Repair of single-stranded DNA nicks, gaps, and loops in mammalian cells.

We studied the ability of mammalian cells to repair single-stranded nicks, gaps, and loops in DNA duplexes. Heteroduplexes prepared from derivatives of the shuttle vector pSV2neo were introduced into monkey COS cells. After replication, the plasmids were recovered and used to transform Escherichia coli. Plasmid DNA from the recovered colonies was tested for repair at each of six different sites. We observed that mammalian cells are capable of repairing single-stranded gaps and free single-stranded ends most efficiently. Regions containing twin loops were recognized, and one of the loops was excised. Portions of the molecules containing small single loops were also repaired. Markers which were 58 nucleotides apart were corepaired with nearly 100% efficiency, while markers which were 1,000 nucleotides or more apart were never corepaired. The mechanisms involved in heteroduplex repair in mammalian cells seem to be similar to those involved in repairing DNA lesions caused by physical and chemical agents.     

Analysis by electron microscopy of the variable segment in the maxi-circle of kinetoplast DNA from Trypanosoma brucei

The 20.5-kbp maxi-circle from the kinetoplast DNA of Trypanosoma brucei contains a 5-kbp segment which is not cut by most restriction endonucleases and which varies in size in closely-related trypanosome strains (Borst, P., Fase-Fowler, F., Hoeijmakers, J.H.J. and Frasch, A.C.C. (1980) Biochim. Biophys. Acta 610, 197–210). We have now analysed partial denaturation maps of the linearized maxi-circles by electron microscopy and find that the variable segment is not more AT-rich than the remainder of the maxi-circle. Early denaturation begins at two separate regions of the maxi-circle outside the variable region and one of these corresponds with the position of the gene for the large (12 S) ribosomal RNA. Denaturation-renaturation of maxi-circles leads to the formation of partially mismatched duplexes that look like underwound loops in electron micrographs. These loops are only found in the variable region and they vary in size and appearance. Under our renaturation conditions single-stranded maxi-circle DNA is devoid of secondary structure and this suggests that the underwound loops arise by misalignment of straight tandem repeats in the DNA. We have also analysed heteroduplexes between maxi-circles from two closely related T. brucei strains that differ by 1 kbp in the size of their variable segment. Most molecules had no underwound loops and contained mismatched regions in the variable segment only. The appearance of these regions is diverse, varying from fully duplex with two single-stranded loops to molecules with a heterogeneous array of smaller loops. The total size of single-stranded DNA in the heteroduplexes may be as high as 1.2 μm, i.e., a factor 4 higher than the size difference between the heteroduplex partners. We conclude that the variable region consists of imperfect tandem repeats of a sequence that evolves rapidly. This region might contain the origin of maxi-circle replication.     

Efficient repair of large DNA loops in Saccharomyces cerevisiae

Small looped mispairs are efficiently corrected by mismatch repair. The situation with larger loops is less clear. Repair activity on large loops has been reported as anywhere from very low to quite efficient. There is also uncertainty about how many loop repair activities exist and whether any are conserved. To help address these issues, we studied large loop repair in Saccharomyces cerevisiae using in vivo and in vitro assays. Transformation of heteroduplexes containing 1, 16 or 38 nt loops led to >90% repair for all three substrates. Repair of the 38 base loop occurred independently of mutations in key genes for mismatch repair (MR) and nucleotide excision repair (NER), unlike other reported loop repair functions in yeast. Correction of the 16 base loop was mostly independent of MR, indicating that large loop repair predominates for this size heterology. Similarities between mammalian and yeast large loop repair were suggested by the inhibitory effects of loop secondary structure and by the role of defined nicks on the relative proportions of loop removal and loop retention products. These observations indicate a robust large loop repair pathway in yeast, distinct from MR and NER, and conserved in mammals.     

Single-stranded DNA gaps, tails and loops are repaired in Escherichia coli

Uniformly methylated heteroduplex plasmids which contained 6 mismatched regions, including loops of 24, 30, 248 and 283 nucleotides, as well as single-stranded gaps and free ends were introduced into a recombination-deficient strain of bacteria, and the products of repair were analyzed. The results indicate that these cells are capable of repairing all of these structures, although with different efficiencies. Repair of single-stranded gaps and free ends, which occurs most efficiently, is always associated with acquisition of information from the uncut strand (unidirectional repair). Regions containing single loops or twin loops were repaired at similar efficiencies. In these cases each of the two strands was capable of acting as the template for repair (bidirectional repair). At sites containing twin or substitution loops, the larger of the loops was removed twice as efficiently as the smaller loop. DNA sequencing of the repaired regions indicated that the repair is precise. The data also suggest that markers separated by only 58 nucleotides do not always segregate together indicating that repair tracts may be relatively short.     

