Repair of DNA containing Fapy.dG and its beta-C-nucleoside analogue by formamidopyrimidine DNA glycosylase and MutY

Fapy.dG is produced in DNA as a result of oxidative stress. Under some conditions Fapy.dG is formed in greater yields than 8-oxodG from a common chemical precursor. Recently, Fapy.dG and its C-nucleoside analogue were incorporated in chemically synthesized oligonucleotides at defined sites. Like 8-oxodG, Fapy.dG instructs DNA polymerase to misincorporate dA opposite it in vitro. The interactions of DNA containing Fapy.dG or the nonhydrolyzable analogue with Fpg and MutY are described. Fpg excises Fapy.dG (K(M) = 2.0 nM, k(cat) = 0.14 min(-1)) opposite dC approximately 17-fold more efficiently than when mispaired with dA, which is misinserted by DNA polymerase in vitro. Fpg also prefers to bind duplexes containing Fapy.dG.dC or beta-C-Fapy.dG.dC compared to those in which the lesion is opposite dA. MutY incises dA when it is opposite Fapy.dG and strongly binds duplexes containing the lesion or beta-C-Fapy.dG. Incision from Fapy.dG.dA is faster than from dG.dA mispairs but slower than from DNA containing 8-oxodG opposite dA. These data demonstrate that Fapy.dG closely resembles the interactions of 8-oxodG with two members of the GO repair pathway in vitro. The similar effects of Fapy.dG and 8-oxodG on DNA polymerase and repair enzymes in vitro raise the question as to whether Fapy.dG elicits similar effects in vivo.     

Interaction of DNA containing Fapy.dA or its C-nucleoside analogues with base excision repair enzymes. Implications for mutagenesis and enzyme inhibition

Fapy.dA is produced in DNA as a result of oxidative stress. Recently, this lesion and its C-nucleoside analogues were incorporated in chemically synthesized oligonucleotides at defined sites. The interaction of DNA containing Fapy.dA or nonhydrolyzable analogues with Fpg and MutY is described. Fpg efficiently excises Fapy.dA (K(m) = 1.2 nM, k(cat) = 0.12 min(-1)) opposite T. The lesion is removed as efficiently from duplexes containing Fapy.dA:dA or Fapy.dA:dG base pairs. Multiple turnovers are observed for the repair of Fapy.dA mispairs in a short period of time, indicating that the enzyme does not remain bound to the product duplex. MutY does not incise dA from a duplex containing this nucleotide opposite Fapy.dA, nor does it exhibit an increased level of binding compared to DNA composed solely of native base pairs. MutY also does not incise Fapy.dA when the lesion is opposite dG. These data suggest that Fapy.dA could be deleterious to the genome. Fpg strongly binds duplexes containing the beta-C-nucleoside analogue of Fapy.dA (beta-C-Fapy.dA) opposite all native nucleotides (K(D) < 27 nM), as well as the alpha-C-nucleoside (alpha-C-Fapy.dA) opposite dC (K(D) = 7.1 +/- 1.5 nM). A duplex containing a beta-C-Fapy.dA:T base pair is an effective inhibitor (K(I) = 3.5 +/- 0.3 nM) of repair of Fapy.dA by Fpg, suggesting the C-nucleoside may have useful therapeutic properties.     

The active site of the Escherichia coli MutY DNA adenine glycosylase.

Escherichia coli MutY is an adenine DNA glycosylase active on DNA substrates containing A/G, A/C, or A/8-oxoG mismatches. Although MutY can form a covalent intermediate with its DNA substrates, its possession of 3' apurinic lyase activity is controversial. To study the reaction mechanism of MutY, the conserved Asp-138 was mutated to Asn and the reactivity of this mutant MutY protein determined. The glycosylase activity was completely abolished in the D138N MutY mutant. The D138N mutant and wild-type MutY protein also possessed different DNA binding activities with various mismatches. Several lysine residues were identified in the proximity of the active site by analyzing the imino-covalent MutY-DNA intermediate. Mutation of Lys-157 and Lys-158 both individually and combined, had no effect on MutY activities but the K142A mutant protein was unable to form Schiff base intermediates with DNA substrates. However, the MutY K142A mutant could still bind DNA substrates and had adenine glycosylase activity. Surprisingly, the K142A mutant MutY, but not the wild-type enzyme, could promote a beta/delta-elimination on apurinic DNA. Our results suggest that Asp-138 acts as a general base to deprotonate either the epsilon-amine group of Lys-142 or to activate a water molecule and the resulting apurinic DNA then reacts with Lys-142 to form the Schiff base intermediate with DNA. With the K142A mutant, Asp-138 activates a water molecule to attack the C1' of the adenosine; the resulting apurinic DNA is cleaved through beta/delta-elimination without Schiff base formation.     

