The structure of guanosine-thymidine mismatches in B-DNA at 2.5-A resolution

The structure of the deoxyoligomer d(C-G-C-G-A-A-T-T-T-G-C-G) was determined at 2.5-A resolution by single crystal x-ray diffraction techniques. The final R factor is 18% with the location of 71 water molecules. The oligomer crystallizes in a B-DNA-type conformation, with two strands interacting to form a dodecamer duplex. The double helix consists of four A X T and six G X C Watson-Crick base pairs and two G X T mismatches. The G X T pairs adopt a "wobble" structure with the thymine projecting into the major groove and the guanine into the minor groove. The mispairs are accommodated in the normal double helix by small adjustments in the conformation of the sugar phosphate backbone. A comparison with the isomorphous parent compound containing only Watson-Crick base pairs shows that any changes in the structure induced by the presence of G X T mispairs are highly localized. The global conformation of the duplex is conserved. The G X T mismatch has already been studied by x-ray techniques in A and Z helices where similar results were found. The geometry of the mispair is essentially identical in all structures so far examined, irrespective of the DNA conformation. The hydration is also similar with solvent molecules bridging the functional groups of the bases via hydrogen bonds. Hydration may be an important factor in stabilizing G X T mismatches. A characteristic of Watson-Crick paired A X T and G X C bases is the pseudo 2-fold symmetry axis in the plane of the base pairs. The G X T wobble base pair is pronouncedly asymmetric. This asymmetry, coupled with the disposition of functional groups in the major and minor grooves, provides a number of features which may contribute to the recognition of the mismatch by repair enzymes.     

Relative specificities in binding of Watson-Crick base pairs by third strand residues in a DNA pyrimidine triplex motif.

The specificity of binding of Watson-Crick base pairs by third strand nucleic acid residues via triple helix formation was investigated in a DNA pyrimidine triplex motif by thermal melting experiments. The host duplex was of the type A10-X-A10: T10-Y-T10, and the third strand T10-Z-T10, giving rise to 16 possible triplexes with Z:XY inserts, 4 duplexes with the Watson-Crick base pairs (XY) and 12 duplexes with mismatch pairs (XZ), all of whose stabilities were compared. Two Z:XY combinations confirm the primary binding of AT and GC target pairs in homopurine.homopyrimidine sequences by T and C residues, respectively. All other Z:XY combinations in the T:AT environment result in triplex destabilization. While some related observations have been reported, the present experiments differ importantly in that they were performed in a T:AT nearest neighbor environment and at physiological ionic strength and pH, all of which were previously untested. The conclusions now drawn also differ substantially from those in previous studies. Thus, by evaluating the depression in Tm due to base triplet mismatches strictly in terms of third strand residue affinity and specificity for the target base pair, it is shown that none of the triplet combinations that destabilize qualify for inclusion in the third strand binding code for the pyrimidine triplex motif. Hence, none of the mismatch triplets afford a general way of circumventing the requirement for homopurine.homopyrimidine targets when third strands are predominated by pyrimidines, as others have suggested. At the same time, the applicability of third strand binding is emphasized by the finding that triplexes are equally or much more sensitive to base triplet mismatches than are Watson-Crick duplexes to base pair mismatches.     

Exonuclease proofreading by human mitochondrial DNA polymerase

We have examined the ability of the human mitochondrial DNA polymerase to correct errors in DNA sequence using single turnover kinetic methods. The rate of excision of single-stranded DNA ranged from 0.07 to 0.17 x s(-1), depending on the identity of the 3'-base. Excision of the 3'-terminal base from correctly base paired DNA occurred at a rate of 0.05 x s(-1), indicating that the cost of proofreading is minimal, as defined by the ratio of the k(exo) for correctly base-paired DNA divided by the rate of forward polymerization (0.05/37 = 0.14%). Excision of duplex DNA containing 1-7 mismatches was biphasic, and the rate and amplitude of the fast phase increased with the number of mismatches, reaching a maximum of 9 x s(-1). We showed that transfer of DNA from the polymerase to the exonuclease active site and back again occurs through an intramolecular reaction, allowing for a complete cycle of reactions for error correction. For DNA containing a buried mismatch (T:T followed by C:G base pairs), the 3' base was removed at a rate of 3 x s(-1). The addition of nucleotide to the reaction that is identical to the 3' base increased the rate of excision 7-fold to 21 x s(-1). We propose that the free nucleotide enhances the rate of transfer of the DNA to the exonuclease active site by interrupting the correct 3' base pair through interaction with the template base. The exonuclease contribution to fidelity is minimal if the calculation is based on hydrolysis of a single mismatch: (k(exo) + k(pol,over))/(k(pol,over)) = 10, but this value increases to approximately 200 when examining error correction in the presence of nucleotides.     

Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies.

We investigated the effects of various primer-template mismatches on DNA amplification of an HIV-1 gag region by the polymerase chain reaction (PCR). Single internal mismatches had no significant effect on PCR product yield while those at the 3'-terminal base had varied effects. A:G, G:A, and C:C mismatches reduced overall PCR product yield about 100-fold, A:A mismatches about 20-fold. All other 3'-terminal mismatches were efficiently amplified, although the G:G mismatches appeared to be more sensitive to sequence context and dNTP concentrations than other mismatches. It should be noted that mismatches of T with either G, C, or T had a minimal effect on PCR product yield. Double mismatches within the last four bases of a primer-template duplex where one of the mismatches is at the 3' terminal nucleotide, in general, reduced PCR product yield dramatically. The presence of a mismatched T at the 3'-terminus, however, allowed significant amplification even when coupled with an adjacent mismatch. Furthermore, even two mismatched Ts at the 3'-terminus allowed efficient amplification.     

DNA substrate structural requirements for the exonuclease and polymerase activities of procaryotic and phage DNA polymerases

A DNA duplex covalently cross-linked between specific bases has been prepared. This and similar duplexes are substrates for the polymerase and exonuclease activities of the Klenow fragment of Escherichia coli DNA polymerase I and T4 and T7 DNA polymerases. The action of Klenow fragment on these duplexes indicates that the polymerase site does not require that the DNA duplex undergo strand separation for activity, whereas the exonuclease site requires that at least four base pairs of the primer strand must melt out for the exonucleolytic removal of nucleotides from the primer terminus. The exonucleolytic action of T4 and T7 DNA polymerases requires that only two and three bases respectively melt out for excision of nucleotides from the primer terminus. Klenow fragment and T4 DNA polymerase are able to polymerize onto duplexes incapable of strand separation, whereas T7 DNA polymerase seems to require that the primer terminus be at least three bases from the cross-linked base pair. A DNA duplex with a biotin covalently linked to a specific base has been prepared. In the presence of the biotin binding protein avidin, the exonucleolytic activity of Klenow fragment requires that the primer terminus be at least 15 base pairs downstream from the base with the biotin-avidin complex. On the other hand, the polymerase activity of Klenow fragment required that the primer terminus be at least six base pairs downstream from the base with the biotin-avidin complex. These results suggest that the polymerase and exonuclease sites of Klenow are physically separate in solution and exhibit different substrate structural requirements for activity.     

DNA triple-helix formation at target sites containing duplex mismatches

We have studied the formation of DNA triple helices at target sites that contain mismatches in the duplex target. Fluorescence melting studies were used to examine a series of parallel triple helices that contain all 64 N.XZ triplet combinations at the centre (where N, X and Z are each of the four natural DNA bases in turn). Similar experiments were also performed with N=bis-amino-U (BAU) (for stable recognition of AT base pairs) and N=S (for recognition of TA inversions). We find that the introduction of a duplex mismatch destabilises the C+.GZ, T.AZ and G.TZ triplets. A similar effect is seen with BAU.AZ triplets. In contrast, other base combinations, based on non-standard triplets such as C.AZ, T.TZ, G.CZ and A.CZ are stabilised by the presence of a duplex mismatch. In each case S binds to sites containing duplex mismatches better than the corresponding Watson-Crick base pairs.     

Wobble dG X dT pairing in right-handed DNA: solution conformation of the d(C-G-T-G-A-A-T-T-C-G-C-G) duplex deduced from distance geometry analysis of nuclear Overhauser effect spectra

