Mispairs In DNA Research Articles

HMutSα is Protected from Ubiquitin-proteasome-dependent Degradation by Atypical Protein Kinase Cζ Phosphorylation

The hMutS alpha (hMSH2-hMSH6) protein heterodimer plays a critical role in the detection of DNA mispairs in the mismatch repair (MMR) process. We recently reported that hMutS alpha proteins were degraded by the ubiquitin-proteasome pathway in a cell-type-dependent manner, indicating that one or several regulator(s) may interfere with hMutS alpha protein ubiquitination and degradation. On the other hand, we and others have shown that protein kinase C (PKC) is involved as a positive regulator of MMR activity. Here, we provide evidence that the atypical PKC zeta regulates ubiquitination, degradation, and levels of hMutS alpha proteins. Using both PKC zeta-transfected U937 and PKC zeta siRNA-transfected MRC-5 cell lines, we found that PKC zeta protein expression was correlated with that of hMutS alpha as well as with MMR activity, but was inversely correlated with hMutS alpha protein ubiquitination and degradation. Interestingly, PKC zeta interacts with hMSH2 and hMSH6 proteins and phosphorylates both. Moreover, in an in vitro assay PKCzeta mediates phosphorylation events decreasing hMutS alpha protein degradation via the ubiquitin-proteasome pathway. Altogether, our results indicate that PKC zeta modulates hMutS alpha stability and protein levels, and suggest a role for PKC zeta in genome stability by regulating MMR activity.

P53 in cytoplasm may enhance the accuracy of DNA synthesis by human immunodeficiency virus type 1 reverse transcriptase.

The tumor suppressor protein p53 displays 3' --> 5' exonuclease activity and can provide a proofreading function for DNA polymerases. Reverse transcriptase (RT) of human immunodeficiency virus (HIV)-1 is responsible for the conversion of the viral genomic ssRNA into the proviral DNA in the cytoplasm. The relatively low fidelity of HIV-1 RT was implicated as a dominant factor contributing to the genetic variability of the virus. The lack of intrinsic 3' --> 5' exonuclease activity, the formation of 3'-mispaired DNA and the subsequent extension of this DNA were shown to be determinants for the low fidelity of HIV-1 RT. It was of interest to analyse whether the cytoplasmic proteins may affect the accuracy of DNA synthesis by RT. We investigated the fidelity of DNA synthesis by HIV-1 RT with and without exonucleolytic proofreading provided by cytoplasmic fraction of LCC2 cells expressing high level of wild-type functional p53. Two basic features related to fidelity of DNA synthesis were studied: the misinsertion and mispair extension. The misincorporation of noncomplementary deoxynucleotides into nascent DNA and subsequent mispair extension by HIV-1 RT were substantially decreased in the presence of cytoplasmic fraction of LCC2 cells with both RNA/DNA and DNA/DNA template-primers with the same target sequence. The mispair extension frequencies obtained with the HIV-1 RT in the presence of cytoplasmic fraction of LCC2 cells were significantly lower (about 2.8-15-fold) than those detected with the purified enzyme. In addition, the productive interaction between polymerization (by HIV-1 RT) and exonuclease (by p53 in cytoplasm) activities was observed; p53 preferentially hydrolyses mispaired 3'-termini, permitting subsequent extension of the correctly paired 3'-terminus by HIV-1 RT. The data suggest that p53 in cytoplasm may affect the accuracy of DNA replication and the mutation spectra of HIV-1 RT by acting as an external proofreader. Furthermore, the decrease in error-prone DNA synthesis with RT in the presence of external exonuclease, provided by cytoplasmic p53, may partially account for lower mutation rate of HIV-1 observed in vivo.

Encoded errors: mutations and rearrangements mediated by misalignment at repetitive DNA sequences.