Methyl-directed repair of frameshift heteroduplexes in cell extracts from Escherichia coli.

The methyl-directed DNA repair efficiency of a series of M13mp9 frameshift heteroduplexes 1, 2, or 3 unpaired bases was determined by using an in vitro DNA mismatch repair assay. Repair of hemimethylated frameshift heteroduplexes in vitro was directed to the unmethylated strand; was dependent on MutH, MutL, and MutS; and was equally efficient on base insertions and deletions. However, fully methylated frameshift heteroduplexes were resistant to repair, while totally unmethylated substrates were repaired with no strand bias. Hemimethylated 1-, 2-, or 3-base insertion and deletion heteroduplexes were repaired by the methyl-directed mismatch repair pathway as efficiently as the G.T mismatch. These results are consistent with earlier in vivo studies and demonstrate the involvement of methyl-directed DNA repair in the efficient prevention of frameshift mutations.     

The large loop repair and mismatch repair pathways of Saccharomyces cerevisiae act on distinct substrates during meiosis

During meiotic recombination in the yeast Saccharomyces cerevisiae, heteroduplex DNA is formed when single-stranded DNAs from two homologs anneal as a consequence of strand invasion. If the two DNA strands differ in sequence, a mismatch will be generated. Mismatches in heteroduplex DNA are recognized and repaired efficiently by meiotic DNA mismatch repair systems. Components of two meiotic systems, mismatch repair (MMR) and large loop repair (LLR), have been identified previously, but the substrate range of these repair systems has never been defined. To determine the substrates for the MMR and LLR repair pathways, we constructed insertion mutations at HIS4 that form loops of varying sizes when complexed with wild-type HIS4 sequence during meiotic heteroduplex DNA formation. We compared the frequency of repair during meiosis in wild-type diploids and in diploids lacking components of either MMR or LLR. We find that the LLR pathway does not act on single-stranded DNA loops of <16 nucleotides in length. We also find that the MMR pathway can act on loops up to 17, but not >19, nucleotides in length, indicating that the two pathways overlap slightly in their substrate range during meiosis. Our data reveal differences in mitotic and meiotic MMR and LLR; these may be due to alterations in the functioning of each complex or result from subtle sequence context influences on repair of the various mismatches examined.     

Sepcific fragmentation of DNA heteroduplex molecules of two bacteriophage lambda mutants with endonuclease Si from Aspergillus oryzae.

Heteroduplex DNA molecules of two bacteriophage mutants (lambda b2 and lambda i434ct68) were obtained by the method of molecular hybridization. These heteroduplexes possessed two types of loops formed as a result of: a) deletion in one of the DNA strands; and b) substitution of a DNA fragment for nonhomological one. The digestion of heteroduplexes with single-stranded specific nuclease SI from Aspergillus oryzae produced two fragments at 37 degrees C and three ones at 55 degrees C. The separation of fragments and determination of their molecular weight were carried out by means of electrophoresis in agarose. The molecular weights both measured and preliminarily calculated proved to be close. One of the fragments was identificated by its biological activity in CaCl2-dependent infectious system with helperphage.     

A rapid and efficient method for region- and strand-specific mutagenesis of cloned DNA.

The single-stranded viral DNA of an M13 phage recombinant containing the early promoter region of SV40 was hybridized with linear, double-stranded replicative form DNA of a related M13 phage containing a short deletion in the cloned SV40 sequence. The heteroduplexes formed between these DNA molecules contained a short, defined single-stranded region in an otherwise duplex molecule. These heteroduplexes were treated with sodium bisulphite to deaminate exposed unpaired cytosines to uracil residues. The single-stranded region was filled in with DNA polymerase I, which incorporates adenine opposite the mutated uracils, and the DNA then transfected into the M13 host JM103 . Viral DNA from the resultant plaques was used for the rapid dideoxy-DNA sequencing procedure; all of the plaques studied contained point mutations within the desired area. This method allows the very rapid and efficient generation of region-directed point mutants which can be quickly sequenced.     

