Biomedical subjects

A S Yang

Publications and source records attributed to A S Yang.

At least 19 recordsLinked to original sources

PubMed2000

An integrated approach to the analysis and modeling of protein sequences and structures. I. Protein structural alignment and a quantitative measure for protein structural distance.

We have devised and implemented in PrISM (protein informatics system for modeling) a new measure of protein structural relationships, the protein structural distance (PSD). The PSD is designed to describe relationships between protein structures in quantitative rather than descriptive terms and is applicable both when two structures are very similar, and when they are very different. It is calculated with a structural alignment procedure that uses double dynamic programming to align secondary structure elements and an iterative rigid body superposition that minimizes the root-mean-square deviation of C(alpha) atoms. The alignment algorithm, as implemented on a modest workstation, is computationally efficient, allowing for large-scale structural comparisons. PSD scores for more than one and a half million pairs of proteins were calculated and compared to the discrete classification of proteins in the SCOP database. The PSD scores, which were obtained automatically, are in large part consistent with the manually derived classifications in SCOP. Discrepancies do arise, however, due, in part, to the fact that SCOP uses criteria other than structural similarity to derive classifications while the PrISM procedure is exclusively structure based. Analysis of PSD scores suggests that there is a continuous aspect of protein conformation space, even though various classification schemes are extremely useful. The use of a continuous measure for structural distance between all pairs of proteins allows us, as described in the two accompanying papers to derive sequence/structure relationships in a more quantitative way than has previously been possible. An important strength of the approach implemented in PrISM is its ability to address many different kinds of queries interactively, making its structural comparison procedure a convenient computational tool that complements structural classification databases such as SCOP and CATH.

Algorithms↗

PubMed2000

An integrated approach to the analysis and modeling of protein sequences and structures. II. On the relationship between sequence and structural similarity for proteins that are not obviously related in sequence.

Here, we discuss the relationship between protein sequence and protein structural similarity. It is established that a protein structural distance (PSD) of 2.0 is a threshold above which two proteins are unlikely to have a detectable pairwise sequence relationship. A precise correlation is established between the level of sequence similarity, defined by a normalized Smith-Waterman score, and the probability that two proteins will have a similar structure (defined by pairwise PSD<2). This correlation can be used in evaluating the likelihood for success in a comparative modeling procedure. We establish the existence of a correlation between sequence and structural similarity for pairs of proteins that are related in structure but whose sequence relationship is not detectable using standard pairwise sequence alignments. Although it is well known that there is a close relationship between sequence and structural similarity for pairwise sequence identities greater than about 30 %, there has been little discussion as to the possible existence of such a relationship for pairs of proteins in or below the twilight zone of sequence similarity (<25 % pairwise sequence identity). Possible implications of our results for the evolution of protein structure are discussed.

Algorithms↗

PubMed2000

An integrated approach to the analysis and modeling of protein sequences and structures. III. A comparative study of sequence conservation in protein structural families using multiple structural alignments.