Structure of purine-purine mispairs during misincorporation and extension by Escherichia coli DNA polymerase I

The mechanism by which purine-purine mispairs are formed and extended was examined with the high-fidelity Klenow fragment of Escherichia coli DNA polymerase I with the proofreading exonuclease activity inactivated. The structures of the purine-purine mispairs were examined by comparing the kinetics of mispair formation with adenine versus 7-deazaadenine and guanine versus 7-deazaguanine at four positions in the DNA, the incoming dNTP, the template base, and both positions of the terminal base pair. A decrease in rate associated with a 7-deazapurine substitution would suggest that the nucleotide is in a syn conformation in a Hoogsteen base pair with the opposite base. During mispair formation, the k(pol)/K(d) values for the insertion of dATP opposite A (dATP/A) as well as dATP/G and dGTP/G were decreased greater than 10-fold with the deazapurine in the dNTP. These results suggest that during mispair formation the newly forming base pair is in a Hoogsteen geometry with the incoming dNTP in the syn conformation and the template base in the anti conformation. During mispair extension, the only decrease in k(pol)/K(d) was associated with the G/G base pair in which 7-deazaguanine was in the template strand. These results as well as previous results [McCain et al. (2005) Biochemistry 44, 5647-5659] in which a hydrogen bond was found between the 3-position of guanine at the primer terminus and Arg668 during G/A and G/G mispair extension indicate that the conformation of the purine at the primer terminus is in the anti conformation during mispair extension. These results suggest that purine-purine mispairs are formed via a Hoogsteen geometry in which the dNTP is in the syn conformation and the template is in the anti conformation. During extension, however, the conformation of the primer terminus changes to an anti configuration while the template base may be in either the syn or anti conformations.     

A fidelity mechanism in DNA polymerase lambda promotes error‐free bypass of 8‐oxo‐dG

8‐oxo‐7,8‐dihydroxy‐2′‐deoxyguanosine (8‐oxo‐dG) has high mutagenic potential as it is prone to mispair with deoxyadenine (dA). In order to maintain genomic integrity, post‐replicative 8‐oxo‐dG:dA mispairs are removed through DNA polymerase lambda (Pol λ)‐dependent MUTYH‐initiated base excision repair (BER). Here, we describe seven novel crystal structures and kinetic data that fully characterize 8‐oxo‐dG bypass by Pol λ. We demonstrate that Pol λ has a flexible active site that can tolerate 8‐oxo‐dG in either the anti‐ or syn‐conformation. Importantly, we show that discrimination against the pro‐mutagenic syn‐conformation occurs at the extension step and identify the residue responsible for this selectivity. This residue acts as a kinetic switch, shunting repair toward long‐patch BER upon correct dCMP incorporation, thus enhancing repair efficiency. Moreover, this switch also provides a potential mechanism to increase repair fidelity of MUTYH‐initiated BER.     

Defective human MutY phosphorylation exists in colorectal cancer cell lines with wild-type MutY alleles

Oxidative DNA damage can generate a variety of cytotoxic DNA lesions such as 8-oxoguanine (8-oxoG), which is one of the most mutagenic bases formed from oxidation of genomic DNA because 8-oxoG can readily mispair with either cytosine or adenine. If unrepaired, further replication of A.8-oxoG mispairs results in C:G to A:T transversions, a form of genomic instability. We reported previously that repair of A.8-oxoG mispairs was defective and that 8-oxoG levels were elevated in several microsatellite stable human colorectal cancer cell lines lacking MutY mutations (human MutY homolog gene, hmyh, MYH MutY homolog protein). In this report, we provide biochemical evidence that the defective repair of A.8-oxoG may be due, at least in part, to defective phosphorylation of the MutY protein in these cell lines. In MutY-defective cell extracts, but not extracts with functional MutY, A.8-oxoG repair was increased by incubation with protein kinases A and C (PKA and PKC) and caesin kinase II. Treatment of these defective cells, but not cells with functional MutY, with phorbol-12-myristate-13-acetate also increased the cellular A.8-oxoG repair activity and decreased the elevated 8-oxoG levels. We show that MutY is serine-phosphorylated in vitro by the action of PKC and in the MutY-defective cells by phorbol-12-myristate-13-acetate but that MutY is already phosphorylated at baseline in proficient cell lines. Finally, using antibody-isolated MutY protein, we show that MutY can be directly phosphorylated by PKC that directly increases the level of MutY catalyzed A.8-oxoG repair.     

