DNA topoisomerase and recombinase activities in Nae I restriction endonuclease.

Nae I endonuclease must bind to two DNA sequences for cleavage. Examination of the amino acid sequence of Nae I uncovered similarity to the active site of human DNA ligase I, except for leucine 43 in Nae I instead of the lysine essential for ligase activity. Changing leucine 43 to lysine 43 (L43K) changed Nae I activity: Nae I-L43K relaxed supercoiled DNA to yield DNA topoisomers and recombined DNA to give dimeric molecules. Interruption of the reactions of Nae I and Nae I-L43K with DNA demonstrated transient protein-DNA covalent complexes. These findings imply coupled endonuclease and ligase domains and link Nae I endonuclease to the topoisomerase and recombinase protein families.

Mutagenesis by incorporation of alkylated nucleotides.

The cellular DNA precursor pool was shown to be a target for N-methyl-N-nitrosourea, a potent mutagen and carcinogen. O6medGTP, a product of this interaction, was chemically synthesized and shown to be incorporated into DNA in vitro by Klenow E. coli pol I and phage T4 DNA polymerases. O6medGTP incorporated predominantly opposite T template residues and to a lower extent opposite C. At some loci incorporation of O6medGTP caused DNA synthesis arrest. The significance of the behavior of O6medGTP for mutagenesis in vivo is discussed.

Molecular mechanisms of chemical mutagenesis: 9-aminoacridine inhibits DNA replication in vitro by destabilizing the DNA growing point and interacting with the DNA polymerase.

9-Aminoacridine was found to inhibit dNTP incorporation into DNA homopolymer duplexes by phage T4 DNA polymerase in vitro. Systematic variation of the molar ratio of 9-aminoacridine to DNA, to DNA polymerase, and to DNA precursors demonstrated that this inhibition at 9-aminoacridine concentrations below 10 microM was mainly due to interaction of 9-aminoacridine with the DNA and suggested that the basis for the preferential inhibition of incorrect precursor incorporation was destabilization of the DNA growing point. Consistent with destabilization, 9-aminoacridine stimulated the hydrolysis of correctly base paired DNA by the 3'-5' exonuclease activity of phage T4 DNA polymerase. This is the first indication to my knowledge that an intercalating dye destabilizes the DNA growing point, whereas it raises the overall Tm of the DNA. At 9-aminoacridine concentrations above 10 microM overall incorporation of dNTPs was inhibited by 9-aminoacridine interaction with the DNA polymerase. A possible explanation for the induction of both deletion and addition frameshift mutations by 9-aminoacridine during DNA biosynthesis is discussed in light of growing-point destabilization.

Changing endonuclease EcoRII Tyr308 to Phe abolishes cleavage but not recognition: possible homology with the Int-family of recombinases.

Endonuclease EcoRII is one of a group of type II restriction enzymes, including Nael, Narl, BspMI, HpaII, and SacII, that require binding of an enhancer sequence to cleave DNA. Comparison of the EcoRII amino-acid sequence with the amino-acid consensus motifs that differentiate between recombinase families uncovered similarity between a 29 amino-acid sequence in the carboxyl end of EcoRII and the motif defining the integrase family of recombinases. This similarity implied that EcoRII tyrosine 308 should be involved in catalyzing hydrolysis of the scissile bond. Site-directed mutagenesis was used to mutate Tyr308 to Phe. The phenylalanine-substituted enzyme could not cleave T5 DNA under conditions in which wild-type enzyme completely cleaved this DNA. The Tyr308 to Phe mutation abolished cleavage activity but not specific binding to DNA. No evidence was found for the existence during the cleavage reaction of a covalent linkage between Tyr308 and DNA.

Mutagenic frequencies of site-specifically located O6-methylguanine in wild-type Escherichia coli and in a strain deficient in ada-methyltransferase.

The adaptive response of Escherichia coli involves protection of the cells against the toxic and mutagenic consequences of exposure to high doses of a methylating agent by prior exposure to low doses of the agent. Ada protein, a major repair activity for O6-methylguanine, is activated to positively control the adaptive response; O6-methylguanine is one of the major mutagenic lesions produced by methylating agents. We investigated the mutation frequency of wild-type Escherichia coli and strains containing the ada-5 mutation in response to site-specifically synthesized O6-methylguanine under conditions in which the adaptive response was not induced. Site-directed mutagenesis and oligonucleotide self-selection techniques were used to isolate the progeny of M13mp18 DNAs constructed to contain O6-methylguanine at any of eight different positions. The progeny were isolated from E. coli strains isogeneic except for deficiency in Ada-methyltransferase repair, UvrABC excision repair, or both. The resulting O6-methylguanine mutation levels at each position were determined by using differential oligonucleotide hybridization. We found that the wild type had up to a 2.6-fold higher mutation frequency than ada-5 mutants. In addition, the mutation frequency varied with the position of the O6-methylguanine in the DNA in the wild type but not in ada-5 mutants; O6-methylguanine lesions at the 5' ends of runs of consecutive guanines gave the highest mutation frequencies. Determination of the mutation frequency of O6-methylguanine in wild-type and mutS cells showed that mismatch repair can affect O6-methylguanine mutation levels.