We report below on features of the three-dimensional structure of the d(C-G-T-G-A-A-T-T-C-G-C-G) self-complementary duplex (designated 12-mer GT) containing symmetrical G X T mismatches in the interior of the helix. The majority of the base and sugar protons in the 12-mer GT duplex were assigned by two-dimensional nuclear Overhauser effect (NOESY) spectra in H2O and D2O solution. A set of 92 short (less than 4.5-A) proton-proton distances defined by lower and upper bounds for one symmetrical half of the 12-mer GT duplex were estimated from NOESY data sets recorded as a function of mixing time. These experimental distances combined with nucleotide bond length parameters were embedded into Cartesian space; several trial structures were refined to minimize bond geometry and van der Waals and chirality error. Confidence in this approach is based on the similarity of the refined structures for the solution conformation of the 12-mer GT duplex. The G and T bases pair through two imino-carbonyl hydrogen bonds, and stacking is maintained between the G X T wobble pair and adjacent Watson-Crick G X C pairs. The experimental distance information is restricted to base and sugar protons, and hence structural features such as base pair overlap, glycosidic torsion angles, and sugar pucker are well-defined by this combination of NMR and distance geometry methods. By contrast, we are unable to define the torsion angles about the bonds C3'-O3'-P-O5'-C5'-C4' in the backbone of the nucleic acid.     

Enhanced base-pair opening in the adenine tract of a RNA double helix

Proton exchange and nuclear magnetic resonance spectroscopy are being used to characterize the kinetics and energetics of base-pair opening in two nucleic acid double helices. One is the RNA duplex 5'-r(GCGAUAAAAAGGCC)-3'/5'-r(GGCCUUUUUAUCGC)-3', which contains a central tract of five AU base pairs. The other is the homologous DNA duplex with a central tract of five AT base pairs. The rates and the equilibrium constants of the opening reaction of each base pair are measured from the dependence of the exchange rates of imino protons on ammonia concentration, at 10 °C. The results reveal that the tract of AU base pairs in the RNA duplex differs from the homologous tract of AT base pairs in DNA in several ways. The rates of opening of AU base pairs in RNA are high and increase progressively along the tract, reaching their largest values at the 3'-end of the tract. In contrast, the opening rates of AT base pairs in DNA are much lower than those of AU base pairs. Within the tract, the largest opening rate is observed for the AT base pair at the 5'-end of the tract. These differences in opening kinetics are paralleled by differences in the stabilities of individual base pairs. All AU base pairs in the RNA are less stable than the AT base pairs in the DNA. The presence of the tract enhances these differences by increasing the stability of AT base pairs in DNA while decreasing the stability of AU base pairs in RNA. Due to these divergent trends, along the tracts, the AU base pairs become progressively less stable than AT base pairs. These findings demonstrate that tracts of AU base pairs in RNA have specific dynamic and energetic signatures that distinguish them from similar tracts of AT base pairs in DNA.     

Structure of the DNA interstrand cross-link of 4,5',8-trimethylpsoralen.

4,5',8-Trimethylpsoralen (TMP) cross-links a 5' TpA or a 5' ApT site by photoreacting with one thymine moiety in each DNA strand. We are interested in whether psoralen interstrand cross-links all share one structure or whether there are significant differences. In this paper, we employed a rapid method for probing the structure of the cross-link by making a series of TMP cross-linked duplexes containing specific base-pair mismatches. The relative stability provided by a base pair can be correlated with neighboring base pairs by comparing the extents of gel retardation when base-pair mismatches happen in each position. From our studies, we infer that with respect to the furan-side strand, the 5'T.A base pair of the two T.A base pairs in the TpA site is not hydrogen bonded. Immediately on each side of the cross-linked TpA site is a highly stabilized base pair. Next, a region of decreased stability occurs in each arm of a cross-linked duplex and these base pairs of least stability are located farther away from the cross-linked thymines as the lengths of the arms of the cross-linked helix increase. Finally, even in 7 M urea at 49 degrees C the cross-linked helix is hydrogen bonded at both ends of a duplex of 22 base pairs. We propose that the structures of interstrand cross-links in DNA vary appreciably with the DNA sequence, the length of the DNA duplex, and the structures of the DNA cross-linking agents.     

