Complete conformational family of 2',3'-didehydro-2',3'-dideoxyguanosine: quantum chemical and electron density topological study

Comprehensive conformational analysis of the biologically active nucleoside 2',3'-didehydro-2',3'-dideoxyaguanosine (d4G) has been performed at the MP2/6-311++G(d,p)//DFT B3LYP/6-31G(d,p) level of theory. The energetic, geometrical and polar characteristics of twenty d4G conformers as well as their conformational equilibrium were investigated. The electron density topological analysis allowed us to establish that the d4G molecule is stabilized by nine types of intramolecular interactions: O5'H...N3, O5'H...C8, C8H...O5', C2'H...N3, C5'H1...N3, C5'H2...N3, C8H...H1C5', C8H...H2'C5' and N2H1...O5'. The obtained results of conformational analysis permit us to think that d4G may be a terminator of the DNA chain synthesis in the 5'-3' direction. Thus it can be inferred that d4G competes with canonical 2'-deoxyaguanosine in binding an active site of the corresponding enzyme.     

Conformational capacity of 2',3'-didehydro-2',3'-dideoxyadenosine as a key to understanding its biological activity: results of quantum chemical modelling

Comprehensive conformational analysis of the biologically active nucleoside 2',3'-didehydro-2',3'-dideoxyadenosine (d4A) has been performed at the MP2/6-311++G(d,p)//DFT B3LYP/6-31G(d,p) level of theory. The energetic, geometrical and polar characteristics of twenty one d4A conformers as well as their conformational equilibrium were investigated. The electron density topological analysis allowed us to establish that the d4A molecule is stabilized by eight types of intramolecular interactions: O5'H...N3, O5'H...C8, C8H...O5', C2'H...N3, C5'H1...N3, C5'H2...N3 Ta C8H...H1/2C5'. The obtained results of conformational analysis lead us to think that d4A may be a terminator of the DNA chain sythesis in the 5'-3' direction. Thus it can be inferred that d4A competes with canonical 2'-deoxyadenosine in binding an active site of the corresponding enzyme.     

Single-strand DNA triple-helix formation

Chemical modification studies provide evidence that single-stranded oligodeoxyribonucleotides can form stable intrastrand triple helices. Two oligonucleotides of opposite polarity were synthesized, each composed of a homopurine-homopyrimidine hairpin stem linked to a pyrimidine sequence which is capable of folding back on the hairpin stem and forming specific Hoogsteen hydrogen bonds. Using potassium permanganate as a chemical modification reagent, we have found that two oligodeoxyribonucleotides of sequence composition type 5'-(purine)8(N)4(pyrimidine)8(N)6(pyrimidine)8-3' and 5'-(pyrimidine)8N6(pyrimidine)8N4(purine)8-3' undergo dramatic structural changes consistent with intrastrand DNA triple-helix formation induced by lowering the pH or raising the Mg2+ concentration. The intrastrand DNA triple helix is sensitive to base mismatches.     

Enhancing the stability of oligonucleotide duplexes. The effect of adding 1-(2'-deoxy-beta-d-ribofuranosyl)-5-nitroindole

We studied the properties of DNA duplexes containing 5-nitroindole (N) in one of the chains. We synthesized 8-membered oligos with N at the 5' or at the 3' end: 5'-d(NXGACCGTC)-3' or 5'-d(GACCGTCXN)-3', where X is one of the four natural bases, making all four kinds of oligos with and without N. We also prepared 11-membered oligos complementary to the above octanucleotides: 5'-d(TGACGGTCYZT)-3' and 5'-d(TZYGACGGTCT)-3', where Y and Z are A, G, C, or T. The stability of duplexes obtained with these oligos was assessed by melting, and the thermodynamic parameters delta H, delta S, and Tm were calculated. Comparison of the melting curves for modified and nonmodified duplexes demonstrated that the presence of N at the 5' end of one chain raises the Tm by 6.6 degrees C on average; if N is at the 3' end of the same chain, the Tm increases by about 3 degrees C.     

Mutants of ETS domain PU.1 and GGAA/T recognition: free energies and kinetics.