A distinct TthMutY bifunctional glycosylase that hydrolyzes not only adenine but also thymine opposite 8-oxoguanine in the hyperthermophilic bacterium, Thermus thermophilus

Oxidative damage represents a major threat to genomic stability because the major product of DNA oxidation, 8-oxoguanine (GO), frequently mispairs with adenine during replication. We were interested in finding out how hyperthermophilic bacteria under goes the process of excising mispaired adenine from A/GO to deal with genomic oxidative damage. Herein we report the properties of an Escherichia coli MutY (EcMutY) homolog, TthMutY, derived from a hyperthermophile Thermus thermophilus. TthMutY preferentially excises on A/GO and G/GO mispairs and has additional activities on T/GO and A/G mismatches. TthMutY has significant sequence homology to the A/G and T/G mismatch recognition motifs, respectively, of MutY and Mig.MthI. A substitution from Tyr112 to Ser or Ala (Y112S and Y112A) in the putative thymine-binding site of TthMutY showed significant decrease in DNA glycosylase activity. A mutant form of TthMutY, R134K, could form a Schiff base with DNA and fully retained its DNA glycosylase activity against A/GO and A/G mispair. Interestingly, although TthMutY cannot form a trapped complex with substrate in the presence of NaBH(4), it expressed AP lyase activity, suggesting Tyr112 in TthMutY may be the key residue for AP lyase activity. These results suggest that TthMutY may be an example of a novel class of bifunctional A/GO mismatch DNA glycosylase that can also remove thymine from T/GO mispair.     

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.     

Formation of a Schiff base intermediate is not required for the adenine glycosylase activity of Escherichia coli MutY

The mutY gene product of Escherichia coli is a 39-kDa protein that catalyzes the removal of adenine bases mispaired with 2'-deoxyguanosine and 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG) in DNA. Although adenine removal proceeds via monofunctional glycosylase activity, MutY is able to form covalent adducts with substrate DNA in the presence of borohydride, a trait otherwise known to be associated only with enzymes having bifunctional glycosylase/AP lyase activity. To help identify active site residues involved in the formation of MutY-DNA adducts in the presence of borohydride, a series of site-directed mutant forms of MutY were generated. Our data show that Lys 142 is the primary residue involved in cross-link formation. The absence of Lys 142 results in near elimination of the enzyme-DNA adducts formed relative to wild-type, suggesting that this residue is the primary one involved in forming covalent associations with DNA during MutY catalysis. Importantly, the enzymatic activity and DNA binding of the K142A enzyme is nearly identical to the WT enzyme. This shows that formation of the covalent intermediate is not required for adenine removal by MutY. Furthermore, this suggests that the covalent intermediate is formed by reaction of Lys 142 with the OG/G:(AP site) product, and this may be a consequence of MutY's unusually high affinity for the product of its glycosylase action.     

Formation of purine-purine mispairs by Sulfolobus solfataricus DNA polymerase IV

DNA damage that stalls replicative polymerases can be bypassed with the Y-family polymerases. These polymerases have more open active sites that can accommodate modified nucleotides. The lack of protein-DNA interactions that select for Watson-Crick base pairs correlate with the lowered fidelity of replication. Interstrand hydrogen bonds appear to play a larger role in dNTP selectivity. The mechanism by which purine-purine mispairs are formed and extended was examined with Solfolobus solfataricus DNA polymerase IV, a member of the RAD30A subfamily of the Y-family polymerases, as is pol eta. The structures of the purine-purine mispairs were examined by comparing the kinetics of mispair formation with adenine versus 1-deaza- and 7-deazaadenine and guanine versus 7-deazaguanine at four positions in the DNA, the incoming dNTP, the template base, and both positions of the terminal base pair. The time course of insertion of a single dNTP was examined with a polymerase concentration of 50 nM and a DNA concentration of 25 nM with various concentrations of dNTP. The time courses were fitted to a first-order equation, and the first-order rate constants were plotted against the dNTP concentration to produce k pol and K d (dNTP) values. A decrease in k pol/ K d (dNTP) associated with the deazapurine substitution would indicate that the position is involved in a crucial hydrogen bond. During correct base pair formation, the adenine to 1-deazaadenine substitution in both the incoming dNTP and template base resulted in a >1000-fold decrease in k pol/ K d (dNTP), indicating that interstrand hydrogen bonds are important in correcting base pair formation. During formation of purine-purine mispairs, the k pol/ K d (dNTP) values for the insertion of dATP and dGTP opposite 7-deazaadenine and 7-deazaguanine were decreased >10-fold with respect to those of the unmodified nucleotides. In addition, the rate of incorporation of 1-deaza-dATP opposite guanine was decreased 5-fold. These results suggest that during mispair formation the newly forming base pair is in a Hoogsteen geometry with the incoming dNTP in the anti conformation and the template base in the syn conformation. These results indicate that Dpo4 holds the incoming dNTP in the normal anti conformation while allowing the template nucleotide to change conformations to allow reaction to occur. This result may be functionally relevant in the replication of damaged DNA in that the polymerase may allow the template to adopt multiple configurations.     

