Processing of mispaired and unpaired bases in heteroduplex DNA in E. coli

Bacteriophage lambda and phi X 174 DNAs, carrying sequenced mutations, have been used to construct in vitro defined species of heteroduplex DNA. Such heteroduplex DNAs were introduced by transfection, as single copies, into E. coli host cells. The progeny of individual heteroduplex molecules from each infective center was analyzed. The effect of the presence of GATC sequences (phi X 174 system) and of their methylation (lambda system) was tested. The following conclusions can be drawn: some mismatched base pairs trigger the process of mismatch repair, causing a localized strand-to-strand information transfer in heteroduplex DNA: transition mismatches G:T and A:C are efficiently repaired, whereas the six transversion mismatches are not always readily recognized and/or repaired. The recognition of transversion mismatches appears to depend on the neighbouring nucleotide sequence; single unpaired bases (frameshift mutation "mismatches") are recognized and repaired, some equally efficiently on both strands (longer and shorter), some more efficiently on the shorter (-1) strand; large non-homologies (about 800 bases) are not repaired by the Mut H, L, S, U system, but some other process repairs the non-homology with a relatively low efficiency; full methylation of GATC sequences inhibits mismatch repair on the methylated strand: this is the chemical basis of strand discrimination (old/new) in mismatch correction; unmethylated GATC sequences appear to improve mismatch repair of a G:T mismatch in phi X 174 DNA, but there may be some residual mismatch repair in GATC-free phi X 174, at least for some mismatches.(ABSTRACT TRUNCATED AT 250 WORDS)     

One way to do experiments on gene conversion?

Summary Competent cells of B. subtilis were transfected with heteroduplex SPP1 DNA, made by annealing complementary strands of wild type and 21 plaque type mutant DNAs. The frequencies of cells yielding mutant and wild type, only wild type, and only mutant phages were determined by single burst analyses of transfected cells. The data obtained reveal that an effective mechanism is operating in B. subtilis, which converts heterozygous to homozygous molecules prior to their replication. This correction mechanism is asymmetric with regard to the strand which is preferentially corrected in a given heteroduplex pair. The direction of asymmetry thus defined depends on the marker introduced into a particular heteroduplex. The efficiency of correction varies with the markers used and is correlated to the position of markers in the genetic map. From this correlation, the direction of replication of the SPP1 genome is deduced. The frequency distribution of wild type and mutant phages in cells yielding both genotypes indicates that both strands of the input DNA contribute equally to the production of progeny, i.e. DNA replication is symmetric.     

Partial purification of an activity from human cells that promotes homologous pairing and the formation of heteroduplex DNA in the presence of ATP

Summary An activity that can promote homologous pairing and strand transfer between suitable DNA substrates has been partially purified from human skin fibroblasts and from Hela cells. The strand transfer reaction was investigated with DNA substrates consisting of single-stranded circular and duplex linear phage DNA. It requires ATP, and under optimal conditions yields heteroduplex molecules containing one strand from each parental DNA substrate. The reactions appears to be of the same general nature as those mediated by RecA proteins of Escherichia coli and the Rec1 protein of Ustilago maydis.     