The ETS family members display specific DNA binding site preferences. As an example, PU.1 and ETS-1 recognize different DNA sequences with a core element centered over 5'-GGAA-3' and 5'-GGAA/T-3', respectively. To understand the molecular basis of this recognition, we carried out site-directed mutagenesis experiments followed by DNA binding studies that use electrophoretic mobility shift assay (EMSA) and surface plasmon resonance methods. EMSA experiments identified amino acid changes A231S and/or N236Y as being important for PU.1 recognition of both 5'-GGAA-3' and 5'-GGAT-3' containing oligonucleotides. To confirm these data and obtain accurate binding parameters, we performed kinetic studies using surface plasmon resonance on these mutants. The N236Y substitution revealed a weak protein-DNA interaction with the 5'-GGAA-3' containing oligonucleotide caused by a faster release of the protein from the DNA (k(off) tenfold higher than the wild-type protein). With the double mutant A231S-N236Y, we obtained an increase in binding affinity and stability toward both 5'-GGAA-3' and 5'-GGAT-3' containing oligonucleotides. We propose that substitution of alanine for serine introduces an oxygen atom that can accept hydrogen and interact with potential water molecules or other atoms to make an energetically favorable hydrogen bond with both 5'-GGAA-3' and 5'-GGAT-3' oligonucleotides. The free energy of dissociation for the double mutant A231S-N236Y with 5'-GGAA-3' (delta deltaG((A231S-N236Y) - (N236Y)) = -1.2 kcal mol confirm the stabilizing effect of this mutant in the protein-DNA complex formation. We conclude that N236Y mutation relaxes the specificity toward 5'-GGAA-3' and 5'-GGAT-3' sequences, while A231S mutation modulates the degree of specificity toward 5'-GGAA-3' and 5'GGAT-3' sequences. This study explains why wild-type PU.1 does not recognize 5'-GGAT-3' sequences and in addition broadens our understanding of 5'-GGAA/T-3' recognition by ETS protein family members.     

MQ-HCN-based pulse sequences for the measurement of 13C1′-1H1′, 13C1′-15N, 1H1′-15N, 13C1′-13C2′, 1H1′-13C2′, 13C6/8-1H6/8, 13C6/8-15N, 1H6/8-15N, 13C6-13C5, 1H6-13C5 dipolar couplings in 13C, 15N-labeled DNA (and RNA)

A suite of multiple quantum (MQ) HCN-based pulse sequences has been developed for the purpose of collecting dipolar coupling data in labeled nucleic acids. All the pulse sequences are based on the robust MQ-HCN experiment which has been utilized for assignment purposes in labeled nucleic acids for a number of years and provides much-needed resolution for the dipolar coupling measurements. We have attempted to collect multiple couplings centered on the 13C1' and 13C6/8 positions. Six pulse sequences are described, one each for measurement of one-bond 13C1'-1H1' and 13C6/8-1H6/8 couplings, one for measurement of one-bond 13C1'-15N and two-bond 1H1'-15N couplings, one for measurement of one-bond 13C6/8-15N and two-bond 1H6/8-15N couplings, one for measurement of one-bond 13C1'- 13C2' and two-bond 1H1'-13C2' couplings, and one for measurement of one-bond 13C6-13C5 and two-bond 1H6-13C5 couplings in the bases of C and T. These sequences are demonstrated for a labeled 18 bp DNA duplex in a 47 kDa ternary complex of DNA, CBFbeta, and the CBFalpha Runt domain, thus clearly demonstrating the robustness of the pulse sequences even for a very large complex.     

Long-range chromosomal mapping of the carcinoembryonic antigen (CEA) gene family cluster

A long-range physical map of the carcinoembryonic antigen (CEA) gene family cluster, which is located on the long arm of chromosome 19, has been constructed. This was achieved by hybridization analysis of large DNA fragments separated by pulse-field gel electrophoresis and of DNA from human/rodent somatic cell hybrids, as well as the assembly of ordered sets of cosmids for this gene region into contigs. The different approaches yielded very similar results and indicate that the entire gene family is contained within a region located at position 19q13.1-q13.2 between the CYP2A and the D19S15/D19S8 markers. The physical linkage of nine genes belonging to the CEA subgroup and their location with respect to the pregnancy-specific glycoprotein (PSG) subgroup genes have been determined, and the latter are located closer to the telomere. From large groups of ordered cosmid clones, the identity of all known CEA subgroup genes has been confirmed either by hybridization using gene-specific probes or by DNA sequencing. These studies have identified a new member of the CEA subgroup (CGM8), which probably represents a pseudogene due to the existence of two stop codons, one in the leader and one in the N-terminal domain exons. The gene order and orientation, which were determined by hybridization with probes from the 5' and 3' regions of the genes, are as follows: cen/3'-CGM7-5'/3'-CGM2-5'/5'-CEA-3'/5'-NCA-3'/5'-CGM1- 3'/3'-BGP-5'/3'- CGM9-5'/3'-CGM6-5'/5'-CGM8-3'/PSGcluster/qter.     

