Cleavage of mispaired heteroduplex DNA substrates by numerous restriction enzymes

The utility of restriction endonucleases as a tool in molecular biology is in large part due to the high degree of specificity with which they cleave well-characterized DNA recognition sequences. The specificity of restriction endonucleases is not absolute, yet many commonly used assays of biological phenomena and contemporary molecular biology techniques rely on the premise that restriction enzymes will cleave only perfect cognate recognition sites. In vitro, mispaired heteroduplex DNAs are commonly formed, especially subsequent to polymerase chain reaction amplification. We investigated a panel of restriction endonucleases to determine their ability to cleave mispaired heteroduplex DNA substrates. Two straightforward, non-radioactive assays are used to evaluate mispaired heteroduplex DNA cleavage: a PCR amplification method and an oligonucleotide-based assay. These assays demonstrated that most restriction endonucleases are capable of site-specific double-strand cleavage with heteroduplex mispaired DNA substrates, however, certain mispaired substrates do effectively abrogate cleavage to undetectable levels. These data are consistent with mispaired substrate cleavage previously reported for Eco RI and, importantly, extend our knowledge of mispaired heteroduplex substrate cleavage to 13 additional enzymes.     

Conformational properties of Z-forming DNA oligomers bearing terminal unpaired bases.

Three sets of semi-self-complementary deoxyribonucleotide decamers with the sequence XX-(5meCG)4, (5meCG)4-XX, or Y-(5meCG)4-Y, where XX = AA, CC, GG, or TT and Y = A, C, G, or T, were synthesized along with the self-complementary octamer (5meCG)4. The 8-mer duplex readily undergoes a B-to-Z conformational conversion upon increasing the NaCl concentration with a transitional midpoint of approximately 1.1 M NaCl. The 10-mers should form 8-bp duplexes a with core sequence of [(5meCG)4]2 with 5'-XX overhangs, 3'-XX overhangs, or 5',3'-Y/Y mismatches. Circular dichroism was employed to determine the conformations of all oligomers. Salt titrations were performed to measure the effect of overhangs and terminal mismatches on the B-to-Z conversion. In general, the presence of 5'-XX overhangs results in a transition midpoint equal to or slightly higher than the control, whereas the presence of 3'-XX overhangs results in a transition midpoint slightly lower than the control. The 3'-CC and 5'-GG overhangs are exceptions, with transition midpoints much higher than the control. These oligomers apparently form duplexes with 5',3'-C/C or 5',3'-G/G mismatches abutting a [(G5meC)4]2 duplex core. The presence of terminal mismatches in the third set of oligomers results in transition midpoints higher than the control. Ultraviolet absorbance methods were used to evaluate the effect of the various stacking motifs of the 10-mers on the thermodynamics of melting relative to the 8-mer for both B and Z conformations. We found that in both the B and Z conformations, the presence of an overhang stabilizes the [(5meCG)4]2 duplex, with the 5' overhangs having a greater stabilizing effect relative to the 3' overhangs. The presence of 5',3'-Y/Y mismatches also imparts a stabilizing effect on the control 8-mer in both the B and Z conformations. These results are discussed in terms of stacking interactions of the terminal unpaired bases.     

Steady-state ATPase activity of E. coli MutS modulated by its dissociation from heteroduplex DNA

The ability of MutS to recognize mismatched DNA is required to initiate a mismatch repair (MMR) system. ATP binding and hydrolysis are essential in this process, but their role in MMR is still not fully understood. In this study, steady-state ATPase activities of MutS from Escherichia coli were investigated using the spectrophotometric method with a double end-blocked heteroduplex containing gapped bases. The ATPase activities of MutS increased as the number of gapped bases increased in a double end-blocked heteroduplex with 2-8 gapped bases in the chain, indicating that MutS dissociates from DNA when it reaches a scission during movement along the DNA. Since movement of MutS along the chain does not require extensive ATP hydrolysis and the ATPase activity is only enhanced when MutS dissociates from a heteroduplex, these results support the sliding clamp model in which ATP binding by MutS induces the formation of a hydrolysis-independent sliding clamp.     

Characterization of imperfect DNA duplexes containing unpaired bases and non-Watson-Crick base pairs.

