Emerging technologies from the Human Genome Project for understanding susceptibility and risk.

The new technologies from the Human Genome Program provide exceptional opportunities for surveying and measuring human exposure, as well as determining susceptibility on an individual-by-individual basis. These new technologies will soon enable rapid screening of populations at risk, as well as the broader public, for a variety of genes known to be associated with increased risk. These include specific oncogenes, tumor suppressor genes and DNA repair enzymes. Use of these technologies also presents a number of ethical issues, both in screening and in use of the information about individuals. Overall, the use of rapid genotyping technologies will introduce a specificity and possible group identifiers that will present new challenges to the determination of risk within the EPA mandate.

Comparison of polymerase insertion and extension kinetics of a series of O2-alkyldeoxythymidine triphosphates and O4-methyldeoxythymidine triphosphate.

The effect of alkyl group size on ability to act as deoxythymidine triphosphate (dTTP) has been studied for the carcinogen products O2-methyl-, O2-ethyl-, and O2-isopropyl-dTTP by using three types of nucleic acids as template and DNA polymerase I (Pol I) or Klenow fragment as the polymerizing enzymes. Apparent Km and relative Vmax values were determined in primer extension on M13 DNA at a single defined site, in poly[d(A-T)], and in nicked DNA. These data are the basis for calculation of the relative rate of insertion opposite A, relative to dTTP. The insertion rate for any O2-alkyl-dTTP is much higher than for a mismatch between unmodified dNTPs. Unexpectedly, O2-isopropyl-dTTP is more efficiently utilized than O2-methyl-dTTP or O2-ethyl-dTTP on each of the templates. O2-isopropyl-dTTP also substitutes for dTTP over extended times of DNA synthesis at a rate only slightly lower than that of dTTP. Parallel experiments using O4-methyl-dTTP under the same conditions show that it is incorporated opposite A more frequently than is O2-methyl-dTTP. Therefore, both the ring position and the size of the alkyl group influence polymerase recognition. Once formed, all O2-alkyl-T.A termini permit elongation, as does O4-methyl-T.A. In contrast to the relative difficulty of incorporating the O-alkyl-dTTPs, formation of the following normal base pair (C.G) occurs rapidly when dGTP is present. This indicates that a single O-alkyl-T.A pair does not confer significant structural distortion recognized by Pol I.

Formation of interstrand cross-links in chloroacetaldehyde-treated DNA demonstrated by ethidium bromide fluorescence.

Chloroacetaldehyde, the stable metabolite of the human carcinogen vinyl chloride, forms interstrand cross-links in vitro in salmon sperm DNA and in the alternating copolymer, poly(deoxyadenylate-deoxythymidylate) [poly(dA-dT)]. Formation of the cross-link was a function of both time of reaction and concentration of chloroacetaldehyde. Cross-linking in chloroacetaldehyde-treated poly(dA-dT) was detected initially by changes in renaturation hysteresis [Singer et al., Carcinogenesis (Lond.), 5: 1165-1171, 1984]. This has been confirmed and quantitated using the relative fluorescence of ethidium bromide after denaturation and reannealing at 40 degrees C. Three percent cross-linking was detected after 10 min reaction with 20 mM chloroacetaldehyde at 24 degrees C. In DNA the relative fluorescence of ethidium bromide after denaturation and rapid cooling was used to estimate the number of cross-links formed. Three times as much cross-linking occurs in DNA compared to poly(dA-dT) under identical reaction conditions. The postulated structure for an interstrand cross-link in poly(dA-dT) is a hydroxyethyl bridge across the strands between the N6-amino groups of alternate adenine residues. In DNA, other amino groups in the proper configuration can be involved.

The vinyl chloride-derived nucleoside, N2,3-ethenoguanosine, is a highly efficient mutagen in transcription.

N2,3-Ethenoguanine (N2,3-epsilon G) was recently identified in the liver of vinyl chloride-exposed rats. We have now synthesized the nucleoside and the 5'-diphosphate which was copolymerized with CDP. The deoxypolynucleotide complement, synthesized by AMV reverse transcriptase contained, in addition to dG, dC and dT. The total pyrimidine content was approximately equivalent to the N2,3-epsilon G content of the template. Incorporation of dC is neither lethal nor mutagenic, while dT incorporation represents a mutagenic event, occurring with approximately 20% frequency. N2,3-epsilon G X dT base pairs can have two hydrogen bonds with minimal helical distortion, as is also the case for N2,3-epsilon G X C base pairs. N2,3-epsilon G is the only derivative formed in vivo by the human carcinogen, vinyl chloride, that can be shown to have a high probability of causing transitions which could initiate malignant transformation.