Assembling long heteroduplexes by asymmetric polymerase chain reaction and annealing the resulting single-stranded DNAs

We developed an effective protocol for generating high-purity heteroduplexes via annealing single-stranded DNAs (ssDNAs) derived from plasmid DNA by asymmetric polymerase chain reaction (A-PCR). With the addition of dimethyl sulfoxide, a one-step A-PCR procedure can generate ssDNAs stably at a range of reaction temperatures. Several annealing buffers can anneal two ssDNAs into heteroduplexes effectively. We further developed a simple strategy to create d(GATC) hemimethylated heteroduplexes by annealing fully methylated homoduplexes in the presence of excessive unmethylated ssDNAs. The constructed heteroduplexes have been well tested as substrates for mismatch repair in Escherichia coli and, thus, can be used in various biotechnology applications.     

Infectivity of Lambda Heteroduplex Deoxyribonucleic Acid Molecules

Heteroduplex deoxyribonucleic acid (DNA) molecules were formed in vitro by denaturing and renaturing a mixture of DNAs from a variety of lambda strains. The infectivity of these heteroduplexes was studied by using host-controlled modification and restriction to prevent infection by contaminating parental homoduplexes. Either strand was able to protect against degradation by restriction nucleases in vivo. A large proportion of progeny phage, which had been produced after infection with heteroduplexes containing noncomplementary base pairs at multiple loci, retained the genotype of one of the parental homoduplexes. The results indicate that conversion of heteroduplexes to homoduplexes in vivo by a DNA repair mechanism does not occur frequently. Molecules heterozygous for the c17 or vir operator mutations were not infectious; it is suggested that these mutations involve multiple base pairs.     

Correction of large mispaired DNA loops by extracts of Saccharomyces cerevisiae.

Single base mispairs and small loops are corrected by DNA mismatch repair, but little is known about the correction of large loops. In this paper, large loop repair was examined in nuclear extracts of yeast. Biochemical assays showed that repair activity occurred on loops of 16, 27, and 216 bases, whereas a G-T mispair and an 8-base loop were poorly corrected under these conditions. Two modes of loop repair were revealed by comparison of heteroduplexes that contained a site-specific nick or were covalently closed. A nick-stimulated repair mode directs correction to the discontinuous strand, regardless of which strand contains the loop. An alternative mode is nick-independent and preferentially removes the loop. Both outcomes of repair were largely eliminated when DNA replication was inhibited, suggesting a requirement for repair synthesis. Excision tracts of 100-200 nucleotides, spanning the position of the loop, were observed on each strand under conditions of limited DNA repair synthesis. Both repair modes were independent of the mismatch correction genes MSH2, MSH3, MLH1, and PMS1, as judged by activity in mutant extracts. Together the loop specificity and mutant results furnish evidence for a large loop repair pathway in yeast that is distinct from mismatch repair.     

Mutation spectrum following transfection of ultraviolet-irradiated single-stranded or double-stranded shuttle vector DNA into monkey cells

We designed a shuttle vector system that allowed a comparison of the mutation spectrum on the supF target gene after transfection of single-stranded or double-stranded DNA into monkey cells. Single-strand-derived plasmids exhibited a spontaneous mutation frequency tenfold higher than double-strand-derived ones. These spontaneous mutations comprised deletions and point substitutions. This system was applied to the study of ultraviolet-induced mutagenesis. Single-stranded DNA exhibited a lower survival and a higher mutation frequency than double-stranded DNA after identical ultraviolet-irradiation. The use of single-stranded DNA allowed us to confirm and complete the data about the targeting of ultraviolet-induced mutations and the exact nature of the base changes involved. One class of mutations was more frequent after transfection of ultraviolet-irradiated single-stranded DNA than for double-stranded DNA: frameshifts represented 10% of the mutants. Multiple mutations, attributed by some authors to an error-prone excision repair process, have also been observed in the spontaneous and ultraviolet-induced mutation spectra following single-stranded DNA transfection, although it cannot be a direct substrate for excision repair.     