Synthetic duplex DNAs of repeating sequence, such as poly d(TTC).poly d(GAA), were separated into their individual single strands. The various single strands complexed not only, as expected, with their complementary strands, but also with other non-complementary strands. Characterization of such complexes with respect to stoichiometry, Tm values and the dependence of Tm on NaCl concentration showed that a variety of unusual structures could be inferred at physiological salt concentrations. These included extrahelical thymines, G.T oppositions, A.C oppositions and T.C oppositions.     

Differences in susceptibility to restriction by E. coli B between various heteroduplex molecules of bacteriophage FD DNA

Fragments of B-modified bacteriophage fd sB1^o sB2 RF DNA were prepared with the help of purified endonuclease R from Haemophilus parainfluenzae (Hpa II). These were hybridized with unmodified circular single stranded fd DNA. The resulting partial heteroduplex molecules were assayed for infectivity on competent cells of B-restricting and non restricting strains of E. coli. There of such heteroduplexes originating from neighbouring fragments on the physical map of fd RF DNA were shown to be more resistant to Eco B restriction than six others and the unmodified control. It is suggested that the three corresponding vicinal fragments contain essential parts of the Eco B recognition site on this phage DNA.     

Repair of heteroduplex DNA molecules with multibase loops in Escherichia coli.

The fate of heteroduplex molecules containing 5-, 7-, 9-, 192-, 410-, and 514-base loops after transformation of wild-type and various mutant strains of Escherichia coli has been examined. No evidence for repair was obtained for the wild type or for strains with mutations in the following genes: mutS, recA, recBC sbcBC, recD, recF, recJ, recN, recO, recR, recBC sbcBC recF uvrA, recG ruvC, ruvB, lexA3, lexA51, uvrA, nfo xth nth, polA(Ts), or pcnB. These results rule out the involvement of the SOS system and most known recombination and repair pathways. Repair of heteroduplex molecules containing 410- and 514-base loops was observed when a 1-base deletion-insertion mismatch was present nearby. The repair of both the mismatch and the loops was directed by the state of dam methylation of the DNA chains and was dependent on the product of the mutS gene. A high efficiency of repair (95%) was found even when the mismatch and the loops were 1,448 nucleotides apart. We conclude that multibase loops in DNA can be removed only as a consequence of corepair by dam-directed mismatch repair.     

Biochemical characterization of the interaction between the Saccharomyces cerevisiae MSH2-MSH6 complex and mispaired bases in DNA.

The interaction of the Saccharomyces cerevisiae MSH2-MSH6 complex with mispaired bases was analyzed using gel mobility shift assays and surface plasmon resonance methods. Under equilibrium binding conditions, MSH2-MSH6 bound to homoduplex DNA with a K(d) of 3.9 nM and bound oligonucleotide duplexes containing T:G, +1, +2, +4, and +10 insertion/deletion loop (IDL) mispairs with K(d) values of 0.20, 0.25, 11, 3.2, and 0.55 nM, respectively. Competition binding experiments using 65 different substrates revealed a 10-fold range in mispair discrimination. In general, base-base mispairs and a +1 insertion/deletion mispair were recognized better than intermediate sized insertion/deletion mispairs of 2-8 bases. Larger IDL mispairs (>8 bases) were recognized almost as well as the +1 IDL mispair. Recognition of mispairs by MSH2-MSH6 was influenced by sequence context, with the 6-nucleotide region surrounding the mispair being primarily responsible for influencing mispair recognition. Effects of sequences as far away as 15 nucleotides were also observed. Differential effects of ATP on the stability of MSH2-MSH6-mispair complexes suggested that base-base mispairs and the smaller IDL mispairs were recognized by a different binding mode than larger IDL mispairs, consistent with genetic experiments indicating that MSH2-MSH6 functions primarily in the repair of base-base and small IDL mispairs.     

Chemical reactivity of matched cytosine and thymine bases near mismatched and unmatched bases in a heteroduplex between DNA strands with multiple differences.

