechanistic study of the "A rule"

Site-specifically modified oligodeoxynucleotides containing a single natural abasic site or a chemically synthesized (tetrahydrofuran or deoxyribitol) model abasic site were used as templates for primer extension reactions catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I or by calf thymus DNA polymerase alpha. Analysis of the fully extended products of these reactions indicated that both polymerases preferentially incorporate dAMP opposite the natural abasic site and tetrahydrofuran, while DNA templates containing the ring-opened deoxyribitol moiety block translesional synthesis, promoting sequence context-dependent deletions. The frequency of nucleotide insertion opposite the three types of abasic sites follows the order dAMP > dGMP > dCMP > dTMP. The frequency of chain extension was highest when dAMP was positioned opposite a natural abasic site. The frequency of translesional synthesis past abasic sites follows the order tetrahydrofuran > deoxyribose > deoxyribitol. The Klenow fragment promotes blunt end addition of dAMP; this reaction was much less efficient than insertion of dAMP opposite an abasic site. We conclude that the miscoding potential of a natural abasic site in vitro closely resembles that of its tetrahydrofuran analog. Ring-opened abasic sites favor deletions. Studies with polymerase alpha in vitro predict preferential incorporation of dAMP at abasic sites in mammalian cells.