Mutations and rearrangements that occur by misalignment during DNA replication are frequent sources of genetic variation in bacteria. Dislocations between a replicating strand and its template at repetitive DNA sequences underlie the mechanism of these genetic events. Such misalignments can be transient or stable and can involve intramolecular or intermolecular DNA mispairing, even pairing across a replication fork. Paradoxically, these replication 'slippage' events both create and destroy repetitive sequences in bacterial genomes. This review catalogues several types of slippage errors, presents the cellular processes that act to limit them and discusses the consequences of this class of genetic events on the evolution of bacterial genomes and physiology.

Signaling from DNA mispairs to mismatch-repair excision sites despite intervening blockades.

Mismatch-repair (MMR) systems promote genomic stability by correction of DNA replication errors. Thus, MMR proteins--prokaryotic MutS and MutL homodimers or their MutSalpha and MutLalpha heterodimer homologs, plus accessory proteins--specifically couple mismatch recognition to nascent-DNA excision. In vivo excision-initiation signals--specific nicks in some prokaryotes, perhaps growing 3' ends or Okazaki-fragment 5' ends in eukaryotes--are efficiently mimicked in vitro by nicks or gaps in exogenous DNA substrates. In some models for recognition-excision coupling, MutSalpha bound to mismatches is induced by ATP hydrolysis, or simply by binding of ATP, to slide along DNA to excision-initiation sites, perhaps in association with MutLalpha and accessory proteins. In other models, MutSalpha.MutLalpha complexes remain fixed at mismatches and contact distant excision sites by DNA looping. To challenge the hypothesis that recognition complexes remain fixed, we placed biotin-streptavidin blockades between mismatches and pre-existing nicks. In human nuclear extracts, mismatch efficiently provoked the initiation of excision despite the intervening barriers, as predicted. However, excision progress and therefore mismatch correction were prevented.

A residue in MutY important for catalysis identified by photocross-linking and mass spectrometry.

MutY is an adenine glycosylase in the base excision repair (BER) superfamily that is involved in the repair of 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG):A and G:A mispairs in DNA. MutY contains a [4Fe-4S]2+ cluster that is part of a novel DNA binding motif, referred to as the iron-sulfur cluster loop (FCL) motif. This motif is found in a subset of members of the BER glycosylase superfamily, defining the endonuclease III-like subfamily. Site-specific cross-linking was successfully employed to investigate the DNA-protein interface of MutY. The photoreactive nucleotide 4-thiothymidine (4ST) incorporated adjacent to the OG:A mismatch formed a specific cross-link between the substrate DNA and MutY. The amino acid participating in the cross-linking reaction was characterized by positive ion electrospray ionization (ESI) tandem mass spectrometry. This analysis revealed Arg 143 as the site of modification in MutY. Arg 143 and nearby Arg 147 are conserved throughout the endo III-like subfamily. Replacement of Arg 143 and Arg 147 with alanine by site-directed mutagenesis reduces adenine glycosylase activity of MutY toward OG:A and G:A mispairs. In addition, the R143A and R147A enzymes exhibit a reduced affinity for duplexes containing the substrate analogue 2'-deoxy-2'-fluoroadenosine opposite OG and G. Modeling of MutY bound to DNA using an endonuclease III-DNA complex structure shows that these two conserved arginines are located within close proximity to the DNA backbone. The insight from mass spectrometry experiments combined with functional mutagenesis results indicate that these two amino acids in the [4Fe-4S]2+ cluster-containing subfamily play an important role in recognition of the damaged DNA substrate.

Escherichia coli nucleoside diphosphate kinase is a uracil-processing DNA repair nuclease