Analysis of the RNA-editing reaction of ADAR2 with structural and fluorescent analogues of the GluR-B R/G editing site

ADARs are adenosine deaminases responsible for RNA editing reactions that occur in eukaryotic pre-mRNAs, including the pre-mRNAs of glutamate and serotonin receptors. Here we describe the generation and analysis of synthetic ADAR2 substrates that differ in structure around an RNA editing site. We find that five base pairs of duplex secondary structure 5' to the editing site increase the single turnover rate constant for deamination 17-39-fold when compared to substrates lacking this structure. ADAR2 deaminates an adenosine in the sequence context of a natural editing site >90-fold more rapidly and to a higher yield than an adjacent adenosine in the same RNA structure. This reactivity is minimally dependent on the base pairing partner of the edited nucleotide; adenosine at the editing site in the naturally occurring A.C mismatch is deaminated to approximately the same extent and only 4 times faster than adenosine in an A.U base pair at this site. A steady-state rate analysis at a saturating concentration of the most rapidly processed substrate indicates that product formation is linear with time through at least three turnovers with a slope of 13 +/- 1.5 nM.min(-1) at 30 nM ADAR2 for a k(ss) = 0.43 +/- 0.05 min(-1). In addition, ADAR2 induces a 3.3-fold enhancement in fluorescence intensity and a 14 nm blue shift in the emission maximum of a duplex substrate with 2-aminopurine located at the editing site, consistent with a mechanism whereby ADAR2 flips the reactive nucleotide out of the double helix prior to deamination.     

Thermodynamics of DNA duplexes with adjacent G.A mismatches.

The sequence 5'-d(ATGAGCGAAT) forms a very stable self-complementary duplex with four G.A mismatch base pairs (underlined) out of ten total base pairs [Li et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 26-30]. The conformation is in the general B-family and is stabilized by base-pair hydrogen bonding of an unusual type, by favorable base dipole orientations, and by extensive purine-purine stacking at the mismatched sites. We have synthesized 13 decamers with systematic variations in the sequence above to determine how the flanking sequences, the number of G.A mismatches, and the mismatch sequence order (5'-GA-3' or 5'-AG-3') affect the duplex stability. Changing A.T to G.C base pairs in sequences flanking the mismatches stabilizes the duplexes, but only to the extent observed with B-form DNA. The sequence 5'-pyrimidine-GA-purine-3', however, is considerably more stable than 5'-purine-GA-pyrimidine-3'. The most stable sequences with two pairs of adjacent G.A mismatches have thermodynamic parameters for duplex formation that are comparable to those for fully Watson-Crick base-paired duplexes. Similar sequences with single G.A pairs are much less stable than sequences with adjacent G.A mismatches. Reversing the mismatch order from 5'-GA-3' to 5'-AG-3' results in an oligomer that does not form a duplex. These results agree with predictions from the model derived from NMR and molecular mechanics and indicate that the sequence 5'-pyrimidine-GA-purine-3' forms a stable conformational unit that fits quite well into a B-form double helix.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determinants of DNA mismatch recognition within the polymerase domain of the Klenow fragment.

The Klenow fragment of Escherichia coli DNA polymerase I catalyzes template-directed synthesis of DNA and uses a separate 3'-5' exonuclease activity to edit misincorporated bases. The polymerase and exonuclease activities are contained in separate structural domains. In this study, nine Klenow fragment derivatives containing mutations within the polymerase domain were examined for their interaction with model primer-template duplexes. The partitioning of the DNA primer terminus between the polymerase and 3'-5' exonuclease active sites of the mutant proteins was assessed by time-resolved fluorescence anisotropy, utilizing a dansyl fluorophore attached to the DNA. Mutation of N845 or R668 disrupted favorable interactions between the Klenow fragment and a duplex containing a matched terminal base pair but had little effect when the terminus was mismatched. Thus, N845 and R668 are required for recognition of correct terminal base pairs in the DNA substrate. Mutation of N675, R835, R836, or R841 resulted in tighter polymerase site binding of DNA, suggesting that the side chains of these residues induce strain in the DNA and/or protein backbone. A double mutant (N675A/R841A) showed an even greater polymerase site partitioning than was displayed by either single mutation, indicating that such strain is additive. In both groups of mutant proteins, the ability to discriminate between duplexes containing matched or mismatched base pairs was impaired. In contrast, mutation of K758 or Q849 had no effect on partitioning relative to wild type, regardless of DNA mismatch character. These results demonstrate that DNA mismatch recognition is dependent on specific amino acid residues within the polymerase domain and is not governed solely by thermodynamic differences between correct and mismatched base pairs. Moreover, this study suggests a mechanism whereby the Klenow fragment is able to recognize polymerase errors following a misincorporation event, leading to their eventual removal by the 3'-5' exonuclease activity.     