Fluorescence of terbium ion-nucleic acid complexes: a sensitive specific probe for unpaired residues in nucleic acids.

The interaction of the lanthanide cation Tb3+ with the phosphate moieties of non-hydrogen-bonded residues of nucleic acids has been shown to result in substantial enhancement of the fluorescence of this cation. The excitation spectrum for this fluorescence is characteristic of the base moiety of the residue to which the Tb3+ is bound, while the emission spectrum is characteristic of the cation itself. The intensity of the fluorescence enhancement, however, is dependent upon the base of the ligand moiety, with G inducing the strongest enhancement, C and T rather less, and A very little. Base-paired residues of nucleic acids induce no such fluorescence enhancement, even though the cation is more tightly bound to double helical regions than to residues in single strands. The enhancement of Tb3+ fluorescence upon binding to non-hydrogen-bonded residues therefore provides a highly specific conformational probe for such residues. This probe has been exploited successfully for the purpose of analyzing the kinetics of reassociation of DNAs (C0t analysis) and as a specific stain for single-strand DNA bands on polyacrylamide gels.

O6-methylguanine-induced replication blocks.

The ability of Klenow polymerase I, phage T7 polymerase (Sequenase), human polymerase alpha, and human polymerase beta to synthesize past (bypass) O6-methylguanine (O6-meG) lesions was studied in the presence of MgCl2 and MnCl2. An end-labeled 16-mer primer was annealed to the 3' end of gel-purified oligodeoxyribonucleotide templates (45-mers), each containing a single O6-meG in place of one G in the sequence -G1G2CG3G4T-. Extension products were analyzed by denaturing polyacrylamide gel electrophoresis and autoradiography. A fraction of the products extended by Klenow fragment terminated either opposite or one base before O6-meG located at sites 1 and 3. Termination occurred primarily one base before O6-meG located at sites 2 and 4. The remaining fractions that bypassed the lesions represented full-length product. In control reactions, the O6-meG-containing templates were annealed with complementary 45-mers, repaired with O6-alkylguanine DNA-alkyltransferase, annealed with an excess of labeled primer, and extended by Klenow fragment. Full-length extension of > 90% was observed with each template. Primer extension past O6-meG by DNA polymerase alpha and Sequenase was partially blocked in a manner which varied with the site of O6-meG in the template while primer extension by DNA polymerase beta was completely blocked (< 2% full length extension) with O6-meG at sites 1-4. Substitution of MnCl2 for MgCl2 in the reaction mixture greatly increased the bypass of O6-meG by Klenow fragment and DNA polymerase alpha but not Sequenase or DNA polymerase beta. The increased ability of Klenow fragment to bypass O6-meG in the presence of MnCl2 was found to result from an increased incorporation of G (O6-meG at sites 1 and 2) and A (O6-meG at sites 1, 2, and 3) opposite the lesion. The results indicate that O6-meG can block in vitro polymerization by several DNA polymerases and are consistent with the observed cytotoxic effects of methylating agents on mammalian cells.

Location of putative binding and catalytic sites of NaeI by random mutagenesis.

Endonuclease NaeI is a prototype for an unusual group of type II restriction endonucleases that must bind two DNA recognition sequences to cleave DNA. The naeIR gene, expressed from a Ptac promotor construct, was toxic to Escherichia coli in the absence of NaeI-sequence specific methylases. The naeIR gene was mutagenized with N-methyl-N'-nitrosoguanidine; four classes of NaeI variants were isolated in the absence of protecting methylase activity. Class I variants (T60I, E70K) lacked detectable cleavage activity, but displayed good sequence-specific DNA binding. Class II variants (D95N, G141D) displayed 1-5% of the wild-type cleavage activity and normal DNA binding. Class III variants (G131E, G131R, G155D, G245E) displayed significantly attenuated cleavage and binding activities. Class IV variants (G197D, G214R/A219T, G236S, L241P, G245E, G245R, G250E, G270E) lacked both cleavage and binding activities. These results imply two amino acids (Thr-60, Glu-70) essential for catalysis. In addition, two domains are indicated in NaeI: one (Thr-60 to Gly-155) mediates substrate binding and catalysis, the other (Gly-197 to Gly-270) may mediate binding of the activating DNA sequence. Our results are compared with the active site residues of EcoRI, EcoRV, and BamHI.