Escherichia coli nucleoside diphosphate kinase (eNDK) is an XTP:XDP phosphotransferase that plays an important role in the regulation of cellular nucleoside triphosphate concentrations. It is also one of several recently discovered DNases belonging to the NM23/NDK family. E. coli cells disrupted in the ndk gene display a spontaneous mutator phenotype, which has been attributed to the mutagenic effects of imbalanced nucleotide pools and errors made by replicative DNA polymerases. Another explanation for the increased mutation rates is that endk- cells lack the nuclease activity of the NDK protein that is essential for a DNA repair pathway. Here, we show that purified, cloned endk is a DNA repair nuclease whose substrate is uracil misincorporated into DNA. We have identified three new catalytic activities in eNDK that act sequentially to repair the uracil lesion: (i) uracil-DNA glycosylase that excises uracil from single-stranded and from U/A and U/G mispairs in double-stranded DNA; (ii) apyrimidinic endonuclease that cleaves double-stranded DNA as a lyase by forming a covalent enzyme-DNA intermediate complex with the apyrimidinic site created by the glycosylase; and (iii) DNA repair phosphodiesterase that removes 3'-blocking residues from the ends of duplex DNA. All three of these activities, as well as the nucleoside-diphosphate kinase, reside in the same protein. Based on these findings, we propose an editing function for eNDK as a mechanism by which the enzyme prevents mutations in DNA.

Substrate specificity and sequence preference of G:T mismatch repair: incision at G:T, O6-methylguanine:T, and G:U mispairs in DNA by human cell extracts.

Extracts of two human glioma cell lines (lacking O6-methylguanine DNA-methyltransferase) (i.e., A1235 and its alkylation-resistant derivative A1235-MR4) were examined for their ability to execute strand incision at different base mismatches in model (45-bp) DNA. These heteroduplex substrates were of the same sequence except for the presence, at the same site, of one of three mispairs: G:T, O6-methylguanine:T (m6G:T), and G:U. The parental (A1235) extract, when supplemented with ATP and human thymine DNA glycosylase (TDG), acted proficiently on all three substrates, incising immediately 5' to the mismatched thymine or uracil residue. In contrast, the derivative extract, under the same conditions, recognized only the G:U substrate. The activity of the A1235 extract toward the G:T (or m6G:T) substrate was markedly reduced in the absence of ATP, whereas the G:U substrate was incised rapidly by both extracts irrespective of the addition of ATP. These combined data confirm and extend our earlier findings demonstrating that human cells possess two G:T incision activities, one efficient and ATP-dependent and the other inefficient and ATP-independent. The derivative extract lacks the former activity but retains the latter activity. In substrate competition assays, the G:U substrate inhibited the ATP-dependent G:T incision activity to a greater extent than did the G:T substrate itself. Given the well-known substrate preference of TDG for G:U as compared to G:T, this unexpected result implies that TDG may be an integral component of the ATP-dependent G:T incision machinery in human cells. Finally, the base 5' to the mismatched G in the G:T mispair conferred sequence preference on the A1235 extract in the presence of ATP and TDG, with a pyrimidine (especially cytosine) being much favored over a purine. This latter observation suggests that the ATP-dependent G:T incision activity is designed to repair deaminated 5-methycytosine lesions in CpG islands, the methylation of which is linked to control of gene expression.

Characterization of 2-hydroxyadenine DNA glycosylase activity of Escherichia coli MutY protein

Purpose : 2-Hydroxyadenine (2-ohA) is an oxidation product of adenine generated in DNA by ionizing radiation and various chemical oxidants. 2-ohA has mutational potential comparable to that of 8-oxoguanine in bacteria and mammalian cells. Recent studies have shown that 2-ohA is removed from DNA by a human MutY homolog, MYH protein, in vitro. On the other hand, the repair mechanisms for 2-ohA in Escherichia coli are not yet understood. Materials and methods : Gel shift assays were used to assess the binding activity of E. coli full-length MutY protein and its N-terminal (residues 1-226) domain (M25) to 2-ohA/G-, 2-ohA/A-, 2-ohA/C- and 2-ohA/T-containing 24-mer oligonucleotides. Furthermore, whether these proteins specifically cleave 2-ohA-containing duplex oligonucleotides was examined. Results : The purified MutY and M25 proteins had similar binding affinities to 2-ohA/G-, 2-ohA/A- and 2-ohA/C-containing oligonucleotides. MutY protein removed 2-ohA preferentially from 2-ohA/G mispairs. M25 protein showed the reduced catalytic activity for 2-ohA/G-containing oligonucleotides. Conclusions : E. coli MutY protein has a DNA glycosylase activity that removes 2-ohA from 2-ohA/G mispairs in DNA. The C-terminal domain is required for the removal of 2-ohA from DNA, but is not crucial for binding to 2-ohA-containing oligonucleotides.