How much pi-stacking do DNA termini seek? Solution structure of a self-complementary DNA hexamer with trimethoxystilbenes capping the terminal base pairs

The exposed terminal base pairs of DNA duplexes are nonclassical binding sites for small molecules. Instead, small molecules usually prefer intercalation or minor groove binding. Here we report the solution structure of the DNA duplex (TMS-TGCGCA)(2), where TMS denotes trimethoxystilbene carboxamides that are 5'-tethered to the DNA. The stilbenes, for which intercalation is conformationally accessible, stack on the terminal T:A base pairs of an undisturbed B-form duplex. Two conformations, differing by the orientation of the stilbene relative to the terminal base pair, are observed, indicating that the flip rate is slow for the pi-stacked aromatic ring system. The trimethoxystilbene is known to greatly increase base pairing fidelity at the terminus. Here we show that it gauges the size of the T:A base pair by embracing the 2'-methylene group of the terminal dA residue of the unmodified terminus with its methoxy "arms", but that it does not engage the entire base pair in pi-stacking. Mismatched base pairs with their altered geometry will not allow for the same embracing interaction. On the basis of the current structure, a trimethoxychrysene carboxamide is proposed as a ligand with increased pi-stacking surface and possible applications as improved fidelity-enhancing element.     

Energetics of base pair opening in a DNA dodecamer containing an A3T3 tract.

Nuclear magnetic resonance spectroscopy has been used to characterize the kinetics and energetics of opening of base pairs in the DNA dodecamer [d(CGCAAATTTGCG)]2. The dodecamer contains an A3T3 tract that induces intrinsic curvature of the helix axis. Previous studies from this and other laboratories have shown that the kinetics of base pair opening in AnTn tracts is unique: the opening rates of the A.T base pairs in the interior of the tract are much lower than that of the A.T base pair at the 5'-end of the tract. In the present work, we have investigated the energetics of the pathways for opening of the A.T base pairs in the A3T3 tract. The energetic parameters of the activated state(s) are obtained from the temperature dependence of the opening rate constants. The lower opening rates for the A.T base pairs situated in the interior of the tract are shown to originate from higher activation enthalpies which are compensated, in part, by increases in the activation entropies. We have also obtained an energetic characterization of the open state(s) of the A.T base pairs in the dodecamer by measuring the equilibrium constants for base pair opening and their temperature dependence. The results suggest that the transitions from closed to open state(s) in the A.T base pairs of the A3T3 tract are energetically similar.     

Minimal DNA requirement for topoisomerase II-mediated cleavage in vitro

The minimal DNA requirement for topoisomerase II-mediated DNA cleavage in vitro was determined by analyzing the interaction of the enzyme with sets of DNA substrates varying successively by single bases at the 5'- or 3'-end of either strand. A 16-base pair double-stranded region was established as the minimal duplex region required for topoisomerase II cleavage activity. The region was located symmetrically around the 4-base staggered cleavage site. Topoisomerase II-mediated cleavage within the 16-base pair core duplex, however, required single-stranded regions flanking the duplex to either the 5'- or 3'-sides, or an extension at both ends of the duplex with 1 or more base pairs.     

Dissociation kinetics of 19 base paired oligonucleotide-DNA duplexes containing different single mismatched base pairs.

The dissociation kinetics of 19 base paired oligonucleotide-DNA duplex containing a various single mismatched base pair are studied on dried agarose gels. The kinetics of the dissociation are first order under our experimental conditions. The incorporation of a single mismatched base pair destabilizes the DNA duplexes to some extent, the amount depending on the nature of the mismatched base pair. G-T and G-A mismatches slightly destabilize a duplex, while A-A, T-T, C-T and C-A mismatches significantly destabilize it. The activation energy for the overall dissociation processes for these oligonucleotide-DNA duplexes containing 19 base pairs is 52 +/- 2 Kcal mol-1 as determined from the slope of Arrhenius plot.     