The information required to generate a protein structure is contained in its amino acid sequence, but how three-dimensional information is mapped onto a linear sequence is still incompletely understood. Multiple structure alignments of similar protein structures have been used to investigate conserved sequence features but contradictory results have been obtained, due, in large part, to the absence of subjective criteria to be used in the construction of sequence profiles and in the quantitative comparison of alignment results. Here, we report a new procedure for multiple structure alignment and use it to construct structure-based sequence profiles for similar proteins. The definition of "similar" is based on the structural alignment procedure and on the protein structural distance (PSD) described in paper I of this series, which offers an objective measure for protein structure relationships. Our approach is tested in two well-studied groups of proteins; serine proteases and Ig-like proteins. It is demonstrated that the quality of a sequence profile generated by a multiple structure alignment is quite sensitive to the PSD used as a threshold for the inclusion of proteins in the alignment. Specifically, if the proteins included in the aligned set are too distant in structure from one another, there will be a dilution of information and patterns that are relevant to a subset of the proteins are likely to be lost. In order to understand better how the same three-dimensional information can be encoded in seemingly unrelated sequences, structure-based sequence profiles are constructed for subsets of proteins belonging to nine superfolds. We identify patterns of relatively conserved residues in each subset of proteins. It is demonstrated that the most conserved residues are generally located in the regions where tertiary interactions occur and that are relatively conserved in structure. Nevertheless, the conservation patterns are relatively weak in all cases studied, indicating that structure-determining factors that do not require a particular sequential arrangement of amino acids, such as secondary structure propensities and hydrophobic interactions, are important in encoding protein fold information. In general, we find that similar structures can fold without having a set of highly conserved residue clusters or a well-conserved sequence profile; indeed, in some cases there is no apparent conservation pattern common to structures with the same fold. Thus, when a group of proteins exhibits a common and well-defined sequence pattern, it is more likely that these sequences have a close evolutionary relationship rather than the similarities having arisen from the structural requirements of a given fold.

Algorithms↗

PubMed1999

Characterization of the interaction between the Wilson and Menkes disease proteins and the cytoplasmic copper chaperone, HAH1p.

Wilson disease (WD) and Menkes disease (MNK) are inherited disorders of copper metabolism. The genes that mutate to give rise to these disorders encode highly homologous copper transporting ATPases. We use yeast and mammalian two-hybrid systems, along with an in vitro assay to demonstrate a specific, copper-dependent interaction between the six metal-binding domains of the WD and MNK ATPases and the cytoplasmic copper chaperone HAH1. We demonstrate that several metal-binding domains interact independently or in combination with HAH1p, although notably domains five and six of WDp do not. Alteration of either the Met or Thr residue of the HAH1p MTCXXC motif has no observable effect on the copper-dependent interaction, whereas alteration of either of the two Cys residues abolishes the interaction. Mutation of any one of the HAH1p C-terminal Lys residues (Lys(56), Lys(57), or Lys(60)) to Gly does not affect the interaction, although deletion of the 15 C-terminal residues abolishes the interaction. We show that apo-HAH1p can bind in vitro to copper-loaded WDp, suggesting reversibility of copper transfer from HAH1p to WD/MNKp. The in vitro HAH1/WDp interaction is metalospecific; HAH1 preincubated with Cu(2+) or Hg(+) but not with Zn(2+), Cd(2+), Co(2+), Ni(3+), Fe(3+), or Cr(3+) interacted with WDp. Finally, we model the protein-protein interaction and present a theoretical representation of the HAH1p.Cu.WD/MNKp complex.

Adenosine Triphosphatases↗

PubMed1999

Sequence to structure alignment in comparative modeling using PrISM.

PrISM (Protein Informatics System for Modeling) is a protein analysis and modeling system in which informatics, alignment, modeling, and assessment modules are integrated in a computational environment where protein analysis and modeling protocols can be designed and assessed interactively. It can then be used automatically and repetitively in response to a variety of protein analysis and modeling problems. PrISM was used to predict a single model for each of the 43 targets in the CASP3 experiment. In this paper, we present results for 13 target sequences, which we consider to be comparative modeling targets with clearly related structural templates. We emphasize the problem of aligning a target sequence to a template structure with various alignment methods. When more than one alignment method and/or parameter set are applied, the final alignment is chosen on the basis of a model ranking system also used in PrISM's fold recognition module. Advanced sequence-template alignment procedures in PrISM are useful in some cases when standard pairwise dynamic programming algorithm fail to make any reasonable global alignment. The same procedures, however, failed in other cases, corresponding to remotely related query-template pairs that involved extensive insertions and deletions.

Algorithms↗

PubMed1998

Electrostatic contributions to the binding free energy of the lambdacI repressor to DNA.