Abasic sites induce triplet-repeat expansion during DNA replication in vitro.

The occurrence of triplet-repeat expansion (TRE) during transmission of genetic information is involved in many neurological and neuromuscular diseases including Fragile X syndrome and myotonic dystrophy. DNA slippage during replicative synthesis appears to cause TRE. The causes of DNA slippage, however, remain mostly unknown. We investigated the effects of abasic sites on the occurrence of TRE during DNA replication in vitro using Escherichia coli Klenow polymerase I as the model polymerase. Here we show that a single abasic site analog, synthesized in the triplet-repeat tract at the 5' end of the template strand, induced dramatic TRE during DNA synthesis. The amount of TRE induced decreased when the abasic site was moved to the middle of the repeat tract, consistent with effectively decreasing the length of the repeat tract. Placing the abasic site in the primer did not induce TRE. TRE was sequence-dependent. The damage-induced increase in growing strand TRE depended on the sequence of the growing strand repeat as AAT approximately ATT > CAG > CTG. The expansions required replication from a primer complementary to the repeat tract. The expanded tracts were sequenced and contained multiple additions of the original repeat. The results imply that DNA damage can play a significant role in generating TRE in vivo.

Effects of temperature, Mg2+ concentration and mismatches on triplet-repeat expansion during DNA replication in vitro.

The human genome contains many simple tandem repeats that are widely dispersed and highly polymorphic. At least one group of simple tandem repeats, the DNA trinucleotide repeats, can dramaticallyexpand in size during transmission from one generation to the next to cause disease by a process known as dynamic mutation. We investigated the ability of trinucleotide repeats AAT and CAG to expand in size during DNA replication using a minimal in vitro system composed of the repeat tract, with and without unique flanking sequences, and DNA polymerase. Varying Mg2+concentration and temperature gave dramatic expansions of repeat size during DNA replication in vitro. Expansions of up to 1000-fold were observed. Mismatches partially stabilized the repeat tracts against expansion. Expansions were only detected when the primer was complementary to the repeat tract rather than the flanking sequence. The results imply that cellular environment and whether the growing strand contains a nick or gap are important factors for the expansion process in vivo.

Induction of deletion and insertion mutations by 9-aminoacridine. An in vitro model.

The ability of 9-aminoacridine to induce mutagenic lesions during DNA replication in vitro was investigated. The ampicillinase gene of pBR322 was replicated in vitro in the presence of 9-aminoacridine. Transfection of the replicated DNA into Escherichia coli gave Amps mutants. Determination of the base changes in 76 of these mutants indicated that the spectrum of mutations induced by 9-aminoacridine was consistent with its action in vivo. Both large (407-base) and small (1- and 2-base) deletions were induced at repetitive sequences. The frequency of deletion mutations depended on the identity of the base deleted and sequences surrounding the deletions. The characteristics of the frameshift mutations induced were consistent with the interactions of 9-aminoacridine with DNA. These results establish that 9-aminoacridine can induce frameshift mutations during the replication process and provide an in vitro model of frameshift induction for mechanistic studies.

DNA damage at thymine N-3 abolishes base-pairing capacity during DNA synthesis.

3-Methylthymine was synthesized into DNA copolymers and deoxynucleoside triphosphate to study its effect on DNA synthesis by the Klenow fragment of Escherichia coli polymerase I and avian myeloblastosis virus reverse transcriptase. Both polymerases were greatly inhibited by template 3-methylthymine. In response to 3-methylthymine, misincorporation of dTTP increased slightly, but occurred only at low levels consistent with spontaneous misincorporation in vitro. Surprisingly, template 3-methylthymine resulted in a striking decrease in background misincorporation, relative to normal incorporation by the Klenow fragment, of dGTP and, to a lesser extent, of dATP and dCTP. The incorporation of 3-methyl-dTTP into DNA was studied using DNA sequencing technology. The Klenow fragment failed to incorporate 3-methyl-dTTP even at 1 mM. Reverse transcriptase incorporated 3-methyl-dTTP opposite adenine, cytosine, and thymine, but at only about 1/40,000th the efficiency of complementary deoxynucleoside triphosphate incorporation. Furthermore, synthesis generally stalled at sites of 3-methyl-thymine incorporation. From these results, we conclude that damage at the central hydrogen-bonding position of thymine abolishes its base-pairing capabilities during DNA synthesis.