Stabilization of DNA duplex by 2-substituted adenine as a minor groove modifier

2-(1-Naphthalenylethynyl)-2'-deoxyadenosine ((N)A) was synthesized and incorporated into oligodeoxynucleotides. DNA duplexes containing newly designed 5'-(N)AT-3'/3'-T(N)A-5' base pairs are considerably stabilized than unmodified duplexes by stacking interaction of naphthalene rings in the narrow minor groove as characterized by a new emission at longer wavelength and exciton coupled CD signals.     

A mutation in the Mod subunit of EcoP15I restriction enzyme converts the DNA methyltransferase to a site-specific endonuclease

A closer inspection of the amino acid sequence of EcoP15I DNA methyltransferase revealed a region of similarity to the PDXn(D/E)XK catalytic site of type II restriction endonucleases, except for methionine in EcoP15I DNA methyltransferase instead of proline. Substitution of methionine at position 357 by proline converts EcoP15I DNA methyltransferase to a site-specific endonuclease. EcoP15I-M357P DNA methyltransferase specifically binds to the recognition sequence 5'-CAGCAG-3' and cleaves DNA asymmetrically EcoP151-M357P.DNA methyltransferase specifically binds to the recognition sequence 5'-CAGCAG-3' and cleaves DNA asymmetrically, 5'-CAGCAG(N)(10)-3', as indicated by the arrows, in presence of magnesium ions.     

Variation in nucleotide sequences coding for the N-terminal regions of the matrix and nonstructural proteins of influenza A viruses.

Nucleotide sequences have been determined for complementary DNA transcribed from the 3' ends of RNA segments 7 (matrix gene) and 8 (nonstructural gene) from a number of human influenza A viruses isolated over a period of 43 years and representing H0N1, H1N1, H2N2, and H3N2 subtypes. The pattern of nucleotide variation in both genes suggests that RNA segments 7 and 8 were conserved during the reassortment events which were responsible for the antigenic shifts H1N1 leads to H2N2 and H2N2 leads to H3N2. During the 23-year period between the isolation of A/PR/8/34(H0N1) and A/RI/5-/57(H2N2), substitutions have occurred at 7 of 230 nucleotides in RNA segment 7 and 13 of 220 nucleotides in RNA segment 8, and in 20 years A/RI/5-/57(H2N2) to A/Canberra Grammar/77(H3N2) substitutions have occurred at 5 of 230 nucleotides in RNA segment 7 and 12 of 220 nucleotides in RNA segment 8. These give rise to 2 of 67, 5 of 64, 1 of 67, and 5 of 64 amino acid changes, respectively. The number of nucleotide and amino acid changes observed is of the same order of magnitude as that which occurs over a comparable period of drift in RNA segments 4 and 6, which code for the variable antigenic determinants hemagglutinin and neuraminidase.     

Intermolecular recombination between DNAs introduced into mouse L cells is mediated by a nonconservative pathway that leads to crossover products.