A model based on the nonlinear Poisson-Boltzmann (NLPB) equation is used to study the electrostatic contribution to the binding free energy of the lambdacI repressor to its operator DNA. In particular, we use the Poisson-Boltzmann model to calculate the pKa shift of individual ionizable amino acids upon binding. We find that three residues on each monomer, Glu34, Glu83, and the amino terminus, have significant changes in their pKa and titrate between pH 4 and 9. This information is then used to calculate the pH dependence of the binding free energy. We find that the calculated pH dependence of binding accurately reproduces the available experimental data over a range of physiological pH values. The NLPB equation is then used to develop an overall picture of the electrostatics of the lambdacI repressor-operator interaction. We find that long-range Coulombic forces associated with the highly charged nucleic acid provide a strong driving force for the interaction of the protein with the DNA. These favorable electrostatic interactions are opposed, however, by unfavorable changes in the solvation of both the protein and the DNA upon binding. Specifically, the formation of a protein-DNA complex removes both charged and polar groups at the binding interface from solvent while it displaces salt from around the nucleic acid. As a result, the electrostatic contribution to the lambdacI repressor-operator interaction opposes binding by approximately 73 kcal/mol at physiological salt concentrations and neutral pH. A variety of entropic terms also oppose binding. The major force driving the binding process appears to be release of interfacial water from the protein and DNA surfaces upon complexation and, possibly, enhanced packing interactions between the protein and DNA in the interface. When the various nonelectrostatic terms are described with simple models that have been applied previously to other binding processes, a general picture of protein/DNA association emerges in which binding is driven by the nonpolar interactions, whereas specificity results from electrostatic interactions that weaken binding but are necessary components of any protein/DNA complex.

DNA, Viral↗

PubMed1997

Physical properties of rabbit articular cartilage after transection of the anterior cruciate ligament.

The effect of unilateral transection of the anterior cruciate ligament on the confined compression and swelling properties of the distal femoral articular cartilage of skeletally mature rabbits at 9 weeks after surgery was determined. Gross morphological grading of the transected and contralateral control distal femora stained with India ink confirmed that cartilage degeneration had been induced by ligament transection. Osteochondral cores, 1.8 mm in diameter, were harvested from the medial femoral condyles. The modulus, permeability, and electrokinetic (streaming potential) coefficient of the articular cartilage of the osteochondral cores were assessed by confined compression creep experiments. The properties (mean +/- SD) of control cartilage were: confined compression modulus, 0.75 +/- 0.28 MPa; hydraulic permeability, 0.63 +/- 0.28 x 10(-15) m2/Pa*sec; and electrokinetic coefficient, 0.16 +/- 0.31 x 10(-9) V/Pa. In transected knees, the modulus was reduced by 18% (p = 0.04), while the permeability and electrokinetic coefficient were not detectably altered. The change in modulus was accompanied by a trend (p = 0.07) toward a decrease (-11%) in the glycosaminoglycan density within the tissue, a significant increase (p < 0.001) in the water content of the cartilage after equilibration in 1 x phosphate buffered saline from 70.3 +/- 4.1% in control knees to 75.2 +/- 4.0% in transected knees, and little further swelling after tissue equilibration in hypotonic saline. The compressive modulus of the cartilage from both control and transected knees was positively correlated with the density of tissue glycosaminoglycan. The alterations in the physical properties of the articular cartilage after transection of the anterior cruciate ligament in the rabbit show trends similar to those observed in human and other animal models of osteoarthritis and provide further support for the use of this model in the study of cartilage degeneration.

Animals↗

PubMed1996

Methylation inhibitors can increase the rate of cytosine deamination by (cytosine-5)-DNA methyltransferase.