Methyl directed DNA mismatch repair inVibrio cholerae

Mismatches in DNA occur either due to replication error or during recombination between homologous but non-identical DNA sequences or due to chemical modification of bases. The mismatch in DNA, if not repaired, result in high spontaneous mutation frequency. The repair has to be in the newly synthesized strand of the DNA molecule, otherwise the error will be fixed permanently. Three distinct mechanisms have been proposed for the repair of mismatches in DNA in prokaryotic cells and gene functions involved in these repair processes have been identified. The methyl-directed DNA mismatch repair has been examined inVibrio cholerae, a highly pathogenic gram negative bacterium and the causative agent of the diarrhoeal disease cholera. The DNA adenine methyltransferase encoding gene (dam) of this organism which is involved in strand discrimination during the repair process has been cloned and the complete nucleotide sequence has been determined.Vibrio cholerae dam gene codes for a 21.5 kDa protein and can substitute for theEscherichia coli enzyme. Overproduction ofVibrio cholerae Dam protein is neither hypermutable nor lethal both in Escherichia coli andVibrio cholerae. WhileEscherichia coli dam mutants are sensitive to 2-aminopurine,Vibrio cholerae 2-aminopurine sensitive mutants have been isolated with intact GATC methylation activity. The mutator genesmutS andmutL involved in the recognition of mismatch have been cloned, nucleotide sequence determined and their products characterized. Mutants ofmutS andmutL ofVibrio cholerae have been isolated and show high rate of spontaneous mutation frequency. ThemutU gene ofVibrio cholerae, the product of which is a DNA helicase II, codes for a 70 kDa protein. The deduced amino acid sequence of themutU gene hs all the consensus helicase motifs. The DNA cytosine methyltransferase encoding gene (dam) ofVibrio cholerae has also been cloned. Thedcm gene codes for a 53 kDa protein. This gene product might be involved in very short patch (VSP) repair of DNA mismatches. The vsr gene which is directly involved in VSP repair process codes for a 23 kDa protein. Using these information, the status of DNA mismatch repair inVibrio cholerae will be discussed.     

Postreplication repair in E. coli: strand exchange reactions of gapped DNA by RecA protein

Summary We have used a sensitive gel electrophoresis assay to detect the products of Escherichia coli RecA protein catalysed strand exchange reactions between gapped and duplex DNA molecules. We identify structures that correspond to joint molecules formed by homologous pairing, and show that joint molecules are converted by RecA protein into heteroduplex monomers by reciprocal strand exchanges. However, strand exchanges only occur when there is a 3-terminus complementary to the single stranded DNA in the gap. In the absence of a complementary free end, the two DNA molecules pair and short heteroduplex regions are formed by localised interwinding.     

Mutagenesis by aflatoxin in M13 DNA: Base-substitution mechanisms and the origin of strand bias

Summary The two goals of the experiments described here are: (a) to examine whether there is a strand bias in mutagenic processing of bulky lesions in M13 replicative form (RF) DNA, and (b) to examine the mutational mechanisms of metabolically activated aflatoxin. For these experiments, two types of nicked heteroduplex M13 RF DNA molecules (+WT/-am1 and +am1/-WT) in which either the minus (-) or the plus (+) strand carried a gene 1 amber nonsense codon, were constructed. Heteroduplex DNAs were modified in vitro with aflatoxin B₁ activated by hamster liver S9 enzymes, and transfected into SOS(UV)-induced Escherichia coli (Sup^o/uvrA^-/mucAB⁺). Forward mutations in the lacZ -complementing gene segment were scored and sequenced. Results indicated that aflatoxin-induced mutation frequencies in the +WT/-am1 heteroduplex were significantly greater than those in the +am1/-WT heteroduplex, suggesting more efficient mutagenic processing of lesions in the plus strand. These results permit specific suggestions for improved mutation detection in the extensively used M13 forward mutagenesis system. Sequence analysis of point mutations from the +WT/-am1 experiments showed that most substitutions were targeted to plus-strand guanines. Both G-to-A transitions and G-to-T transversions were induced with equal effeciency. Since activated aflatoxin B₁ is known to react almost exclusively with DNA guanines at the N7 position, these results suggest that bulky lesions at guanine N7 position may have the properties of mis-instructional as well as non-instructional lesions.     

Transfection of heteroduplexes containing uracil · guanine or thymine · guanine mispairs into plant cells

We have compared the fate of U · G mispairs or analogous T · G mispairs in DNA heteroduplexes transfected into tobacco protoplasts. The heteroduplex DNA consisted of tomato golden mosaic virus DNA sequences in theEscherichia coli vectors pUC118 or pUC119. After transfection, the mismatched U residues were lost with an efficiency of greater than 95%, probably as a result of the uracil-DNA glycosylase pathway for excision of U residues in any sequence context. In contrast to the preferential removal of the mispaired U residues, biased removal of T residues from analogous heteroduplexes was not seen in the transfected plant cells. Also, we investigated the effect of extensively methylating one strand of the heteroduplex DNA used for transfection. Surprisingly, such methylation resulted in highly biased loss of the mismatched base from the 5-methylcytosine-rich strand of T · G-containing heteroduplexes.Deceased. We dedicate this paper to the memory of this young scientist.     