O-Alkyl deoxythymidines are recognized by DNA polymerase I as deoxythymidine or deoxycytidine.

The O2- and O4-methyldeoxythymidine triphosphates (O-alkyl dTTP) can be used to substitute for dTTP in Escherichia coli DNA polymerase I (Pol I)-catalysed synthesis of poly[deoxyadenosine-deoxythymidine] (dA-dT). When incorporated into the polynucleotide, no detectable perturbation of structure occurred with even 20% O-methyldeoxythymidine in place of dT. However, on replication of such polymers with Pol I, significant amounts of deoxyguanosine triphosphate (dGTP) were incorporated, as well as high levels of deoxyadenosine triphosphate (dATP), indicating tautomer-like behaviour. Higher homologues, such as O4-ethyl (e4) dTTP or O4-isopropyl (ip4) dTTP, could also replace dTTP, but with lower efficiency. Nevertheless, their presence, like O4-methyl (m4) dT substitutions, caused transitions as well as inhibiting enzyme digestion with a variety of 3' nucleases, particularly to the 3'----5' exonuclease activity (proofreading) of polymerases. Further proof of mutagenicity comes from site-directed experiments placing m4dT or e4dT in place of dT at position 587 in am3 of phi X174, in which all revertants sequenced had A----G transitions. This implies that, since m4dT and e4dT are poorly repaired in eukaryotes, it is likely that they will remain in the DNA and lead to effects on enzyme activity, as well as mutations which contribute to the carcinogenicity of N-nitroso compounds.

O4-Methyl-, O4-ethyl-, and O4-isopropylthymidine 5'-triphosphates as analogues of thymidine 5'-triphosphate: kinetics of incorporation by Escherichia coli DNA polymerase I.

O4-Methyl-, O4-ethyl-, and O4-isopropylthymidine 5'-triphosphates, which can be formed by N-nitroso carcinogens, were tested for their ability to substitute for thymidine 5'-triphosphate (dTTP) in synthesis catalyzed by Escherichia coli DNA polymerase I (Pol I) by using activated DNA or synthetic polymers as templates. All could substitute for dTTP for short periods, the rate and extent decreasing with the size of the alkyl group. Because the structure of O4-alkylthymidine does not permit normal hydrogen bond formation with deoxyadenosine, it was inferred that eventual formation of a poor or frayed primer end was responsible for termination of synthesis. Synthesis of polymers at temperatures ranging from 0 to 40 degrees C showed that the extent of incorporation using the O4-alkyl-dTTPs was favored, relative to dTTP, when the terminal helical structure was stabilized by low temperatures. Kmapp values were determined for each O4-alkyldeoxynucleoside 5'-triphosphate. These values were 0.7 microM for dTTP, 5 microM for methyl-dTTP, 11 microM for ethyl-dTTP, and 33 microM for isopropyl-dTTP. O4-Alkyl-dTTPs were tested for their ability to inhibit or compete with dTTP incorporation and found to have a minimal effect, even when present at high concentration. These experiments indicated that Pol I can incorporate deoxynucleotides with O4-alkyl substituents into an ordered DNA structure. A postulated base-pairing scheme with deoxyadenosine is described.

Replication and transcription of polynucleotides containing ethenocytosine, ethenoadenine and their hydrated intermediates.

Replication with Escherichia coli DNA polymerase I (Pol I) and transcription with DNA-dependent RNA polymerase from Escherichia coli or calf thymus, using as templates synthesized ribo- or deoxyribopolynucleotides containing 1,N6-ethenoadenine (epsilon A) or 3,N4-ethenocytosine (epsilon C), showed that only epsilon C could direct significant misincorporation. The hydrated intermediate of epsilon C caused errors only upon transcription, but not upon replication. epsilon A was a very poor mutagen as assessed by replication with Pol I. Transcription of polynucleotides containing epsilon A under error-prone conditions caused frequent A misincorporation which could not be detected in replication assays. It is concluded that epsilon C may lead to point mutations, specifically directing the misincorporation of thymine. The analogous derivative, epsilon A, is bulky and is likely to be bypassed rather than read. This mechanism could cause frameshift mutation, as generally found for other bulky adducts.