The target cytosines of (cytosine-5)-DNA methyltransferases in prokaryotic and eukaryotic DNA show increased rates of C-->T transition mutations compared to non-target cytosines. These mutations are induced either by the spontaneous deamination of 5-mC-->T generating inefficiently repaired G:T rather than G:U mismatches, or by the enzyme-induced C-->U deamination which occurs under conditions of reduced levels of S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy). We tested whether various inhibitors of (cytosine-5)-DNA methyltransferases analogous to AdoMet and AdoHcy would affect the rate of enzyme-induced deamination of the target cytosine by M.HpaII and M.SssI. Interestingly, we found two compounds, sinefungin and 5'-amino-5'-deoxyadenosine, that increased the rate of deamination 10(3)-fold in the presence and 10(4)-fold in the absence of AdoMet and AdoHcy. We have therefore identified the first mutagenic compounds specific for the target sites of (cytosine-5)-DNA methyltransferases. A number of analogs of AdoMet and AdoHcy have been considered as possible antiviral, anticancer, antifungal and antiparasitic agents. Our findings show that chemotherapeutic agents with affinities to the cofactor binding pocket of (cytosine-5)-DNA methyltransferase should be tested for their potential mutagenic effects.

Bacteria↗

PubMed1996

Free energy determinants of secondary structure formation: III. beta-turns and their role in protein folding.

The stability of beta-turns is calculated as a function of sequence and turn type with a Monte Carlo sampling technique. The conformational energy of four internal hydrogen-bonded turn types, I, I', II and II', is obtained by evaluating their gas phase energy with the CHARMM force field and accounting for solvation effects with the Finite Difference Poisson-Boltzmann (FDPB) method. All four turn types are found to be less stable than the coil state, independent of the sequence in the turn. The free-energy penalties associated with turn formation vary between 1.6 kcal/mol and 7.7 kcal/mol, depending on the sequence and turn type. Differences in turn stability arise mainly from intraresidue interactions within the two central residues of the turn. For each combination of the two central residues, except for -Gly-Gly-, the most stable beta-turn type is always found to occur most commonly in native proteins. The fact that a model based on local interactions accounts for the observed preference of specific sequences suggests that long-range tertiary interactions tend to play a secondary role in determining turn conformation. In contrast, for beta-hairpins, long-range interactions appear to dominate. Specifically, due to the right-handed twist of beta-strands, type I' turns for -Gly-Gly- are found to occur with high frequency, even when local energetics would dictate otherwise. The fact that any combination of two residues is found able to adopt a relatively low-energy turn structure explains why the amino acid sequence in turns is highly variable. The calculated free-energy cost of turn formation, when combined with related numbers obtained for alpha-helices and beta-sheets, suggests a model for the initiation of protein folding based on metastable fragments of secondary structure.

Data Interpretation, Statistical↗

PubMed1996

Mechanisms for the involvement of DNA methylation in colon carcinogenesis.

C --> T transitions at CpG sites are the most prevalent mutations found in the p53 tumor suppressor gene in human colon tumors and in the germline (Li-Fraumeni syndrome). All of the mutational hot spots are methylated to 5-methylcytosine, and it has been hypothesized that the majority of these mutations are caused by spontaneous hydrolytic deamination of this base to thymine. We have previously reported that bacterial methyltransferases induce transition mutations at CpG sites by increasing the deamination rate of C --> U when the concentration of the methyl group donor S-adenosylmethionine (AdoMet) drops below its Km, suggesting an alternative mechanism to create these mutations. Unrepaired uracil pairs with adenine during replication, completing the C --> T transition mutation. To determine whether this mechanism could contribute to the development of human colon cancer, we examined the level of DNA (cytosine-5)-methyltransferase (MTase) expression, the concentration of AdoMet, and the activity of uracil-DNA glycosylase in human colon tissues, and searched for the presence of mutations in the MTase gene. Using reverse transcription-PCR methods, we found that average MTase mRNA expression levels were only 3.7-fold elevated in tumor tissues compared with surrounding normal mucosa from the same patient. Also, no mutations were found in conserved regions of the gene in 10 tumors sequenced. High-performance liquid chromatographic analysis of extracts from the same tissues showed that AdoMet concentrations were not reduced below the Km value for the mammalian enzyme, and the concentration ratio of AdoMet:S-adenosylhomocysteine, the breakdown product of AdoMet and the competitive MTase inhibitor, did not differ significantly. Finally, extracts from the tumor tissue efficiently removed uracil from DNA. Therefore, biochemical conditions favoring a mutagenic pathway of C --> U --> T were not found in a target tissue known to undergo a high rate of C --> T transitions at CpG sites.