The chemical reactivity of matched T and C bases to osmium tetroxide and hydroxylamine near mismatched and unmatched bases in a heteroduplex between two strands of DNA with multiple differences was examined. Data was available for matched bases one or two positions away from 24 mismatches. Reactive bases were found near 16 of the mismatches and were usually one or two bases away. This reactivity is consistent with structural studies indicating perturbation of the duplex around mismatches and will allow another mode of study of the effect of mismatches. The reactivity of these bases was found not to be strongly correlated with mismatch type or GC basepair content of the basepairs around the mismatches. Extra reactivity may have been promoted by the presence of either T or C in the mismatch allowing increased reactivity of nearby T or C. The utility of the phenomenon for the detection of mutations is discussed. Unmatched bases in the heteroduplex also gives rise to reactive matched bases nearby.     

Transfer of the MSH2.MSH6 complex from proliferating cell nuclear antigen to mispaired bases in DNA

Proliferating cell nuclear antigen (PCNA) is thought to play a role in DNA mismatch repair at the DNA synthesis step as well as in an earlier step. Studies showing that PCNA interacts with mispair-binding protein complexes, MSH2.MSH3 and MSH2.MSH6, and that PCNA enhances MSH2.MSH6 mispair binding specificity suggest PCNA may be involved in mispair recognition. Here we show that PCNA and MSH2.MSH6 form a stable ternary complex with a homoduplex (G/C) DNA, but MSH2.MSH6 binding to a heteroduplex (G/T) DNA disrupts MSH2.MSH6 binding to PCNA. We also found that the addition of ATP or adenosine 5'-O-(thiotriphosphate) restores MSH2.MSH6 binding to PCNA, presumably by disrupting MSH2.MSH6 binding to the heteroduplex (G/T) DNA. These results support a model in which MSH2.MSH6 binds to PCNA loaded on newly replicated DNA and is transferred from PCNA to mispaired bases in DNA.     

B-Z junctions in supercoiled pRW751 DNA contain unpaired bases or non-Watson-Crick base pairs

Structural distortions on the boundary between right-handed and left-handed DNA segments in negatively supercoiled plasmid pRW751 (a derivative of pBR322 containing (dC-dG)13 and (dC-dG)16 segments) were studied by means of osmium tetroxide, pyridine and glyoxal. These two probes react preferentially with single-stranded DNA, but only the latter requires non-paired bases for the reaction. Nuclease S1 and testing of the inhibition of BamHI cleavage (whose recognition sequences GGATCC lie on the "outer" boundaries between the (dC-dG)n and the pBR322 nucleotide sequence) were used to detect the site-specific chemical modification in pRW751. As a result of glyoxal treatment BamHI cleavage was strongly inhibited in topoisomeric samples whose superhelical density was sufficiently negative to stabilize the (dC-dG)n segments in the left-handed form. Osmium tetroxide, pyridine modification resulted in a similar inhibition of BamHI cleavage and in a formation of nuclease S1 sensitive sites. The results suggest that the "outer" B-Z junctions in pRW751 contain one or few non-paired bases or non-Watson-Crick base pairs.     

Meiotic silencing by unpaired DNA.

The silencing of gene expression by segments of DNA present in excess of the normal number is called cosuppression in plants and quelling in fungi. We describe a related process, meiotic silencing by unpaired DNA (MSUD). DNA unpaired in meiosis causes silencing of all DNA homologous to it, including genes that are themselves paired. A semidominant Neurospora mutant, Sad-1, fails to perform MSUD. Sad-1 suppresses the sexual phenotypes of many ascus-dominant mutants. MSUD may provide insights into the function of genes necessary for meiosis, including genes for which ablation in vegetative life would be lethal. It may also contribute to reproductive isolation of species within the genus Neurospora. The wild-type allele, sad-1(+), encodes a putative RNA-directed RNA polymerase.     

Gene conversion in Escherichia coli: the recF pathway for resolution of heteroduplex DNA.