Mismatch cleavage by single-strand specific nucleases

We have investigated the ability of single-strand specific (sss) nucleases from different sources to cleave single base pair mismatches in heteroduplex DNA templates used for mutation and single-nucleotide polymorphism analysis. The TILLING (Targeting Induced Local Lesions IN Genomes) mismatch cleavage protocol was used with the LI-COR gel detection system to assay cleavage of amplified heteroduplexes derived from a variety of induced mutations and naturally occurring polymorphisms. We found that purified nucleases derived from celery (CEL I), mung bean sprouts and Aspergillus (S₁) were able to specifically cleave nearly all single base pair mismatches tested. Optimal nicking of heteroduplexes for mismatch detection was achieved using higher pH, temperature and divalent cation conditions than are routinely used for digestion of single-stranded DNA. Surprisingly, crude plant extracts performed as well as the highly purified preparations for this application. These observations suggest that diverse members of the S₁ family of sss nucleases act similarly in cleaving non-specifically at bulges in heteroduplexes, and single-base mismatches are the least accessible because they present the smallest single-stranded region for enzyme binding. We conclude that a variety of sss nucleases and extracts can be effectively used for high-throughput mutation and polymorphism discovery.     

Bacterial genes mutL, mutS, and dcm participate in repair of mismatches at 5-methylcytosine sites.

Certain amber mutations in the cI gene of bacteriophage lambda appear to recombine very frequently with nearby mutations. The aberrant mutations included C-to-T transitions at the second cytosine in 5'CC(A/T)GG sequences (which are subject to methylation by bacterial cytosine methylase) and in 5'CCAG and 5'CAGG sequences. Excess cI+ recombinants arising in crosses that utilize these mutations are attributable to the correction of mismatches by a bacterial very-short-patch (VSP) mismatch repair system. In the present study I found that two genes required for methyladenine-directed (long-patch) mismatch repair, mutL and mutS, also functioned in VSP mismatch repair; mutH and mutU (uvrD) were dispensable. VSP mismatch repair was greatly reduced in a dcm Escherichia coli mutant, in which 5-methylcytosine was not methylated. However, mismatches in heteroduplexes prepared from lambda DNA lacking 5-methylcytosine were repaired in dcm+ bacteria. These results indicate that the product of gene dcm has a repair function in addition to its methylase activity.     

Functional characterization of the DNA mismatch binding protein MutS from Haemophilus influenzae

This investigation demonstrates DNA mismatch repair activity in Haemophilus influenzae cell free extracts. The mutS gene as well as purified protein of H. influenzae restored repair activity in complementation assays performed with mutS deficient Escherichia coli strain. The difference in affinity for GT and AC mismatched bases by H. influenzae MutS was reflected in the efficiency with which these DNA heteroduplexes were repaired in vitro, with GT being repaired well and AC the least. Unlike E. coli MutS, the H. influenzae homolog failed to give protein-DNA complex with homoduplex DNA. Interestingly, MutS was found to bind single-stranded DNA but with lesser affinity as compared to heteroduplex DNA. Apart from the nucleotide- and DNA-mediated conformational transitions, as monitored by circular dichroism and limited proteolysis, our data suggest a functional role when H. influenzae MutS encounters single-stranded DNA during exonucleolytic step of DNA repair process. We propose that, conformational changes in H. influenzae MutS not only modulate mismatch recognition but also trigger some of the down stream processes involved in the DNA mismatch repair process.     

Spontaneous Mutations Occur near Dam Recognition Sites in a dam-Escherichia coli Host

The mismatch repair system of Escherichia coli K12 removes mispaired bases from DNA. Mismatch repair can occur on either strand of DNA if it lacks N6-methyladenines within 5'-GATC-3' sequences. In hemimethylated heteroduplexes, repair occurs preferentially on the unmethylated strand. If both strands are fully methylated, repair is inhibited. Mutant (dam-) strains of E. coli defective in the adenine methylase that recognizes 5'-GATC-3' sequences (Dam), and therefore defective in mismatch repair, show increased spontaneous mutation rates compared to otherwise isogenic dam+ hosts. We have isolated and characterized 91 independent mutations that arise as a consequence of the Dam- defect in a plasmid-borne phage P22 repressor gene, mnt. The majority of these mutations are A:T----G:C transitions that occur within six base pairs of the two 5'-GATC-3' sequences in the mnt gene. In contrast, the spectrum of mnt- mutations in a dam+ host is comprised of a majority of insertions of IS elements and deletions that do not cluster near Dam recognition sites. These results show that Dam-directed post-replicative mismatch repair plays a significant role in the rectification of potential transition mutations in vivo, and suggest that sequences associated with Dam recognition sites are particularly prone to replication or repair errors.     