Fibers of RecA protein and complexes of RecA protein and single-stranded phi X174 DNA as visualized by negative-stain electron microscopy.

Monomers of purified RecA protein polymerize into helical fibers whose pitch is 7.2 nm to 7.5 nm and whose diameter is 11 nm. Either short (approximately 0.2 micron), single fibers, or bundles of aligned, longer fibers, can be formed preferentially, by varying the Mg2+ concentration. When RecA protein is bound to circular, single-stranded phi X174 DNA it forms helical fibers of different classes of contour lengths, ranging from 0.98 micron, depending upon the conditions of assembly. Two different helical pitches are found, one of 9.3 nm when the incubation buffer contains, besides the obligatory Mg2+, either ATP gamma S or ATP accompanied by single-strand binding protein, and one of 5.5 nm when the latter additives are omitted. Preformed fibers of the compact type can be converted to open ones of 9.3 nm pitch upon addition of ATP gamma S, even after the removal of unbound RecA. All signs of helicity are obliterated upon glutaraldehyde cross-linking except in those fibers whose assembly has been mediated by ATP gamma S. RecA protein and single-strand binding protein are competitively bound to single-stranded DNA. Composite complexes, however, are not encountered unless ATP gamma S is present. Otherwise, segments of DNA that are coated by one or the other protein are seen as separate regions. When the assembly of complexes of single-stranded DNA and RecA is mediated by single-strand binding protein and ATP, the axial separation between successive bases is 0 X 42 nm, somewhat greater than the axial distance between bases in one strand of duplex DNA in the B form. It is proposed that the bases of the single-stranded DNA in the complex are located near its inner surface, and that base-pairing with double-stranded DNA takes place following invasion of the central cavity of the complex.

O4-Methyl, -ethyl, or -isopropyl substituents on thymidine in poly(dA-dT) all lead to transitions upon replication.

In a previous paper, we reported that O4-methyl dTTP can be incorporated into poly(dA-dT) in place of thymidine without distortion of the helical structure, but on replication it could behave as deoxycytidine and misincorporate dGTP. Only weak interactions are possible for any O4-modified T X A pair. While O4-alkyl T X G pairing should be favored, experiments to detect the ability of Escherichia coli DNA polymerase I (pol I) to utilize the triphosphate as dCTP were ambiguous. dTTPs with larger alkyl groups (ethyl, isopropyl) have now been synthesized and tested for their recognition as dTTP by pol I. Enhanced steric hindrance could be expected, particularly for O4-isopropyl dTTP, which has a three-carbon branched chain. However, both compounds behaved qualitatively like O4-methyl dTTP, being incorporated into poly(dA-dT) and then directing deoxyguanosine misincorporation by pol I. Quantitative comparisons of mutagenicity were not possible because of the finding that, unlike polymers made with O4-methyl dTTP, those made with ethyl or isopropyl dTTP were resistant to hydrolysis by using a variety of nucleases. The frequent misincorporations of dGTP would be expected to produce transitions in vivo. O4-ethyldeoxythymidine is very poorly repaired in vivo, which would also be expected for repair of O4-isopropyldeoxythymidine. Therefore, under suitable conditions, these particular carcinogen products are likely to be initiators of carcinogenesis.

The stereostructure of knots and catenanes produced by phage lambda integrative recombination: implications for mechanism and DNA structure.

We studied the mechanism of recombination by determining the structure of the products of the phage lambda Int system. Electron microscopy of RecA-coated products revealed only knots and catenanes containing a regular right-handed spiral structure. The structure and distribution of products establish that the recombination sites pair by essentially random collision, rather than by tracking. However, the distribution also indicates that the binding of the enzyme must introduce nonrandom components into the reaction and stabilize at least two additional supercoils that become links in the product. Moreover, the regularity of the structures indicates that the strand exchange is accomplished in a very simple way, introducing only a single link into the product. All other links result from the direct conversion of substrate supercoils into knot and catenane links. These supercoils must be in a right-handed, braided form, rather than solenoidally wound as in nucleosomes.