The independent repair of mismatched nucleotides present in heteroduplex DNA has been used to explain gene conversion and map expansion after general genetic recombination. We have constructed and purified heteroduplex plasmid DNAs that contain heteroallelic 10-base-pair insertion-deletion mismatches. These DNA substrates are similar in structure to the heteroduplex DNA intermediates that have been proposed to be produced during the genetic recombination of plasmids. These DNA substrates were transformed into wild-type and mutant Escherichia coli strains, and the fate of the heteroduplex DNA was determined by both restriction mapping and genetic tests. Independent repair events that yielded a wild-type Tetr gene were observed at a frequency of approximately 1% in both wild-type and recB recC sbcB mutant E. coli strains. The independent repair of small insertion-deletion-type mismatches separated by 1,243 base pairs was found to be reduced by recF, recJ, and ssb single mutations in an otherwise wild-type genetic background and reduced by recF, recJ, and recO mutations in a recB recC sbcB genetic background (the ssb mutation was not tested in the latter background). Independent repair of small insertion-deletion-type mismatched nucleotides that were as close as 312 nucleotides apart was observed. There was no apparent bias in favor of the insertion or deletion of mutant sequences.     

The Escherichia coli MutL protein stimulates binding of Vsr and MutS to heteroduplex DNA.

Vsr DNA mismatch endonuclease is the key enzyme of very short patch (VSP) DNA mismatch repair and nicks the T-containing strand at the site of a T-G mismatch in a sequence-dependent manner. MutS is part of the mutHLS repair system and binds to diverse mismatches in DNA. The function of the mutL gene product is currently unclear but mutations in the gene abolish mutHLS -dependent repair. The absence of MutL severely reduces VSP repair but does not abolish it. Purified MutL appears to act catalytically to bind Vsr to its substrate; one-hundredth of an equivalent of MutL is sufficient to bring about a significant effect. MutL enhances binding of MutS to its substrate 6-fold but does so in a stoichiometric manner. Mutational studies indicate that the MutL interaction region lies within the N-terminal 330 amino acids and that the MutL multimerization region is at the C-terminal end. MutL mutant monomeric forms can stimulate MutS binding.     

The E249K mutator mutant of DNA polymerase beta extends mispaired termini

The DNA polymerase beta mutant enzyme, which is altered from glutamic acid to lysine at position 249, exhibits a mutator phenotype in primer extension assays and in the herpes simplex virus-thymidine kinase (HSV-tk) forward mutation assay. The basis for this loss of accuracy was investigated by measurement of misincorporation fidelity in single turnover conditions. For the four misincorporation reactions investigated, the fidelity of the E249K mutant was not significantly different from wild type, implying that the mutator phenotype was not caused by a general inability to distinguish between correct and incorrect bases during the incorporation reaction. However, the discrimination between correct and incorrect substrates by the E249K enzyme occurred less during the conformational change and chemical steps and more during the initial binding step, compared with pol beta wild type. This implies that the E249K mutation alters the kinetic mechanism of nucleotide discrimination without reducing misincorporation fidelity. In a missing base primer extension assay, we observed that the mutant enzyme produced mispairs and extended them. This indicates that the altered fidelity of E249K could be due to loss of discrimination against mispaired primer termini. This was supported by the finding that the E249K enzyme extended a G:A mispair 8-fold more efficiently than wild type and a C:T mispair 4-fold more efficiently. These results demonstrate that an enhanced ability to extend mispairs can produce a mutator phenotype and that the Glu-249 side chain of DNA polymerase beta is critical for mispair extension fidelity.     

In vitro packaging of heteroduplex bacteriophage T7 DNA: evidence for repair of mismatched bases.

Heteroduplex DNA molecules that were wild type or contained combinations of amber, missense, and temperature-sensitive mutations were prepared from bacteriophage T7. These DNA molecules were then encapsulated in in vitro packaging reactions so as to produce infective T7 phage. The genotypes of the phage were examined to determine the degree to which mismatched base pairs in the heteroduplex had been corrected. The data show that conversion of the mismatches took place either during in vitro packaging or immediately after infection of either an Escherichia coli or Shigella sonnei host. The mode of mismatch conversion observed in these experiments was independent of the host mutH, mutL, mutS, and uvrD genes. There was no significant amount of discrimination between markers on either of the two complementary strands. The observed frequency of conversion of a mismatch depended on the genetic marker being monitored and on experimental conditions but was generally in the range between 5 and 30%.     