Detection of DNA base mismatches using DNA intercalators

The most prevalent mechanisms leading to mutations in DNA are direct mis-incorporation of bases during replication and sustained chemical damage. Under normal circumstances, the cell corrects these problems using DNA polymerase proofreading mechanisms as well as the complex repair machinery of the cell. In certain tissues that contain mismatch repair deficiencies, DNA mispairs may accumulate.1, 2, 3, 4 and 5 Even in healthy cells, however, mismatches and lesions can sometimes go unchecked, resulting in permanent alterations in the gene sequence for subsequent generations. Identification of genetic variations (single-nucleotide polymorphisms, SNPs) among individuals and populations has implications in understanding human disease and treatment, as well as the interaction of the environment and multiple genes during evolution.6 Once these SNPs are identified and understood, rapid and reliable detection of them will be critical for the study, diagnosis, and treatment of genetically linked disease.

P53 enhances the fidelity of DNA synthesis by human immunodeficiency virus type 1 reverse transcriptase.

The tumor suppressor protein p53 plays a critical role in the maintenance of genetic integrity. p53 possesses 3'-->5' exonuclease activity, however, the significance of this function in DNA replication process remains elusive. It was suggested that 3'-->5' exonuclease activity of p53 may provide a proofreading function for DNA polymerases. In order to better understand the significance of this activity, the purified wild-type recombinant p53 was further evaluated for substrate specificity and for contribution to the accuracy of DNA synthesis. p53-associated 3'-->5' exonuclease displays 3' terminal nucleotide excision from RNA/DNA template-primer using ribosomal RNA as a template. The data demonstrate that p53 is highly efficient in removing a terminal mispair. Analysis of mispair excision opposite the template adenine residue shows that p53 catalyzes 3' terminal mismatch excision with a specificity of A : G>A : A>A : C. Hence, the observed specificity of mismatch excision indicates that p53 exonucleolytic proofreading preferentially repairs transversion mutations. The influence of the p53 on the accuracy of DNA synthesis was determined with exonuclease-deficient human immunodeficiency virus-1 (HIV-1) reverse transcriptase (RT), a key enzyme in the life cycle of the virus, that contributes significantly to the low accuracy of proviral DNA synthesis. Using an in vitro biochemical assay with recombinant purified HIV-1 RT, p53 and defined RNA/DNA or DNA/DNA template-primers, two basic features related to fidelity of DNA synthesis were studied: the misinsertion and mispair extension. The misincorporation of non-complementary deoxynucleotides into nascent DNA and subsequent mispair extension by HIV-1 RT were substantially decreased in the presence of p53 with both RNA/DNA and DNA/DNA template-primers. In addition, the productive interaction between polymerization (by HIV-1 RT) and exonuclease (by p53) activities was observed; p53 preferentially hydrolyzes mispaired 3'-termini, permitting subsequent extension of the correctly paired 3'-terminus by HIV-1 RT. Taken together the data demonstrate that preferential excision of mismatched nucleotides by 3'-->5' exonuclease activity of wild-type p53 enhances the fidelity of DNA synthesis by HIV-1 RT in vitro, thus providing a biochemical mechanism to reduce mutations caused by incorporation of mismatched nucleotides. The fact that p53 is reactive with both RNA/DNA and DNA/DNA template-primers raises an interesting possibility of the existence of functional cooperation between p53 and HIV-1 RT in cytoplasm during the reverse transcription process, which may be important for maintaining HIV genomic integrity.

Escherichia coli Nth and human hNTH1 DNA glycosylases are involved in removal of 8-oxoguanine from 8-oxoguanine/guanine mispairs in DNA.