We describe experiments designed to measure the efficiency of intermolecular recombination between mutant herpesvirus thymidine kinase (tk) genes introduced into mouse L cells. Recombinants were scored as stable transformants containing a functional tk gene. The two recombination substrates used were ptkB8, a pBR322-based plasmid containing a mutant tk gene, with a BamHI linker in an SphI restriction site that is centrally located within the gene, and mp10tk delta 3' delta 5', an mp10 vector with a tk gene deleted at both the 3' and 5' ends. The only homology shared by the two DNAs is 885 base pairs within the tk gene. To determine whether the double-strand break repair model that has been used to explain recombination in yeast cells (J. W. Szostak, T. L. Orr-Weaver, R. J. Rothstein, and F. W. Stahl, Cell 33:25-35, 1983) can account for recombination during the introduction of these DNAs into mammalian cells, we transformed cells with BamHI-linearized ptkB8 and supercoiled mp10tk delta 3' delta 5' replicative-form DNA. These two DNAs should recombine efficiently according to that model and should generate gene conversion products. In this reaction, the supercoiled DNA acts as the donor of information to repair the cleaved tk gene. Our results indicated that the efficiency of this reaction was very low (less than 10 transformants were obtained per 0.1 microgram of each DNA used in the reaction per 10(6) cells). In contrast, if BamHI-cleaved ptkB8 DNA was cotransformed into cells along with a circular DNA molecule containing a tk gene deleted only at its 3' end or only at its 5' end (mp10tk delta 3' or mp10tk delta 5'), then the efficiency of recombination could be more than 4 orders of magnitude higher than it was with circular mp10tk delta 3' delta 5' DNA. Recombination frequencies were highest when the tk delta 3' or tk delta 5' DNA used was cleaved at the tk deletion junction. Southern analyses of DNA from TK+ transformants generated with BamHI-cleaved ptkB8 and BamHI-cleaved mp10tk delta 3' DNAs indicated that recombination was almost always associated with the reassortment of markers flanking the reconstructed tk DNA. Together, these results are more consistent with the nonconservative single-strand annealing model for recombination that we proposed several years ago (F.-L. Lin, K. Sperle, and N. Sternberg, Mol. Cell. Biol. 4:1020-1034, 1984) than they are with the double-strand break repair model.     

Effects of parathyroid hormone and cyclic AMP analogues on the activity of ornithine decarboxylase and expression of the differentiated phenotype of chondrocytes in culture

Parathyroid hormone (PTH) greatly increased the level of adenosine 3', 5' cyclic monophosphate (cAMP) in rabbit costal chondrocytes in culture 2 minutes after its addition. PTH, as well as N6 O2' dibutyryl adenosine 3', 5' cyclic monophosphate (DBcAMP) and 8 Bromo adenosine 3', 5' cyclic monophosphate (8 Br-cAMP) induced ornithine decarboxylase (ODC; L-ornithine carboxylyase; EC 4.1.1.17), which reached a maximum 4 hours after their addition. Neither cAMP, N6 O2' dibutyryl guanosine 3', 5' cyclic monophosphate (DBcGMP), nor sodium butyrate increased the activity of the enzyme. PTH had no effect on DNA synthesis, while DBcAMP and 8 Br-cAMP decreased DNA synthesis. Expression of the differentiated phenotype of chondrocytes in culture was also induced by PTH, DBcAMP, and 8 Br-cAMP, but not by cAMP, DBcGMP, or sodium butyrate, as judged by morphological change. Glycosaminoglycan synthesis, a characteristic of the cartilage phenotype, began to increase 8 hours after addition of PTH or DBcAMP, reaching a plateau 32 hours after their addition. These findings suggest that PTH induces increase of ODC activity and expression of the differentiated phenotype of chondrocytes through increase of cAMP and that induction of OCD is closely related to expression of the differentiated phenotype of chondrocytes.     

Reaction of acid-activated mitomycin C with calf thymus DNA and model guanines: elucidation of the base-catalyzed degradation of N7-alkylguanine nucleosides