Assessment of mutagenic efficiency of two carcinogen-modified nucleosides, 1,N6-ethenodeoxyadenosine and O4-methyldeoxythymidine, using polymerases of varying fidelity.

Terminal deoxynucleotidyl transferase (TdT) was used to prepare copolymers of dA and 1,N6-ethenodeoxyadenosine (epsilon dA). When used as templates for Escherichia coli DNA polymerase I (Pol I) and compared with poly (dA), normal dTTP incorporation was not significantly affected by the presence of 7% epsilon dA. dGTP misincorporation was only slightly increased and occurred about once for every 500 epsilon dA residues. The error-prone polymerase from avian myeloblastosis virus (AMV reverse transcriptase) increased this error rate 5- to 20-fold to a maximum of 1 dG/25 epsilon dA. No dCTP misincorporation was detected with either polymerase. In transcription with E. coli DNA-dependent RNA polymerase, no errors were revealed by nearest neighbor analysis. Poly (dA) treated with chloroacetaldehyde under conditions producing the same proportion of epsilon dA (without the hydrated form) as the synthesized template behaved in the same manner with a similar low level of misincorporation of dG. Such treatment of alternating poly d(A-T) caused structural changes indicative of crosslinks but did not alter its template properties. Increasing the amount of epsilon dA in either synthesized or modified polymers greatly decreased the template activity without increasing the error rate. It is suggested that epsilon dA generally does not prevent dT incorporation but behaves as a bulky lesion which is bypassed. In contrast to the low mutagenic efficiency of epsilon dA, O4-methyldeoxythymidine (m4dT), in copolymers with dA, directed the misincorporation of 1 dG/12 m4dT with Pol I and 1 dG/3 m4dT with reverse transcriptase. Nearest neighbor analysis of transcripts showed the incorporation of 1 dG/12 m4dT. These data are in agreement with the previous reported mutagenicity of m4dT in alternating poly d(A-T, m4T).

N4-Methoxydeoxycytidine triphosphate is in the imino tautomeric form and substitutes for deoxythymidine triphosphate in primed poly d[A-T] synthesis with E. coli DNA polymerase I.

N4- Methoxydeoxycytidine triphosphate ( mo4dCTP ) substitutes for dTTP in poly d[A-T] synthesis with E. coli DNA polymerase I (Pol I). In parallel experiments using as template-primer, poly d[G-C], no incorporation of [14C] mo4dC was detected. This indicates that this deoxy derivative acts as the imino tautomer, as previously found for the riboderivative . Nearest neighbor analysis of transcripts of poly d[A-T] containing mo4dC shows that the derivative substitutes for only one base. In replication, singlestranded mo4dC -containing polymers gave little misincorporation, including that of dATP which can hydrogen-bond to mo4dC in the imino form, if the methoxy group is anti to the N-3. It is therefore assumed that the methoxy group is constrained anti in a polymer such as d[A-T], but can be in the syn form in singlestranded polymers and not recognized by DNA polymerase. mo4dC destabilizes the poly d[A-T] helix, as indicated by a lowered and less cooperative melting. Steric factors such as adjacent base displacement were invoked for similar findings with the doublestranded r( U61 , mo4C39 ) X r(A).

Determination of the absolute handedness of knots and catenanes of DNA.

DNA winds about itself in a right-handed or left-handed fashion at several structural levels. The double helix is generally right-handed and is given a (+) sign by convention, whereas supercoiling of the helix axis is always (-) in the cell. The winding in higher -order forms such as knots and catenanes is unknown, and this has impeded elucidation of the mechanisms of their formation and resolution by replication, recombination and topoisomerase action. We introduce here a procedure for determining the handedness of DNA winding by inspection of electron micrographs of DNA molecules coated with Escherichia coli RecA protein. We demonstrate the validity of the method and show that DNA topoisomerase I of E. coli generates an equal mixture of (+) and (-) duplex DNA knots, and that one product of recombination by resolvase of transposon Tn3 (refs 8, 9) is a catenane of uniquely (+) sign.