The spectrum of DNA damage caused by reactive oxygen species includes a wide variety of modifications of purine and pyrimidine bases. Among these modified bases, 7,8-dihydro-8-oxoguanine (8-oxoG) is an important mutagenic lesion. Base excision repair is a critical mechanism for preventing mutations by removing the oxidative lesion from the DNA. That the spontaneous mutation frequency of the Escherichia coli mutT mutant is much higher than that of the mutM or mutY mutant indicates a significant potential for mutation due to 8-oxoG incorporation opposite A and G during DNA replication. In fact, the removal of A and G in such a situation by MutY protein would fix rather than prevent mutation. This suggests the need for differential removal of 8-oxoG when incorporated into DNA, versus being generated in situ. In this study we demonstrate that E.coli Nth protein (endonuclease III) has an 8-oxoG DNA glycosylase/AP lyase activity which removes 8-oxoG preferentially from 8-oxoG/G mispairs. The MutM and Nei proteins are also capable of removing 8-oxoG from mispairs. The frequency of spontaneous G:C-->C:G transversions was significantly increased in E.coli CC103mutMnthnei mutants compared with wild-type, mutM, nth, nei, mutMnei, mutMnth and nthnei strains. From these results it is concluded that Nth protein, together with the MutM and Nei proteins, is involved in the repair of 8-oxoG when it is incorporated opposite G. Furthermore, we found that human hNTH1 protein, a homolog of E.coli Nth protein, has similar DNA glycosylase/AP lyase activity that removes 8-oxoG from 8-oxoG/G mispairs.

MSH-MLH complexes formed at a DNA mismatch are disrupted by the PCNA sliding clamp

In the yeast Saccharomyces cerevisiae, mismatch repair (MMR) is initiated by the binding of heterodimeric MutS homolog (MSH) complexes to mismatches that include single nucleotide and loop insertion/deletion mispairs. In in vitro experiments, the mismatch binding specificity of the MSH2-MSH6 heterodimer is eliminated if ATP is present. However, addition of the MutL homolog complex MLH1-PMS1 to binding reactions containing MSH2-MSH6, ATP, and mismatched substrate results in the formation of a stable ternary complex. The stability of this complex suggests that it represents an intermediate in MMR that is subsequently acted upon by other MMR factors. In support of this idea, we found that the replication processivity factor proliferating cell nuclear antigen (PCNA), which plays a critical role in MMR at step(s) prior to DNA resynthesis, disrupted preformed ternary complexes. These observations, in conjunction with experiments performed with streptavidin end-blocked mismatch substrates, suggested that PCNA interacts with an MSH-MLH complex formed on DNA mispairs.

Metal-Stabilized Rare Tautomers and Mispairs of DNA Bases: N6-Metalated Adenine and N4-Metalated Cytosine, Theoretical and Experimental Views

Crystal structure studies indicate that metalation of the exocyclic amino group of cytosine and adenine nucleobases by PtII and HgII entities, respectively, induces protonation of a nucleobase ring nitrogen atom, and hence, causes a proton shift from an exocyclic to an endocyclic N atom. This metal-assisted process thus leads to the generation of rare nucleobase tautomers. In principle, such processes can lead to the stabilization of mispairs. The present study reports the first quantum chemical analysis of the metal-assisted tautomerization. The calculations clearly demonstrate that metalation of the exocyclic amino group of nucleobases significantly increases the protonation energy of the aromatic rings of nucleobases by about 30−34 kcal/mol for the PtII adduct and by about 10−14 kcal/mol for the HgII adduct. The calculations suggest that this kind of metalation could, besides the structural changes of DNA, significantly enhance the probability of formation of mispairs in DNA. In the course of the study...

Separation-of-function mutations in Saccharomyces cerevisiae MSH2 that confer mismatch repair defects but do not affect nonhomologous-tail removal during recombination.