Mitomycin C (MC, 1) forms covalent adducts under acidic activating conditions (pH approximately 4) with deoxyguanosine, d(GpC), and guanine residues of calf thymus DNA. In the case of deoxyguanosine, five adducts arise from a common precursor, N7-(2' beta, 7'-diaminomitosen-1'-yl)-2'-deoxyguanosine (10a; not isolated), which hydrolyzes spontaneously via two pathways: scission of the glycosidic bond to form N7-(2' beta, 7'-diaminomitosen-1' alpha-yl)guanine (5) and its 1' beta-isomer (6) and imidazolium ring opening to generate three 2,6-diamino-4-hydroxy-5-(N-formyl-2' beta, 7'-diaminomitosen-1' beta-yl)pyrimidine (FAPyr) derivatives that are substituted at N6 by isomeric 2'-deoxyribose units [i.e., 1' beta-furanose (7), 1' alpha-furanose (8), and 1' beta-pyranose (9)]. The structures of 5-9 were determined by spectroscopic methods. The same five adducts were obtained from d(GpC), but only the guanine adducts 5 and 6 were formed in DNA. Adducts 7-9 interconvert during high-performance liquid chromatography (HPLC). The unexpected isomerization of the deoxyribose moiety of the initially formed 1' beta-furanose adduct 7 to those of 8 and 9 occurs upon imidazolium ring opening, as discerned by the course of imidazolium cleavage of the simple models N7-ethyl- and N7-methylguanosine and N7-methyl-2'-deoxyguanosine. All ring-opened N7-alkylguanosine derivatives studied here exist as a mixture of distinct N-formyl rotamers, manifested by multiple interconverting peaks on HPLC and in the 1H NMR spectra. In the UV spectra of such derivatives, a new and diagnostic maximum at 218 nm (at pH 7) is observed. Acid-activated MC is found to alkylate preferentially the Gua-N7 position in deoxyguanosine or d(GpC), in contrast to reductively activated MC, which preferentially alkylates the Gua-N2 position. This finding is explained by the different electronic structures of acid- and reduction-activated MC. In DNA, the N7 specificity of acid-activated MC is partially offset by steric factors.     

RNA mimetics: oligoribonucleotide N3'-->P5' phosphoramidates.

The synthesis and properties of novel RNA mimetics, oligoribonucleotide N3'-->P5' phosphoramidates, are described. These oligonucleotides contain 3'-aminoribonucleosides connected via N3'-->P5' phosphoramidate linkages, replacing the native RNA O3'-->P5' phosphodiester counterparts. The key monomers 2'-t-butyldimethylsilyl-3'-(monomethoxytrityl)-amino-5'-phospho ramidi tes were synthesized and used to prepare the oligonucleotide phosphoramidates using a solid phase methodology based on the phosphoramidite transfer reaction. Oligoribophosphoramidates are very resistant to enzymatic hydrolysis by snake venom phosphodiesterase. These compounds form stable duplexes with complementary natural phosphodiester DNA and RNA strands, as well as with 2'-deoxy N3'-->P5' phosphoramidates. The increase in melting temperature, Delta T m, was 5-14 degrees C relative to the 2'-deoxy phosphoramidates for decanucleotides. Also, the thermal stability of the ribophosphoramidatehomoduplex was noticeably higher (Delta T m +9.5 degrees C) than that for the isosequential 2'-deoxy phosphoramidate complex. Furthermore, the oligopyrimidine ribo N3'-->P5' phosphoramidate formed an extremely stable triplex with an oligopurine/oligopyrimidine DNA duplex with Delta T m +14.3 degrees C relative to the 2'-deoxy N3'-->P5' phosphoramidate counterpart. The properties of the oligoribonucleotide N3'-->P5' phosphoramidates indicate that these compounds can be used as hydrolytically stable structural and functional RNA mimetics.     

The stereochemistry of trans-4-hydroxynonenal-derived exocyclic 1,N2-2'-deoxyguanosine adducts modulates formation of interstrand cross-links in the 5'-CpG-3' sequence

The trans-4-hydroxynonenal (HNE)-derived exocyclic 1, N(2)-dG adduct with (6S,8R,11S) stereochemistry forms interstrand N(2)-dG-N(2)-dG cross-links in the 5'-CpG-3' DNA sequence context, but the corresponding adduct possessing (6R,8S,11R) stereochemistry does not. Both exist primarily as diastereomeric cyclic hemiacetals when placed into duplex DNA [Huang, H., Wang, H., Qi, N., Kozekova, A., Rizzo, C. J., and Stone, M. P. (2008) J. Am. Chem. Soc. 130, 10898-10906]. To explore the structural basis for this difference, the HNE-derived diastereomeric (6S,8R,11S) and (6R,8S,11R) cyclic hemiacetals were examined with respect to conformation when incorporated into 5'-d(GCTAGC XAGTCC)-3' x 5'-d(GGACTCGCTAGC)-3', containing the 5'-CpX-3' sequence [X = (6S,8R,11S)- or (6R,8S,11R)-HNE-dG]. At neutral pH, both adducts exhibited minimal structural perturbations to the DNA duplex that were localized to the site of the adduction at X(7) x C(18) and its neighboring base pair, A(8) x T(17). Both the (6S,8R,11S) and (6R,8S,11R) cyclic hemiacetals were located within the minor groove of the duplex. However, the respective orientations of the two cyclic hemiacetals within the minor groove were dependent upon (6S) versus (6R) stereochemistry. The (6S,8R,11S) cyclic hemiacetal was oriented in the 5'-direction, while the (6R,8S,11R) cyclic hemiacetal was oriented in the 3'-direction. These cyclic hemiacetals effectively mask the reactive aldehydes necessary for initiation of interstrand cross-link formation. From the refined structures of the two cyclic hemiacetals, the conformations of the corresponding diastereomeric aldehydes were predicted, using molecular mechanics calculations. Potential energy minimizations of the duplexes containing the two diastereomeric aldehydes predicted that the (6S,8R,11S) aldehyde was oriented in the 5'-direction while the (6R,8S,11R) aldehyde was oriented in the 3'-direction. These stereochemical differences in orientation suggest a kinetic basis that explains, in part, why the (6S,8R,11S) stereoisomer forms interchain cross-links in the 5'-CpG-3' sequence whereas the (6R,8S,11R) stereoisomer does not.     