Base Sequence↗

PubMed1996

The rate of CpG mutation in Alu repetitive elements within the p53 tumor suppressor gene in the primate germline.

Cytosine to thymine transition mutations at the CpG dinucleotide are the most common point mutations in cancer and genetic disease. We calculated the in vivo rate of CpG mutation in the primate germline by deriving a primordial consensus sequence for an Alu repetitive element which inserted into intron 6 of the primate p53 gene 35 to 55 million years ago. Comparison of this primordial sequence to the Alu sequence in intron 6 of present-day primates was used to determine the nature and rate of mutations which occurred during evolution. We estimate the half-life of a CpG nucleotide to be 24 to 60 million years, and the rate constant for mutation at this dinucleotide to be 1.2 x 1O(-8) to 2.9 x 1O(-8) years(-1). These results were confirmed by the analysis of a second Alu sequence in intron 10 of the p53 gene. The in vivo mutation rate is at least 1250-fold slower than the in vitro chemical rate of 5-methylcytosine deamination in double-stranded DNA, showing that current estimates of CpG mutation repair have been significantly underestimated. Furthermore, the mutability of the CpG dinucleotide has led to the depletion of this dinucleotide from the vertebrate genome, and calculations in this study suggest that current levels of the CpG dinucleotide in the primate genome are very close to a steady state equilibrium in which the rate of CpG mutation is equal to the rate of CpG formation by random mutation.

Animals↗

PubMed1996

Thermal access to amplified chemical potential and the determination of equilibrium constants in protein solutions at subfreezing temperatures.

During rapid cooling of ferric heme protein solutions containing fluoride, locally concentrated ligand cannot fully equilibrate with heme before the temperature drops below 200 K and into the range where energy is insufficient for exchange with iron-bound water. When temperature is then jumped above 200 K, exchange of fluoride for bound water is activated. Between 200 and 240 K, further fluoride complex formation takes place over several minutes; its extent is measured along the kinetic curve by reimmersing the sample into liquid nitrogen and taking EPR spectra. Kinetic curves for replacement of iron-bound water by fluoride in horse aquo-ferrimyoglobin and human aquo-ferrihemoglobin, and corresponding equilibrium constants have been obtained at temperatures between 200 and 240 K. The reaction rates are affected by sucrose. Results indicate that the kinetics of exchange of fluoride for heme-bound water at subfreezing temperatures is protein specific and not diffusion-controlled, and is not affected by the phase transition of ice which takes place at subfreezing temperature. Free energy changes accompanying these reactions are largely continuous as the systems pass from above to below freezing.

Animals↗

PubMed1995

A mutant HpaII methyltransferase functions as a mutator enzyme.

DNA (cytosine-5)-methyltransferases can cause deamination of cytosine when the cofactor S-adenosylmethionine (AdoMet) is limiting and thus function as sequence-specific C-->U mutator enzymes. Here we explored whether mutations causing inactivation of the cofactor binding activity of the HpaII methyltransferase, thus mimicking conditions of limiting AdoMet concentration, could convert a DNA methyltransferase to a C-->U mutator enzyme. We created two mutator enzymes from the HpaII methyltransferase (F38S and G40D) which both showed enhanced cytosine deamination activities in vitro and in vivo. Interestingly, the G:U mispairs generated by these enzymes were not repaired completely in bacteria equipped with uracil-DNA glycosylase-initiated repair machinery, giving rise to a potent mutator phenotype. This is the first report showing the creation of mutator enzymes from a DNA methyltransferase and the demonstration of their mutagenicity in living cells.