DNA-mediated charge transport as a probe of MutY/DNA interaction

MutY is an Escherichia coli DNA repair enzyme that binds to 8-oxo-G:A and G:A mismatches and catalyzes the deglycosylation of the mismatched 2'-deoxyadenosine. We have applied DNA-mediated charge transport to probe the interaction of MutY with its DNA substrate. Oligonucleotides synthesized with a tethered rhodium intercalator and guanine doublets placed before and after the MutY binding site are used to assay for base flipping activity by MutY. On the basis of this assay, we find no evidence that MutY uses progressive base flipping as a means to find its binding site; protein binding does not perturb long-range DNA charge transport. DNA-mediated charge transport can be utilized to promote protein-DNA cross-linking from a distance. Long-range oxidation of 8-oxo-G within the MutY binding site using tethered rhodium intercalators promoted cross-linking and yielded information on MutY side chains that interact with this base. On the basis of photooxidative cross-linking of the wild type but not K142A mutant, it is evident that, within the protein complex, lysine 142 makes important contacts with 8-oxo-G.     

Mispairs with Watson‐Crick base‐pair geometry observed in ternary complexes of an RB69 DNA polymerase variant

Recent structures of DNA polymerase complexes with dGMPCPP/dT and dCTP/dA mispairs at the insertion site have shown that they adopt Watson‐Crick geometry in the presence of Mn²⁺ indicating that the tautomeric or ionization state of the base has changed. To see whether the tautomeric or ionization state of base‐pair could be affected by its microenvironment, we determined 10 structures of an RB69 DNA polymerase quadruple mutant with dG/dT or dT/dG mispairs at position n‐1 to n‐5 of the Primer/Template duplex. Different shapes of the mispairs, including Watson‐Crick geometry, have been observed, strongly suggesting that the local environment of base‐pairs plays an important role in their tautomeric or ionization states.     

Catalytic and DNA binding properties of the ogg1 protein of Saccharomyces cerevisiae: comparison between the wild type and the K241R and K241Q active-site mutant proteins

The Ogg1 protein of Saccharomyces cerevisiae belongs to a family of DNA glycosylases and apurinic/apyrimidinic site (AP) lyases, the signature of which is the alpha-helix-hairpin-alpha-helix-Gly/Pro-Asp (HhH-GPD) active site motif together with a conserved catalytic lysine residue, to which we refer as the HhH-GPD/K family. In the yeast Ogg1 protein, yOgg1, the HhH-GPD/K motif spans residues 225-260 and the conserved lysine is K241. In this study, we have purified the K241R and K241Q mutant proteins and compared their catalytic and DNA binding properties to that of the wild-type yOgg1. The results show that the K241R mutation greatly impairs both the DNA glycosylase and the AP lyase activities of yOgg1. Specificity constants for cleavage of a 34mer oligodeoxyribonucleotide containing a 7,8-dihydro-8-oxoguanine (8-OxoG) paired with a cytosine, [8-OxoG.C], are 56 x 10(-)(3) and 5 x 10(-)(3) min(-)(1) nM(-)(1) for the wild-type and the K241R protein, respectively. On the other hand, the K241Q mutation abolishes the DNA glycosylase and AP lyase activities of yOgg1. In contrast, the K241R and K241Q proteins have conserved wild-type DNA binding properties. K(dapp) values for binding of [8-OxoG.C] are 6.9, 7.4, and 4.8 nM for the wild-type, K241R, and K241Q proteins, respectively. The results also show that AP site analogues such as 1, 3-propanediol (Pr), tetrahydrofuran (F), or cyclopentanol (Cy) are not substrates but constitute good inhibitors of the wild-type yOgg1. Therefore, we have used a 59mer [Pr.C] duplex to further analyze the DNA binding properties of the wild-type, K241R, and K241Q proteins. Hydroxyl radical footprints of the wild-type yOgg1 show strong protection of six nucleotides centered around the Pr lesion in the damaged strand. On the complementary strand, only the cytosine placed opposite Pr was strongly protected. The same footprints were observed with the K241R and K241Q proteins, confirming their wild-type DNA binding properties. These results indicate that the K241Q mutant protein can be used to study interactions between yOgg1 and DNA containing metabolizable substrates such as 8-OxoG or an AP site.     