Formation of a cleavasome: enhancer DNA-2 stabilizes an active conformation of NaeI dimer.

Cleavage of DNA by NaeI-type restriction enzymes is stimulated by a DNA element with affinity for the activator site of the enzyme: a cleavage-enhancer DNA element. Measurements of the mobility of NaeI activity in comparison with protein standards on gel permeation columns and glycerol gradients demonstrated that NaeI, without enhancer, can form a 70,000 MW dimer. The dimer, however, is inactive: it could not cleave the "resistant" NaeI site in M13mp18 DNA in the absence of enhancer. In cleavage assays, enhancer stimulated either DNA nicking or DNA cleavage, depending upon NaeI concentration, and reduced the NaeI concentration required for the transition from nicking to cleavage activity. A gel mobility-shift assay of the interaction of NaeI with enhancer showed the formation of two complexes. Results using different sized DNAs and different percentage acrylamide gels for gel mobility-shift analysis implied that the two complexes were caused by NaeI monomer and dimer structures rather than one and two DNA binding. Dimer formation increased with the affinity of enhancer for NaeI. UV cross-linking "captured" the NaeI-enhancer complex; electrophoretic analysis of the cross-linked products showed NaeI dimer bound to enhancer. These results imply a model for cleavage enhancement in which enhancer binding stabilizes an active NaeI dimer conformation ("cleavasome") that cleaves both DNA strands before dissociating.

Changing a leucine to a lysine residue makes NaeI endonuclease hypersensitive to DNA intercalative drugs.

A single amino acid change transforms restriction enzyme NaeI to a topoisomerase and recombinase (NaeI-L43K) that shows no sequence similarity to these protein families. This transformation appears to result from coupled endonuclease and ligase domains. To further elucidate the relationship between NaeI-L43K and the topoisomerase protein family, we studied the effect of the topoisomerase inhibitors on NaeI-L43K activity. The intercalative drugs amsacrine, ellipticine, and daunorubicin inhibited NaeI-L43K, whereas the nonintercalating drugs camptothecin, VP-16, and oxolinic acid did not. Ethidium bromide also inhibited NaeI-L43K, implying that intercalation is responsible for its inhibition. The effects of the intercalative drugs on the DNA cleavage steps of NaeI and NaeI-L43K were compared. The drugs hardly inhibited DNA cleavage by wild type NaeI but completely inhibited DNA cleavage by NaeI-L43K. This difference in inhibition demonstrates that the L43K amino acid change sensitized NaeI to these drugs. Low concentrations of the intercalative drugs, except for ethidium bromide, enhance production of topoisomerase--DNA covalent intermediates but inhibited production of the NaeI-L43K--DNA covalent intermediate. These results imply some unique differences between DNA relaxation by NaeI-L43K and DNA topoisomerase. Concomitant with studying inhibition of the cleavage intermediate, NaeI-L43K was found to covalently bond with the 5' end of the cleaved DNA strand.

Effects on NaeI-DNA recognition of the leucine to lysine substitution that transforms restriction endonuclease NaeI to a topoisomerase: a model for restriction endonuclease evolution.

Substituting lysine for leucine at position 43 (L43K) transforms NaeI from restriction endonuclease to topoisomerase and makes NaeI hypersensitive to intercalative anticancer drugs. Here we investigated DNA recognition by Nael-L43K. Using DNA competition and gel retardation assays, NaeI-L43K showed reduced affinity for DNA substrate and the ability to bind both single- and double-stranded DNA with a definite preference for the former. Sedimentation studies showed that under native conditions NaeI-L43K, like NaeI, is a dimer. Introduction of mismatched bases into double-stranded DNA significantly increased that DNA's ability to inhibit NaeI-L43K. Wild-type NaeI showed no detectable binding of either single-stranded DNA or mismatched DNA over the concentration range studied. These results demonstrate that the L43K substitution caused a significant change in recognition specificity by NaeI and imply that NaeI-L43K's topoisomerase activity is related to its ability to bind single-stranded and distorted regions in DNA. A mechanism is proposed for the evolution of the NaeI restriction-modification system from a topoisomerase/ligase by a mutation that abolished religation activity and provided a needed change in DNA recognition.

O6-methylguanine and A.C and G.T mismatches cause asymmetric structural defects in DNA that are affected by DNA sequence.