Strand targeting signal(s) for in vivo mutation avoidance by post-replication mismatch repair in Escherichia coli

Summary The involvement of GATC sites in directing mismatch correction for the elimination of replication errors in Escherichia coli was investigated in vivo by analyzing mutation rates for a gene carried on a series of related plasmids that contain 2, 1 and 0 such sites. This gene encoding chloramphenicol acetyl transferase (Cat protein) was inactivated by a point mutation. In vivo mutations restoring resistance to chloramphenicol were scored in mismatch repair proficient (mut ⁺) and deficient (mutHLS^-) strains. In mut ⁺ cells, reduction of GATC sites from 2 to 0 increased mutation rates approximately 10-fold. Removal of the GATC site distal to the cat ^- mutation increased the rate of mutation less than 2-fold, indicating that mismatch repair can proceed normally with a single site. The mutation rate increased 3-fold after removal of the GATC site proximal to the mutation. In the absence of a GATC site, mutL^- and mutS^- strains exhibited a 2- to 3-fold increased mutation rate as compared to isogenic mutH^- and mut ⁺ strains. This indicates that 50%–70% of replication errors can be corrected in a mutLS-dependent way in the absence of any GATC site to target mismatch correction to newly synthesized DNA strands. Other strand targeting signals, possibly single strand discontinuities, might be used in mutLS-dependent repair     

Analysis of single-stranded DNA stability and damage-induced strand loss in mammalian cells using SV40-based shuttle vectors

The fate and stability of fully or partially single-stranded DNA molecules transfected into mammalian cells have been analysed. For this, we constructed a simian virus 40 (SV40)-based shuttle vector containing the f1 bacteriophage replication origin in the two possible orientations (pi SVF1-A and pi SVF1-B). This vector contains the SV40 origin of replication, the late viral genes and DNA sequences for replication and selection in Escherichia coli. It also carries the lacO sequence, which permits the analysis of plasmid stability. Single-stranded DNA from pi SVF1-A and pi SVF1-B were produced in bacteria and annealed in vitro to form a heteroduplex molecule. We showed that, in monkey kidney COS7 cells, single-stranded vectors replicate to form duplex molecules. After transfection of the three forms of molecules (single-stranded, heteroduplex or double-stranded), replicated DNA was rescued in E. coli. Vector stability was analysed by checking for plasmid rearrangements and screening for lacO mutants. The single-stranded pi SVF1 has a lower rearrangement level, while the spontaneous mutation frequency (on the lacO target) is in the same range as for the double-stranded vector. In contrast, the level of spontaneous mutagenesis is higher for the heteroduplex than for the single- and double-stranded forms. In addition, we found that replication of heteroduplex with one strand containing ultraviolet light-induced lesions yields progeny molecules in which the irradiated strand is mostly lost. This result indicates for the first time the specific loss of the damaged strand in mammalian cells.     

RecA-mediated joint molecule formation between O-methylated RNA/DNA hairpins and single-stranded targets

Chimeric oligonucleotides consisting of one DNA strand paired with an O-methylated RNA strand interrupted by six DNA residues have been used in gene targeting experiments. Here we demonstrate that these hairpins can form a heteroduplex (or joint molecule) with single-stranded DNA targets in a reaction mediated by the E. coli RecA protein. One end of the double hairpin may unwind to form a 14-base-RecA filament which initiates the reaction. Chimeric oligonucleotides containing only O-methylated RNA residues on one strand or truncated hairpins lacking this 14-base segment did not participate in RecA-driven heteroduplex formation under these reaction conditions. The results presented here represent a first step in studying one facet of a strategy which uses O-methylated RNA residues as participants in homologous pairing events. Received: 2 June 1997 / Accepted: 29 September 1997     

Chromatographically Fractionated Complementary Strands of Bacillus subtilis Deoxyribonucleic Acid: Transformation of Hybrids