Hemimethylation prevents DNA replication in E. coli

The DNA adenine methylase of E. coli methylates adenines at GATC sequences. Strains deficient in this methylase are transformed poorly by methylated plasmids that depend on either the pBR322 or the chromosomal origins for replication. We show here that hemimethylated plasmids also transform dam- bacteria poorly but that unmethylated plasmids transform them at high frequencies. Hemimethylated daughter molecules accumulate after the transformation of dam- strains by fully methylated plasmids, suggesting that hemimethylation prevents DNA replication. We also show that plasmids purified from dam+ bacteria are hemimethylated at certain sites. These results can explain why newly formed daughter molecules are not substrates for an immediate reinitiation of DNA replication in wild-type E. coli.     

Recombination following transformation of Escherichia coli by heteroduplex plasmid DNA molecules

Circular heteroduplex DNA molecules introduced into Escherichia coli-competent cells are converted to new recombinant plasmids as a result of enzymatic actions in vivo. A pair of plasmids with partial sequence homology were each linearized at a different position with restriction enzymes, and the termini were made flush with the single-strand-specific S1 nuclease. Duplex molecules were then formed by melting and annealing these plasmid DNAs together. In contrast to linear homoduplex molecules, heteroduplexes circularize and therefore transform E. coli efficiently. Unique DNA sequences on each of the parental strands in the transforming heteroduplexes can be selectively incorporated or deleted as a result of in vivo enzymatic activities in transformed cells. This method permits the generation of new recombinant sequences in vivo without relying solely on the presence of convenient restriction sites for manipulation of DNA fragments in vitro.     

recB recC-dependent processing of heteroduplex DNA stimulates recombination of an adjacent gene in Escherichia coli.

The effect of DNA mismatched repair on the genetic recombination of a gene adjacent to the mismatch site (MS) was tested by using four mismatch configurations. An MS was constructed in a well-characterized plasmid recombination substrate, and recombination with a resident compatible plasmid was measured after transformation of the mismatched plasmid into Escherichia coli. The mismatched plasmids were constructed such that one of the DNA strands was methylated by the DNA adenine methylase (Dam), while the other strand was unmethylated. The processing of a hemimethylated single-base-pair mismatch had no effect on the recombination of the adjacent gene, suggesting that the most efficient (Dam-instructed) mismatch repair process does not secondarily promote genetic recombination. However, mismatches that could form an ordered secondary structure resembling a cruciform increased the recombination of this adjacent gene at least 20-fold. An identical mismatch that could not form an ordered secondary structure had no effect in this system. The increased frequency of recombination observed was found to require the recB or recC gene product or both. Furthermore, the recombination appeared unidirectional, in that the cruciform-containing plasmid did not produce stable transformants. Our results support a model in which the cruciform-containing plasmid can participate in recombination with the resident plasmid but is unable to produce stable transformant progeny. A proposed role for the RecBCD enzyme (ExoV) in this process is discussed.     

Site-specific cleavage of DNA by E. coli DNA gyrase.

E. coli DNA gyrase, which catalyzes the supercoiling of DNA, cleaves DNA site-specifically when oxolinic acid and sodium dodecylsulfate are added to the reaction. We studied the structure of the gyrasecleaved DNA because of its implications for the reaction mechanism and biological role of gyrase. Gyrase made a staggered cut, creating DNA termini with a free 3' hydroxyl and a 5' extension that provided a template primer for DNA polymerase. The cleaved DNA was resistant to labeling with T4 polynucleotide kinase even after treatment with proteinase K. Thus the denatured enzyme that remains attached to cleaved DNA is covalently bonded to both 5' terminal extensions. The 5' extensions of many gyrase cleavage fragments from phi X174, SV40 and Col E1 DNA were partially sequenced using repair with E. coli DNA polymerase I. No unique sequence existed within the cohesive ends, but G was the predominant first base incorporated by DNA polymerase I. The cohesive and sequences of four gyrase sites were determined, and they demonstrated a four base 5' extension. The dinucleotide TG, straddling the gyrase cut on one DNA strand, provided the only common bases within a 100 bp region surrounding the cleavage sites. Analysis of other cleavage fragments showed that cutting between a TG doublet is common to most, or all, gyrase cleavages. Other bases common to some of the sequenced sites were clustered nonrandomly around the TG doublet, and may be variable components of the cleavage sequence. This diverse recognition sequence with common elements is a pattern shared with several other specific nucleic acid-protein interactions.