Mismatched and modified base pairs are central to questions of DNA mutation and repair. NMR and X-ray crystallography of mispairs indicate little to no local helical distortion, but these techniques are not sensitive to more global distortions of the DNA molecule. We used polyacrylamide gel electrophoresis and thermal denaturation to examine A.C, G.T, and O6-methylG.T and O6-methylG.C mismatches synthesized in place of either of two adjacent G.C base pairs in synthetic DNA duplexes. Substitution for G.C at either position decreased the stability of the duplex; O6-methylguanine was more destabilizing in place of the 5'G than in place of the 3'G. Comparisons between polymers synthesized so that lesions occurred regularly spaced on the same side of the helix and polymers synthesized so that the lesions alternated from side to side on the helix showed that these lesions introduced helical distortion composed of (i) a symmetric frictional component, probably caused by localized bubble formation, and (ii) an asymmetric component indicative of a more global effect on the DNA molecule. Comparisons between these effects at the two adjacent positions show that the extent of structural perturbation depends on sequence context.

Nonidentical DNA-binding sites of endonuclease NaeI recognize different families of sequences flanking the recognition site.

NaeI endonuclease uses a two-site binding mechanism to cleave substrate DNA: reaction-rate studies imply that occupancy of the second DNA site causes an allosteric change in the protein that enables DNA cleavage at the first site [Conrad, M., & Topal, M. D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9707-9711]. Measurements of relative binding affinities for 14-base-pair DNA fragments containing the NaeI recognition sequence GCCGGC and various flanking sequences showed that the two DNA-binding sites are not identical. G.C-rich flanking sequences were preferred by the activator binding site, whereas A.T-rich flanking sequences were preferred by the substrate binding site: GGGTGCCGGCAGGG was preferred 8-fold more by the activator site but 14-fold less by the substrate site than TTTCGCCGGCGTTT. Substitution of pyrimidine or 7-deazapurine for purine immediately 3' to GCCGGC reduced DNA affinity for only the activator site by up to 26-fold, implying that the activator DNA-binding site requires N-7 base contacts immediately flanking GCCGGC. The implications of nonidentical DNA-binding sites, one of which binds a specific DNA site to allosterically activate the other, are discussed.

Modified DNA fragments activate NaeI cleavage of refractory DNA sites.

Endonuclease NaeI cleaves DNA using a two-site mechanism. The DNA-binding sites are nonidentical: they recognize different families of flanking sequences. A unique NaeI site that is resistant to cleavage resides in M13 double-stranded DNA. NaeI can be activated to cleave this site by small DNA fragments containing one or more NaeI sites. These activators are not practical for genetic engineering because unphosphorylated activators that are consumed during the cleavage of substrate give ends that may interfere with subsequent ligations. We show that a DNA fragment containing phosphorothioate linkages at the NaeI scissile bonds (S-activator) is not cleaved by NaeI, even though this S-activator binds to the substrate site. The S-activator activates NaeI to cleave M13 DNA under conditions that completely exhaust unsubstituted activator. These results demonstrate that activation is not coupled to cleavage of activator, that NaeI reverts to its inactive state soon after dissociation of the EA complex, and that S-activator makes for a nondepletable activator during prolonged incubations.

Step-wise DNA relaxation and decatenation by NaeI-43K.

Nae I protein was originally isolated for its restriction endonuclease properties. Nae I was later discovered to either relax or cleave supercoiled DNA, depending upon whether Nae I position 43 contains a lysine (43K) or leucine (43L) respectively. Nae I-43K DNA relaxation activity appears to be the product of coupling separate endonuclease and ligase domains within the same polypeptide. Whereas Nae I relaxes supercoiled DNA like a topoisomerase, even forming a transient covalent intermediate with the substrate DNA, Nae I shows no obvious sequence similarity to the topoisomerases. To further characterize the topoisomerase activity of Nae I, we report here that Nae I-43K changes the linking number of a single negatively supercoiled topoisomer of pBR322 by units of one and therefore is a type I topoisomerase. Positively supercoiled pBR322 was resistant to Nae I-43K. At low salt concentration Nae I-43K was processive; non-saturating amounts of enzyme relaxed a fraction of the DNA. At high salt concentration the same non-saturating amounts of Nae I-43K partially relaxed all the DNA in a step-wise fashion to give a Gaussian distribution of topoisomers, demonstrating a switch from a processive to a distributive mode of action. Nae I-43K decatenated kinetoplast DNA containing nicked circles, implying that Nae I-43K can cleave opposite a nick. The products of the reaction are decatenated nicked circles under both processive and distributive conditions. The behavior of Nae I-43K is consistent with that of a prokaryotic type I topoisomerase.