Substrate recognition by Escherichia coli MutY using substrate analogs.

The Escherichia coli adenine glycosylase MutY is involved in the repair of 7,8-dihydro-8-oxo-2"-deoxyguanosine (OG):A and G:A mispairs in DNA. Our approach toward understanding recognition and processing of DNA damage by MutY has been to use substrate analogs that retain the recognition properties of the substrate mispair but are resistant to the glycosylase activity of MutY. This approach provides stable MutY-DNA complexes that are amenable to structural and biochemical characterization. In this work, the interaction of MutY with the 2"-deoxyadenosine analogs 2"-deoxy-2"-fluoroadenosine (FA), 2"-deoxyaristeromycin (R) and 2"-deoxyformycin A (F) was investigated. MutY binds to duplexes containing the FA, R or F analogs opposite G and OG within DNA with high affinity; however, no enzymatic processing of these duplexes is observed. The specific nature of the interaction of MutY with an OG:FA duplex was demonstrated by MPE-Fe(II) hydroxyl radical footprinting experiments which showed a nine base pair region of protection by MutY surrounding the mispair. DMS footprinting experiments with an OG:A duplex revealed that a specific G residue located on the OG-containing strand was protected from DMS in the presence of MutY. In contrast, a G residue flanking the substrate analogs R, F or FA was observed to be hypersensitive to DMS in the presence of MutY. These results suggest a major conformational change in the DNA helix upon binding of MutY that exposes the substrate analog-containing strand. This finding is consistent with a nucleotide flipping mechanism for damage recognition by MutY. This work demonstrates that duplex substrates for MutY containing FA, R or F instead of A are excellent substrate mimics that may be used to provide insight into the recognition by MutY of damaged and mismatched base pairs within DNA.     

A repair system for 8-oxo-7,8-dihydrodeoxyguanine.

Active oxygen species can damage DNA and may play a role in aging and carcinogenesis. We have tested MutY glycosylase for activity on undamaged mispairs as well as mispairs formed with the oxidatively damaged substrates, 8-oxo-7,8-dihydrodeoxyguanine (GO) or 8-oxo-7,8-dihydrodeoxyadenine (AO). MutY acts as a glycosylase on four of the heteroduplexes tested, A/G, A/GO, A/C, and A/AO, removing the undamaged adenine from each substrate. Genetic data suggest that the primary substrate for MutY glycosylase in vivo is the A/GO mispair. We present biochemical evidence demonstrating that MutY glycosylase is an important part of a repair system that includes the MutM and MutT proteins. The GO repair system is dedicated to the repair of the oxidatively damaged guanine and the mutations it can induce.     

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.     

8-oxodGTP incorporation by DNA polymerase beta is modified by active-site residue Asn279

To understand how the active site of a DNA polymerase might modulate the coding of 8-oxo-7,8-dihydrodeoxyguanine (8-oxodG), we performed steady-state kinetic analyses using wild-type DNA polymerase beta (pol beta) and two active-site mutants. We compared the coding of these polymerases by calculating the ratio of efficiencies for incorporation of dATP and dCTP opposite 8-oxodG and for incorporation of 8-oxodGTP opposite dA and dC. For wild-type pol beta, there is a 2:1 preference for incorporation of dCTP over dATP opposite 8-oxodG using a 5'-phosphorylated 4-base gap substrate. Mutation of either Asn279 or Arg283 to alanine has almost no effect on the ratio. 8-OxodGTP is preferentially incorporated opposite a template dA (24:1) by wild-type pol beta; mutation of Asn279 to alanine results dramatic change whereby there is preferential incorporation of 8-oxodGTP opposite dC (14:1). This suggests that interactions of 8-oxodGTP with Asn279 in the polymerase active site may alter the conformation of 8-oxodGTP and therefore alter its misincorporation.     