Yeast Msh2p forms complexes with Msh3p and Msh6p to repair DNA mispairs that arise during DNA replication. In addition to their role in mismatch repair (MMR), the MSH2 and MSH3 gene products are required to remove 3' nonhomologous DNA tails during genetic recombination. The mismatch repair genes MSH6, MLH1, and PMS1, whose products interact with Msh2p, are not required in this process. We have identified mutations in MSH2 that do not disrupt genetic recombination but confer a strong defect in mismatch repair. Twenty-four msh2 mutations that conferred a dominant negative phenotype for mismatch repair were isolated. A subset of these mutations mapped to residues in Msh2p that were analogous to mutations identified in human nonpolyposis colorectal cancer msh2 kindreds. Approximately half of the these MMR-defective mutations retained wild-type or nearly wild-type activity for the removal of nonhomologous DNA tails during genetic recombination. The identification of mutations in MSH2 that disrupt mismatch repair without affecting recombination provides a first step in dissecting the Msh-effector protein complexes that are thought to play different roles during DNA repair and genetic recombination.

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.

Evidence that MutY is a monofunctional glycosylase capable of forming a covalent Schiff base intermediate with substrate DNA.

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. DNA strand cleavage via beta-elimination (beta-lyase) activity coupled with MutY's removal of misincorporated adenine bases was sought using both qualitative and quantitative methods. The qualitative assays demonstrate formation of a Schiff base intermediate which is characteristic of DNA glycosylases catalyzing a concomitant beta-lyase reaction. Borohydride reduction of the Schiff base results in the formation of a covalent DNA-MutY adduct which is easily detected in SDS-PAGE experiments. However, quantitative activity assays which monitor DNA strand scission accompanying base release suggest MutY behaves as a simple monofunctional glycosylase. Treatment with base effects DNA strand cleavage at apurinic/apyrimidinic (AP) sites arising via simple glycosylase activity. The amount of cleaved DNA in MutY reactions treated with base is much greater than that in non-base treated reactions, indicating that AP site generation by MutY is not associated with a concomitant beta-lyase step. As standards, identical assays were performed with a known monofunctional enzyme (uracil DNA glycosylase) and a known bifunctional glycosylase/lyase (FPG), the results of which were used in comparison with those of the MutY experiments. The apparent inconsistency between the data obtained for MutY by the qualitative and quantitative methods underscores the current debate surrounding the catalytic activity of this enzyme, and a detailed explanation of this controversy is proposed. The work presented here lays ground for the identification of specific active site residues responsible for the chemical mechanism of MutY enzyme catalysis.

Regulation of apoptotic cell death in the pyloric glands of the canine stomach.

In gastrointestinal epithelia, apoptosis has been thought to play a part in the shedding of postmitotic cells into the lumen. However, we have found that apoptosis more frequently in the generative cell (G) zone (the lower one third of the pit) than in the luminal zone (the upper one third of the pit) and the gland zone in the canine pyloric gland. To analyse the regulation of apoptotic cell death in each zone, we labelled S-phase cells by single and repeated injections of bromodeoxyuridine (BrdU) i.v. at intervals of 8 h. We found that 30% of apoptoses in the G zone were flash-labelled by BrdU and might derive from cells in or just after the S phase. The incidence of apoptosis and mitotic index did not change significantly after repeated injections of BrdU until the 49-h point, when the incidence of apoptosis increased and the mitotic index decreased significantly in the G zone, while the incidence of apoptosis decreased in the luminal zone. The BrdU-induced increase of apoptosis and cell-cycle arrest at the 49-h point may be caused by enhanced DNA mispairs that are elicited by incorporation of BrdU, in particular using the template of BrdU incorporated DNA. Apoptosis in the luminal zone may be down-regulated by reduced cell production in the G-zone.

Higher fidelity of RNA-dependent DNA mispair extension by M184V drug-resistant than wild-type reverse transcriptase of human immunodeficiency virus type 1.

Reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) has low fidelity compared with RTs of other retroviruses and cellular DNA polymerases. We and others have previously found that the fidelity of DNA-dependent DNA polymerization (DDDP) of M184V-mutated HIV-1 RT is significantly higher than that of wild-type RT. Viruses containing the M184V substitution are highly resistant to (-)-2'-dideoxy-3'-thiacytidine (3TC) in vitro and in patients treated with 3TC monotherapy. It was of interest to determine the fidelity of RNA-dependent DNA polymerization (RDDP) of M184V RT compared with wild-type because this step occurs first in reverse transcription; errors made during this step may be copied in subsequent polymerization steps. Using an in vitro mispaired primer extension assay, M184V-mutated RT exhibited 3-49-fold decreased frequency of mispair extension compared with wild-type RT. Fidelity differences between M184V and wild-type RT were most marked in extension of A:G (49-fold) and A:C (16-fold) mispairs, with only a marginal (3-fold) decrease in the extension of A:A mispairs. RT containing a methionine to isoleucine (M184I) mutation showed only slight increases in RDDP fidelity compared with wild-type, ranging from 1.5- to 6-fold increases. Of the three RTs tested, wild-type RT was the most error-prone, with mispair extension frequencies ranging from 6.674 x 10(-1) to 7.454 x10(-2).

Triplet repeat instability and DNA topology: an expansion model based on statistical mechanics.

The variance of writhe, the contribution of writhe to supercoiling, and the free energies of supercoiling were calculated for (CTG.CAG)n and (CGG.CCG)n triplet repeat sequences (TRS) by statistical mechanics from the bending and torsional moduli previously determined. Expansions of these sequences are inherited by non-mendelian transmission and are linked with several hereditary neuromuscular diseases. The variance of writhe was greater for the TRS than for random B-DNA. For random B-DNA, (CGG)n, and (CTG)n, the contribution of writhe to supercoiling was 70, 78, and 79%, whereas the free energy of supercoiling at a length of 10 kilobase pairs was 1040.RT, 760.RT, and 685.RT, respectively. These data indicate that the TRS are preferential sites for the partitioning of supercoiling. Calculations of the differences in free energy of supercoiling between the TRS and random B-DNA revealed a local minimum at approximately 520 base pairs. Human medical genetic studies have shown that individuals carrying up to 180-200 copies of TRS (540-600 base pairs, premutations) in the fragile X or myotonic dystrophy gene loci are usually asymptomatic, whereas large expansions (>200 repeats, full mutations), which lead to disease, are observed in their offspring. Therefore, the length corresponding to the local minimum in free energy of supercoiling correlates with the genetic breakpoint between premutation and full mutation. We propose that (a) TRS instability is mediated by DNA mispairing caused by the accumulation of supercoiling within the repeats, and (b) the expansions that take place at the premutation to full mutation threshold are associated with increased mispairing caused by the optimal partitioning of writhe within the TRS at this length.

Metal-Stabilized Rare Tautomers of Nucleobases. 6. Imino Tautomer of Adenine in a Mixed-Nucleobase Complex of Mercury(II).

(1,3-Dimethyluracil-5-yl)mercury(II), (1,3-DimeU-C5)Hg(II), reacts with the model nucleobase 9-methyladenine (9-MeA) to give the mixed-nucleobase complex [Hg(1,3-DimeU-C5)(9-MeA-N6)]NO(3).H(2)O. Hg(II) binds to the adenine via the N6 position with N1 being protonated. The neutral adenine nucleobase is therefore present in its imino form, which represents the rare tautomer form of this nucleobase. The X-ray crystal structure analysis reveals an anti orientation of the (1,3-DimeU-C5)Hg(II) entity with respect to N1 of 9-MeA and substantial differences in nucleobase geometry as compared to the amino tautomer form of 9-MeA. These differences refer to both the pyrimidine and the imidazole rings of the purine base. The possible relevance of this compound with regard to nucleobase cross-linking in DNA and nucleobase mispairing is discussed. Attempts to demonstrate mispairing between the metalated rare tautomer and "the wrong" bases 1-methylcytosine (1-MeC) and 9-ethylguanine (9-EtGH) in DMSO-d(6) proved unsuccessful due to a ligand exchange process at the heavy metal.