Base Sequence↗

PubMed1995

Methylation of the 5' CpG island of the p16/CDKN2 tumor suppressor gene in normal and transformed human tissues correlates with gene silencing.

Loss of heterozygosity on 9p21, where the p16/CDKN2 tumor suppressor and the p15INK4B cell cycle regulator genes are located, is a common genetic alteration in bladder cancer. However, it has been difficult to demonstrate homozygous deletions and intragenic mutations in either of these two genes in primary transitional cell carcinomas (TCC) of the bladder. Similarly, colon cancer-derived cell lines have shown no homozygous deletions of the p16/CDKN2 locus in contrast to a wide variety of tumor-derived cell lines. We have investigated abnormal methylation of the 5' CpG islands of the p16/CDKN2 and p15INK4B genes as an alternative mechanism of inactivation of these genes in bladder and colon cancers. De novo methylation of the 5' CpG island of p16/CDKN2 was observed in 12 of 18 (67%) uncultured bladder TCCs and in 2 of 3 (67%) bladder cell lines. In contrast, only 1 of 10 (10%) colon carcinomas showed methylation of the 5' CpG island of p16/CDKN2. It was striking to find that this region was extensively methylated and the gene not expressed in the normal colonic mucosa of 6 of 10 (60%) patients with colon cancer, whereas 5 of the corresponding colon tumors showed no methylation and high levels of p16/CDKN2 expression. Our data show a significant correlation (P = 0.00001, two-sided) between the absence of p16/CDKN2 expression and methylation of its 5' CpG island in bladder tumors, cell lines, and normal colon mucosa. In contrast, no association was observed between expression and methylation status of the 5' CpG island of p15INK4B. Our results suggest that the p16/CDKN2 tumor suppressor gene may be inactivated by methylation of its 5' CpG island in TCCs of the bladder. We also present evidence of methylation of the 5' CpG island in this autosomal gene in normal colonic tissue.

Base Sequence↗

PubMed1995

Free energy determinants of secondary structure formation: I. alpha-Helices.

The Zimm-Bragg parameters s and sigma are calculated for the helix-coil transition of poly-L-alanine. The theoretical approach involves evaluating gas phase conformational energies for both coil and helical states using the CHARMM potential function and accounting for solvation effects with various continuum solvation models. Conformational free energies are then incorporated into a formalism developed by Go et al. for the calculation of s and sigma. Calculated values for both s and sigma as well as the enthalpy change associated with helix formation are in good agreement with experimental data when the Finite Difference Poisson-Boltzmann (FDPB) method is used to treat solvent effects. The driving force for the helix-coil transition is analyzed in terms of individual free energy components. Hydrogen bond formation is found to contribute little to helix stability because the internal hydrogen bonding energy is largely canceled by the large free energy cost associated with removing polar groups from water. The entropic cost associated with fixing backbone dihedral angles in the helical conformation is found to be approximately 7 e.u./residue (about 2 kcal/mol at room temperature). The major driving force favoring helix formation can be associated with interactions including enhanced van der Waals interactions in the close-packed helix conformation and the hydrophobic effect. These contribute about 2 kcal/mol favoring the helical state. The differences in helical propensities between alanine and glycine are attributed primarily to hydrophobic and packing interactions involving the C beta with a smaller contribution arising from increased conformational freedom for glycine in the coil state. The description of helix formation presented here is consistent with previous conclusions regarding tertiary structure formation which suggest that hydrophobic and close-packed interactions provide stability while hydrogen bond formation constitutes a structural constraint imposed by the high free energy cost associated with burying unsatisfied hydrogen bonding groups. alpha-Helix formation may thus be viewed as a form of hydrophobic collapse constrained by the requirement that polar groups be either exposed to solvent or form hydrogen bonds. More generally it appears from this study that for a folding model to be a realistic, it must properly account for the chemical nature of the polypeptide chain, particularly the solvation energetics of amide groups.

Amino Acid Sequence↗

PubMed1995

Free energy determinants of secondary structure formation: II. Antiparallel beta-sheets.