Escherichia coli endonuclease III is not an endonuclease but a beta-elimination catalyst.

The oligonucleotide [5'-32P]pdT8d(-)dTn, containing an apurinic/apyrimidinic (AP) site [d(-)], yields three radioactive products when incubated at alkaline pH: two of them, forming a doublet approximately at the level of pdT8dA when analysed by polyacrylamide-gel electrophoresis, are the result of the beta-elimination reaction, whereas the third is pdT8p resulting from beta delta-elimination. The incubation of [5'-32P]pdT8d(-)dTn, hybridized with poly(dA), with E. coli endonuclease III yields two radioactive products which have the same electrophoretic behaviour as the doublet obtained by alkaline beta-elimination. The oligonucleotide pdT8d(-) is degraded by the 3'-5' exonuclease activity of T4 DNA polymerase as well as pdT8dA, showing that a base-free deoxyribose at the 3' end is not an obstacle for this activity. The radioactive products from [5'-32P]pdT8d(-)dTn cleaved by alkaline beta-elimination or by E. coli endonuclease III are not degraded by the 3'-5' exonuclease activity of T4 DNA polymerase. When DNA containing AP sites labelled with 32P 5' to the base-free deoxyribose labelled with 3H in the 1' and 2' positions is degraded by E. coli endonuclease VI (exonuclease III) and snake venom phosphodiesterase, the two radionuclides are found exclusively in deoxyribose 5-phosphate and the 3H/32P ratio in this sugar phosphate is the same as in the substrate DNA. When DNA containing these doubly-labelled AP sites is degraded by alkaline treatment or with Lys-Trp-Lys, followed by E. coli endonuclease VI (exonuclease III), some 3H is found in a volatile compound (probably 3H2O) whereas the 3H/32P ratio is decreased in the resulting sugar phosphate which has a chromatographic behaviour different from that of deoxyribose 5-phosphate. Treatment of the DNA containing doubly-labelled AP sites with E. coli endonuclease III, then with E. coli endonuclease VI (exonuclease III), also results in the loss of 3H and the formation of a sugar phosphate with a lower 3H/32P ratio that behaves chromatographically as the beta-elimination product digested with E. coli endonuclease VI (exonuclease III). From these data, we conclude that E. coli endonuclease III cleaves the phosphodiester bond 3' to the AP site, but that the cleavage is not a hydrolysis leaving a base-free deoxyribose at the 3' end as it has been so far assumed. The cleavage might be the result of a beta-elimination analogous to the one produced by an alkaline pH or Lys-Trp-Lys. Thus it would seem that E. coli 'endonuclease III' is, after all, not an endonuclease.     