The annealing properties as measured by the restoration of transforming activity and hypochromicity of methylated albumin-kieselguhr (MAK)-fractionated complementary strands of Bacillus subtilis deoxyribonucleic acid (DNA) are presented. Temperature-absorbance measurements performed on annealed mixtures of various L and H strand fractions indicated the existence of a complementarity gradient between the two MAK peaks. The markers purA16, leu-8, metB(5), thr-5, and the linked marker hisB(2)-try-2 exhibited different bimodal distributions on MAK columns. The transforming efficiency of heteroduplex mixtures, prepared by cross-annealing resolved complementary strands of wild-type and recipient DNA, was compared. The transforming efficiency of the wild-type L and H strands was equal in one preparation and unequal in a second preparation. It was found that in the second strand preparation the heteroduplex DNA containing the H strand from wild type was more efficient for all of the markers tested. The variations in transforming efficiencies of the complementary strands in heteroduplex molecules reported here and by others are due in part to strands of unequal length and probably to the self-annealing property of the H strands. At present, no conclusion could be made regarding the existence of strand selection bias during integration of donor DNA in competent B. subtilis cells.     

Excision and repair of mismatched base pairs in transformation of Streptococcus pneumoniae

Summary The use of heteroduplex DNA molecules as donors in pneumococcal transformation makes it possible to follow the fate of each DNA strand. The integration efficiency of each strand depends strongly upon the single base changes it carries. The function (hex) which reduces drastically the transformation yield of markers referred to as low efficiency (LE) tends to remove either donor strand without respect to which one is introduced. In the case of high efficiency (HE) markers the reduction in the transformation yield involves the elimination of only one donor strand. For a given locus it can be either one depending upon the mutation. The reduction in transformation yield can be less drastic for HE markers than for both strands of the LE markers. These data are discussed in terms of differences in the affinity for mismatched base pairs.We have studied the transfer of information from each donor DNA strand to the recipient genome, on the basis of differences in the rates of phenotypic expression of a given marker introduced on opposite strands. Results show that, as in the case of LE markers, the information from HE markers, when introduced on the strand recognized by the hex function, is transmitted to both strands of the recipient molecule. Correction of the recipient strand to homozygosis probably accounts for this information transfer. These results, together with earlier investigations, strongly suggest that the hex function is an excision-repair system acting on donor-recipient base pair mismatches.     

Large non-homology in heteroduplex DNA is processed differently than single base pair mismatches

Summary Unmethylated DNA heteroduplexes with a large single stranded loop in one strand have been prepared from separated strands of DNA from two different strains of bacteriophage , one of which has a 800 base pair IS1 insertion in the cI gene. The results of transfections with these heteroduplexes into wild-type and mismatch repair deficient bacteria indicate that such large non-homologies are not repaired by the Escherichia coli mismatch repair system. However, the results do suggest that some process can act to repair such large non-homologies in heteroduplex DNA. Transfections of a series of recombination and excision repair deficient mutants suggest that known excision or recombination repair systems of E. coli are not responsible for the repair. Repair of large non-homologies may play a role in gene conversion involving large insertion or deletion mutations.     

Sequence‐dependent cleavage of mismatched DNA by Ban I restriction endonuclease

Restriction enzymes have previously shown the ability to cleave DNA substrates with mismatched base(s) in recognition sequences; in this study, Ban I endonuclease demonstrated this same ability. Single base substitutions were introduced, and fragments containing various types of unpaired base(s) (heteroduplex fragments) within the Ban I endonuclease recognition sequence, 5′‐G|GPyPuCC‐3′, were generated. Each of the heteroduplex fragments was treated with Ban I endonuclease and analyzed by denaturing gradient gel electrophoresis. Our results showed that heteroduplex fragments containing mismatched bases at either the first or third position of the Ban I recognition sequence or, because of the symmetrical structure of the sequence, the sixth or fourth position on the opposite strand were cleaved by the enzyme. Furthermore, these cleaved fragments contained at least one strand corresponding to the original Ban I recognition sequence. Fragments with mismatches formed by an A (noncanonical , nc ) opposite a purine (canonical , ca ) or a T (nc ) opposite a pyrimidine (ca ) were cleaved more efficiently than other types of mismatched bases. These results may help elucidate the mechanisms by which DNA and protein interact during the process of DNA cleavage by Ban I endonuclease.     