NMR studies of the exocyclic 1,N6-ethenodeoxyadenosine adduct (epsilon dA) opposite deoxyguanosine in a DNA duplex. Epsilon dA(syn).dG(anti) pairing at the lesion site

Proton NMR studies are reported on the complementary d(C-A-T-G-G-G-T-A-C).d(G-T-A-C-epsilon A-C-A-T-G) nonanucleotide duplex (designated epsilon dA.dG 9-mer duplex), which contains exocyclic adduct 1,N6-ethenodeoxyadenosine positioned opposite deoxyguanosine in the center of the helix. The present study focuses on the alignment of dG5 and epsilon dA14 at the lesion site in the epsilon dA.dG 9-mer duplex at neutral pH. This alignment has been characterized by monitoring the NOEs originating from the NH1 proton of dG5 and the H2, H5, and H7/H8 protons of epsilon dA14 in the central d(G4-G5-G6).d(C13-epsilon A14-C15) trinucleotide segment of the epsilon dA.dG 9-mer duplex. These NOE patterns establish that epsilon dA14 adopts a syn glycosidic torsion angle that positions the exocyclic ring toward the major groove edge while all the other bases including dG5 adopt anti glycosidic torsion angles. We detect a set of intra- and interstrand NOEs between protons (exchangeable and nonexchangeable) on adjacent residues in the d(G4-G5-G6).d(C13-epsilon A14-C15) trinucleotide segment which establish formation of right-handed helical conformations on both strands and stacking of the dG5(anti).epsilon dA14(syn) pair between stable dG4.dC15 and dG6.dC13 pairs. The energy-minimized conformation of the central d(G4-G5-G6).d(C13-epsilon A14-C15) segment establishes that the dG5(anti).epsilon dA14(syn) alignment is stabilized by two hydrogen bonds from the NH1 and NH2-2 of dG5(anti) to N9 and N1 of epsilon dA14(syn), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Investigation of putative active-site lysine residues in hydroxymethylbilane synthase. Preparation and characterization of mutants in which (a) Lys-55, (b) Lys-59 and (c) both Lys-55 and Lys-59 have been replaced by glutamine.

A new construct carrying the hemC gene was transformed into Escherichia coli, resulting in approx. 1000-fold over-expression of hydroxymethylbilane synthase (HMBS). This construct was used to generate HMBS in which (a) Lys-55, (b) Lys-59 and (c) both Lys-55 and Lys-59 were replaced by glutamine (K55Q, K59Q and K55Q-K59Q respectively). All three modified enzymes are chromatographically separable from wild-type enzyme. Kinetic studies showed that the substitution K55Q has little effect whereas K59Q causes a 25-fold decrease in Kapp. cat./Kapp. m. Treatment of K55Q, K59Q and K55Q-K59Q separately with pyridoxal 5'-phosphate and NaBH4 resulted in incomplete and non-specific reaction with the remaining lysine residues. Pyridoxal modification of Lys-59 in the K55Q mutant caused greater enzymic inactivation than similar modification of Lys-55 in K59Q. The results in sum show that, though Lys-55 and Lys-59 may be at or near the active site, neither is indispensable for the catalytic activity of HMBS.     

Towards a better understanding of the unusual conformations of the alternating guanine-adenine repeat strands of DNA

Alternating guanine-adenine strands of DNA are known to self-associate into a parallel-stranded homoduplex at neutral pH, fold into an ordered single-stranded structure at acid pH, and adopt yet another ordered single-stranded conformer in aqueous ethanol. The unusual conformers melt cooperatively and exhibit distinct circular dichroism spectra suggestive of a substantial conformational order, but their molecular structures are not known yet. Here, we have probed the molecular structures using guanine and adenine analogs lacking the N7 atom, and thus unable of Hoogsteen pairing, or those restrained in the less-frequent syn glycosidic orientation. The studies showed that the syn glycosidic orientation of dA residues promoted the neutral homoduplex, whereas the syn orientation of dG was incompatible with the homoduplex. In addition, Hoogsteen pairing of dA seemed to be a crucial property of the homoduplex whereas dG did not pair in this way. The situation was the same in both single-stranded conformers with the dG residues. On the other hand, the presence of N7 was important with dA but its syn geometry was not favorable. The present data can be used as restraints to model the unusual molecular structures of the alternating guanine-adenine strands of DNA.