Differential incision of bulky carcinogen-DNA adducts by the UvrABC nuclease: comparison of incision rates and the interactions of Uvr subunits with lesions of different structures

The UvrABC nuclease system from Escherichia coli removes DNA damages induced by a wide range of chemical carcinogens with variable efficiencies. The interactions with UvrABC proteins of the following three lesions site-specifically positioned in DNA, and of known conformations, were investigated: (i) adducts derived from the binding of the (-)-(7S,8R,9R,10S) enantiomer of 7,8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(-)-anti-BPDE] by cis-covalent addition to N(2)-2'-deoxyguanosine [(-)-cis-anti-BP-N(2)-dG], (ii) an adduct derived from the binding of the (+)-(1R,2S,3S,4R) enantiomer of 1,2-dihydroxy-3,4-epoxy-1,2,3, 4-tetrahydro-5-methylchrysene [(+)-anti-5-MeCDE] by trans addition to N(2)-2'-deoxyguanosine [(+)-trans-anti-MC-N(2)-dG], and (iii) a C8-2'-deoxyguanosine adduct (C8-AP-dG) formed by reductively activated 1-nitropyrene (1-NP). The influence of these three different adducts on UvrA binding affinities, formation of UvrB-DNA complexes by quantitative gel mobility shift analyses, and the rates of UvrABC incision were investigated. The binding affinities of UvrA varied among the three adducts. UvrA bound to the DNA adduct (+)-trans-anti-MC-N(2)-dG with the highest affinity (K(d) = 17 +/- 2 nM) and to the DNA containing C8-AP-dG with the least affinity (K(d) = 28 +/- 1 nM). The extent of complex formation with UvrB was also the lowest with the C8-AP-dG adduct. 5' Incisions occurred at the eighth phosphate from the modified guanine. The major 3' incision site corresponded to the fifth phosphodiester bond for all three adducts. However, additional 3' incisions were observed at the fourth and sixth phosphates in the case of the C8-AP-dG adduct, whereas in the case of the (-)-cis-anti-BP-N(2)-dG and (+)-trans-anti-MC-N(2)-dG lesions additional 3' cleavage occurred at the sixth and seventh phosphodiester bonds. Both the initial rate and the extent of 5' and 3' incisions revealed that C8-AP-dG was repaired less efficiently in comparison to the (-)-cis-anti-BP-N(2)-dG and (+)-trans-anti-MC-N(2)-dG containing DNA adducts. Our study showed that UvrA recognizes conformational changes induced by structurally different lesions and that in certain cases the binding affinities of UvrA and UvrB can be correlated with the incision rates. The size of the bubble formed around the damaged site with mismatched bases also appears to influence the incision rates. A particularly noteworthy finding in this study is that UvrABC repair of a substrate with no base opposite C8-AP-dG was quite inefficient as compared to the same adduct with a C opposite it. These findings are discussed in terms of the available NMR solution structures.     

Isolation and computer-aided characterization of MmeI, a type II restriction endonuclease from Methylophilus methylotrophus.

A Type II restriction endonuclease, MmeI, has been purified from the obligate methylotroph, Methylophilus methylotrophus. The enzyme was shown to have the non-palindromic recognition sequence 5'-T C C Pu A C (N)20-3', 3'-A G G Py T G (N)18-5' and to cleave (as indicated) on the 3' side, generating a two nucleotide 3' projection. Determination of the recognition sequence was achieved using two new computer programs; RECOG, which predicts recognition sequences from the pattern of restriction fragments obtained from DNAs of known sequence, and GELSIM, which generates graphical simulations of DNA band patterns obtained by gel electrophoresis of restriction digests of sequenced DNA molecules.     

Purification and characterization of bacteriophage N4-induced DNA polymerase

Coliphage N4 replication is independent of most host DNA replication functions except for the 5'----3' exonuclease activity of polA, DNA ligase, DNA gyrase, and ribonucleotide reductase (Guinta, D., Stambouly, J., Falco, S. C., Rist, J. K., and Rothman-Denes, L. B. (1986) Virology 150, 33-44). It is therefore expected that N4 codes for most of the functions required for replication of its genome. In this paper we report the purification of the N4-coded DNA polymerase from N4-infected cell extracts by following its activity on a gapped template and in an in vitro complementation system for N4 DNA replication (Rist, J. K., Pearle, M., Sugino, A., and Rothman-Denes, L. B. (1986) J. Biol. Chem. 261, 10506-10510). The enzyme is composed of one polypeptide, Mr 87,000. It is most active on templates containing short gaps synthesizing DNA with high fidelity in a quasi-processive manner. A strong 3'----5' exonuclease activity is associated with the DNA polymerase polypeptide. No 5'----3' exonuclease or strand-displacing activities were detected.