PCRless library mutagenesis via oligonucleotide recombination in yeast

The directed evolution of biomolecules with new functions is largely performed in vitro, with PCR mutagenesis followed by high-throughput assays for desired activities. As synthetic biology creates impetus for generating biomolecules that function in living cells, new technologies are needed for performing mutagenesis and selection for directed evolution in vivo. Homologous recombination, routinely exploited for targeted gene alteration, is an attractive tool for in vivo library mutagenesis, yet surprisingly is not routinely used for this purpose. Here, we report the design and characterization of a yeast-based system for library mutagenesis of protein loops via oligonucleotide recombination. In this system, a linear vector is co-transformed with single-stranded mutagenic oligonucleotides. Using repair of nonsense codons engineered in three different active-site loops in the selectable marker TRP1 as a model system, we first optimized the recombination efficiency. Single-loop recombination was highly efficient, averaging 5%, or 4.0×10(5) recombinants. Multiple loops could be simultaneously mutagenized, although the efficiencies dropped to 0.2%, or 6.0×10(3) recombinants, for two loops and 0.01% efficiency, or 1.5×10(2) recombinants, for three loops. Finally, the utility of this system for directed evolution was tested explicitly by selecting functional variants from a mock library of 1:10(6) wild-type:nonsense codons. Sequencing showed that oligonucleotide recombination readily covered this large library, mutating not only the target codon but also encoded silent mutations on either side of the library cassette. Together these results establish oligonucleotide recombination as a simple and powerful library mutagenesis technique and advance efforts to engineer the cell for fully in vivo directed evolution.     

Evidence that error-prone DNA repair converts dibenzo[a,l]pyrene-induced depurinating lesions into mutations: formation, clonal proliferation and regression of initiated cells carrying H-ras oncogene mutations in early preneoplasia

Initiation of skin tumors in mice is associated with the formation of oncogenic mutations in the H-ras gene. Mice treated on the dorsal skin with the potent polycyclic aromatic hydrocarbon (PAH) carcinogen dibenzo[a,l]pyrene (DB[a,l]P) form papillomas carrying the H-ras codon 61 (CAA to CTA) mutations. These mutations are induced in early preneoplastic skin within 1 day after DB[a,l]P treatment (Oncogene 16 (1998) 3203-3210) and appear to be related to DB[a,l]P-Ade-depurinating adducts (Proc. Natl. Acad. Sci. U. S. A. 92 (1995) 10422-10426). The rapid kinetics of mutation induction suggests that abasic sites generated from base depurination may undergo error-prone excision repair in pre-S-phase cells to induce these mutations. Analysis of mutations in the H-ras exon 1 and 2 region in DB[a,l]P-treated early preneoplastic skin indicated great changes in mutation spectra in the preneoplastic period. The initial spectra contained abundant A-->G mutations, which frequently occurred 3' to a putative conserved sequence (TGN-doublet). These mutations appeared to be induced initially as mismatched (G.T) heteroduplexes and then converted into double-stranded mutations by one round of replication. Unlike the A-->G mutations found in DB[a, l]P-treated skin (which forms 99% depurinating adducts), A-->G mutations found in anti-DB[a,l]P-diol epoxide-treated skin (forms 97% stable adducts) did not appear to be G.T heteroduplexes. These results, therefore, suggest that under these conditions, the repair errors occurred only from abasic sites but not from stable adducts. Initiated cells carrying specific oncogenic mutations, formed presumably by misrepair, underwent rapid clonal expansion and regression (transient clonoplasia). The multiplication of initiated stem cells during transient clonoplasia may be a factor determining the tumor-initiating potential of some PAH carcinogens.     

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.     

Determining the influence of structure on hybridization using oligonucleotide arrays.

We have studied the effects of structure on nucleic acid heteroduplex formation by analyzing hybridization of tRNAphe to a complete set of complementary oligonucleotides, ranging from single nucleotides to dodecanucleotides. The analysis points to features in tRNA that determine heteroduplex yield. All heteroduplexes that give high yield include both double-stranded stems as well as single-stranded regions. Bases in the single-stranded regions are stacked onto the stems, and heteroduplexes terminate at potential interfaces for coaxial stacking. Heteroduplex formation is disfavored by sharp turns or a lack of helical order in single-stranded regions, competition from bases displaced from a stem, and stable tertiary interactions. The study is relevant to duplex formation on oligonucleotide microarrays and to antisense technologies.