Role of SSB protein in RecA promoted branch migration reactions

Summary The RecA protein ofEscherichia coli is essential for genetic recombination and postreplicational repair of DNA. In vitro, RecA protein promotes strand transfer reactions between full length linear duplex and single stranded circular DNA of X174 to form heteroduplex replicative form II-like structures (Cox and Lehman 1981a). In a similar way, it transfers one strand of a short duplex restriction fragment to a single stranded circle. Both reactions require RecA and single strand binding protein (SSB) in amounts sufficient to saturate the ssDNA. The rate and extent of strand transfer is enhanced considerably when SSB is added after preincubation of the DNA with RecA protein. In contrast, SSB protein is not required for RecA protein catalysed reciprocal strand exchanges between regions of duplex DNA. These results indicate that while SSB is necessary for efficient transfer between linear duplex and ssDNA to form a single heteroduplex, it is not required for branch migration reactions between duplex molecules that form two heteroduplexes.Abbreviations SSB single strand binding protein - ssDNA single stranded DNA - X phage X174 - bp base pairs - ATP[S] adenosine 5-O-(gamma-thiotriphosphate)     

Escherichia coli mutY-dependent mismatch repair involves DNA polymerase I and a short repair tract

Repair of heteroduplex DNA containing an A/G mismatch in a mutL background requires the Escherichia coli mutY gene function. The mutY-dependent in vitro repair of A/G mismatches is accompanied by repair DNA synthesis on the DNA strand bearing mispaired adenines. The size of the mufY-dependent repair tract was measured by the specific incorporation of -[³²P]dCTP into different restriction fragments of the repaired DNA. The repair tract is shorter than 12 nucleotides and longer than 5 nucleotides and is localized to the 3 side of the mismatched adenine. This repair synthesis is carried out by DNA polymerase I.     

RecA-mediated strand exchange reactions between duplex DNA molecules containing damaged bases, deletions, and insertions

RecA protein from Escherichia coli promotes homologous pairing and strand exchange between duplex DNA molecules if one is partially single-stranded. Using linear duplexes and circles with a single-stranded gap as the substrates, this reaction generates nicked circular heteroduplex DNA and linear molecules with single-stranded ends. The completion of strand exchange can be demonstrated by the production of nicked circular heteroduplex DNA detected by gel electrophoresis and autoradiography using radiolabeled linear molecules. When the effect of ultraviolet damage to the substrate DNA was tested, strand exchange was found to pass 30 or more pyrimidine dimers in each duplex. In contrast, exchanges were blocked or severely slowed by interstrand cross-links and monoadducts produced by psoralen and 360 nm light. Deletions and insertions of from 4 to 38 base pairs in the DNA substrates had little effect on the production of nicked circular heteroduplex DNA. However, those of 120 base pairs, or greater, reduced the product yield to a level below the threshold of detection. These results contrast with those obtained in related three-stranded reactions (Bianchi, M. E., and Radding, C. M. (1984) Cell 35, 511-520), in which stable heteroduplex products with 500 or 1300 unpaired bases were obtained when the insert was located within a single-stranded circular substrate.     

Identification of template strand in heteroduplex DNA directing cell-free enzyme synthesis

Summary Cell-free synthesis of galactose enzymes was directed by heteroduplex DNA carrying an amber mutation in thegal T gene on one DNA strand, the wildtype configuration on the complementary strand. Comparison with the corresponding homoduplex DNA preparations shows that the presence of the wildtype base sequence in the l-strand is necessary and sufficient for the synthesis of active enzyme. Single stranded DNA of either configuration is inactive in this system.     

Complexes formed between the restriction endonuclease EcoK and heteroduplex DNA

The complexes between the Escherichia coli K restriction endonuclease and heteroduplex DNA (one strand methylated and one unmethylated) have been characterized and shown to have different properties from those formed with unmodified DNA. The nature of the heteroduplex complex appears to commit the enzyme to its methylase mode.