In vitro mutagenesis in the lacI gene of Escherichia coli: fate of 3'-terminal mispairs versus internal base mispairs in a transfection assay

The fate of G.T mismatches and frameshifts, present at the 3'-terminus of primer-template or internally, has been studied with a combined transfection and electrophoretic assay following in vitro polymerization by DNA polymerase I (Klenow enzyme) of Escherichia coli. Several synthetic oligodeoxynucleotide primers were synthesized and annealed to uracil-containing single-stranded DNA of M13 phage bearing the lacI gene, to produce 1-3 consecutive G.T mismatches in the middle of the duplex region or at the 3'-OH end of the primer. Additional mismatched primer-templates were prepared, in which the primer had a deleted nucleotide, an extra nucleotide or both G.T mismatch and an extra nucleotide. The extension or degradation of these primers during in vitro DNA synthesis in the presence of all 4 dNTPs ('complete' reaction) or in the absence of dATP ('-A' reaction) was monitored by gel electrophoresis. Duplex DNA products were used in a transfection assay and the nucleotide changes in i-mutant progeny were determined by sequence analysis. The results suggest that whereas a single 3'-terminal G.T mismatch is relatively stable in chain elongation by Klenow enzyme, multiple terminal G.T mismatches are degraded by the 3'-exonuclease activity of this polymerase prior to primer extension. This editing activity is increased with the number of 3'-terminal mispairs. Single, double and triple T----C base substitutions were efficiently recovered when the mismatches occurred internally. Also, single-base eliminations or additions were readily recovered when the mutagenic primers contained an internal base deletion or addition, respectively. When products of the '-A' misincorporation reaction (catalyzed by Klenow enzyme) were assayed by transfection, base substitutions (exclusively T----C), but no frameshifts, were recovered. The results indicate that the absence of multiple tandem base substitutions among i- mutants recovered following primer elongation under mutagenic 'minus' conditions was due to the efficient action of the 3'-exonuclease activity of the Klenow enzyme on multiple terminal mismatches during in vitro polymerization, rather than to in vivo events (lack of expression or occurrence of mismatch repair) in the M13-lacI transfection assay.     

Joining of short DNA oligonucleotides with base pair mismatches by T4 DNA ligase

Oligonucleotide-directed mutagenesis is a widely used method for studying enzymes and improving their properties. The number of mutants that can be obtained with this method is limited by the number of synthetic 25-30mer oligonucleotides containing the mutation mismatch, becoming impracticably large with increasing size of a mutant library. To make this approach more practical, shorter mismatching oligonucleotides (7-12mer) might be employed. However, the introduction of these oligonucleotides in dsDNA poses the problem of sealing a DNA nick containing 5'-terminal base pair mismatches. In the present work we studied the ability of T4 DNA ligase to catalyze this reaction. It was found that T4 DNA ligase effectively joins short oligonucleotides, yielding dsDNA containing up to five adjacent mismatches. The end-joining rate of mismatching oligonucleotides is limited by the formation of the phosphodiester bond, decreasing with an increase in the number of mismatching base pairs at the 5'-end of the oligonucleotide substrate. However, in the case of a 3 bp mismatch, the rate is higher than that obtained with a 2 bp mismatch. Increasing the matching length with the number of mismatching base pairs fixed, or moving the mismatching motif downstream with respect to the joining site increases the rate of ligation. The ligation rate increases with the molar ratio [oligonucleotide:dsDNA]; however, at high excess of the oligonucleotide, inhibition of joining was observed. In conclusion, 9mer oligonucleotides containing a 3 bp mismatch are found optimal substrates to introduce mutations in dsDNA, opening perspectives for the application of T4 DNA ligase in mutagenesis protocols.     

The mechanism of U insertion/deletion RNA editing in kinetoplastid mitochondria.

Recent advances in in vitrosystems and identification of putative enzymatic activities have led to the acceptance of a modified 'enzyme cascade' model for U insertion/deletion RNA editing in kinetoplastid mitochondria. Models involving the transfer of uridines (Us) from the 3'-end of gRNA to the editing site appear to be untenable. Two types of in vitrosystems have been reported: (i) a gRNA-independent U insertion activity that is dependent on the secondary structure of the mRNA; (ii) a gRNA-dependent U insertion activity that requires addition of a gRNA that can form an anchor duplex with the pre-edited mRNA and which contains guiding A and G nucleotides to base pair with the added Us. In the case of the gRNA-mediated reaction, the precise site of cleavage is at the end of the gRNA-mRNA anchor duplex, as predicted by the original model. The model has been modified to include the addition of multiple Us to the 3'-end of the 5'-cleavage fragment, followed by the formation of base pairs with the guiding nucleotides and trimming back of the single-stranded oligo(U) 3'-overhang. The two fragments, which are held together by the gRNA 'splint', are then ligated. Circumstantial in vitroevidence for involvement of an RNA ligase and an endoribonuclease, which are components of a 20S complex, was obtained. Efforts are underway in several laboratories to isolate and characterize specific components of the editing machinery.