The factors that determine the stability of antiparallel beta-sheets are considered via a theoretical analysis of conformational free energies. A series of idealized model polyalanine beta-sheets are built with constraints such that the angular geometry of hydrogen bonding varies in the range observed in proteins while hydrogen bonding distance remains fixed. The conformations of the sheets generated in this way have a broad distribution of twist angles ranging from highly twisted left-handed to highly twisted right-handed orientations. The association free energies of the sheets are calculated with a gas phase CHARMM potential and FDPB/gamma solvation models. Left-handed structures are found to be less stable than right handed structures due to intrachain steric hindrance in isolated left-handed strands. This explains why antiparallel beta-sheets in proteins are invariably twisted in the right-handed direction. The free energy surface for right-handed sheets shows particular preference for conformations ranging from flat to those that exhibit a pronounced right-handed twist. This suggests that antiparallel beta-sheets can adopt a variety of right-handed conformations, a result that is consistent with observations on known proteins. In parallel with our study of alpha-helices we find that van der Waals and hydrophobic interactions are the primary factor stabilizing polyalanine beta-sheets, while electrostatic interactions including hydrogen bonding are found to be destabilizing. However, in contrast to helices, the net change in conformational free energy involving only backbone-backbone interactions (including beta-carbons) is not sufficient to overcome the loss in configurational entropy that accompanies sheet formation. Rather we suggest that cross-strand non-polar side-chain-side-chain interactions are essential for sheet formation, explaining why large non-polar amino acids have the greatest sheet forming propensities. Thus, sheet propensities involve pairwise interactions and are expected to be context dependent, as has been observed in recent experiments.

Amino Acid Sequence↗

PubMed1995

Base excision repair of U:G mismatches at a mutational hotspot in the p53 gene is more efficient than base excision repair of T:G mismatches in extracts of human colon tumors.

Approximately 50% of mutations that inactivate the p53 tumor suppressor gene in the germline and in colon tumors are C to T transitions at methylation sites (CpG sites). These mutations are believed to be caused by an endogenous mechanism and spontaneous deamination of 5-methyl-cytosine to T is likely to contribute significantly to this high mutation rate. The resulting T:G mismatches created by this process have been hypothesized to be less efficiently repaired than U:G mismatches formed by deamination of C. We have, therefore, performed the first study to directly compare rates of T:G versus U:G base excision repair at identical sites observed to be mutated in the p53 gene using extracts of human normal colon mucosa and colon carcinoma tissue. Mismatched U was excised up to 6000-fold more efficiently than T, suggesting that differences in repair efficiencies are the major source of C to T transition mutations at CpG sites in human tissues. The data also suggests that T:G mismatches are repaired by additional mechanisms in human cells.

Base Sequence↗

PubMed1995

HhaI and HpaII DNA methyltransferases bind DNA mismatches, methylate uracil and block DNA repair.

The hydrolytic deamination of 5-methylcytosine (5-mC) to thymine (T) is believed to be responsible for the high mutability of the CpG dinucleotide in DNA. We have shown a possible alternate mechanism for mutagenesis at CpG in which HpaII DNA-(cytosine-5) methyltransferase (M.HpaII) can enzymatically deaminate cytosine (C) to uracil (U) in DNA [Shen, J.-C., Rideout, W.M., III and Jones, P.A., Cell, 71, 1073-1080, (1992)]. Both the hydrolytic deamination of 5-mC and enzymatic deamination of C create premutagenic DNA mismatches (G:U and G:T) with the guanine (G) originally paired to the normal C. Surprisingly, we found that DNA-(cytosine-5) methyltransferases have higher affinities for these DNA mismatches than for their normal G:C targets and are capable of transferring a methyl group to the 5-position of U, creating T at low efficiencies. This binding by methyltransferase to mismatches at the recognition site prevented repair of G:U mismatches by uracil DNA glycosylase in vitro.

Base Sequence↗