Binding of benzo[a]pyrene 7,8-diol-9,10-epoxides to DNA, RNA, and protein of mouse skin occurs with high stereoselectivity.

The formation, stereostructure, and cellular reactions of the 7,8-diol-9,10-epoxide metabolites of the carcinogen benzo[a]pyrene have been examined after topical application of benzo[a]pyrene to the skin of mice. In this known target tissue, polymer adducts from diastereomeric diol epoxides, (+)-(7S, 8R, 9R, 10R) and (+)-(7R, 8S, 9R, 10R), were formed stereospecifically from their corresponding 7,8-dihydrodiols. Both diol epoxides bind with proteins, RNA, and DNA in vivo. For the nucleic acids, binding occurs preferentially at the 2-amino group of guanine in cellular RNA and DNA in vivo. Methods for establishing the structure of the cellular adducts as well as the possible biological implications of their formation are discussed.

Homologous recombination between single-stranded DNA and chromosomal genes in Saccharomyces cerevisiae.

Transformation of Saccharomyces cerevisiae strains was examined by using the URA3 and TRP1 genes cloned into M13 vectors in the absence of sequences capable of promoting autonomous replication. These constructs transform S. cerevisiae cells to prototrophy by homologous recombination with the resident mutant gene. Single-stranded DNA was found to transform S. cerevisiae cells at efficiencies greater than that of double-stranded DNA. No conversion of single-stranded transforming DNA into duplex forms could be detected during the transformation process, and we conclude that single-stranded DNA may participate directly in recombination with chromosomal sequences. Transformation with single-stranded DNA gave rise to both gene conversion and reciprocal exchange events. Cotransformation with competing heterologous single-stranded DNA specifically inhibited transformation by single-stranded DNA, suggesting that one of the components in the transformation-recombination process has a preferential affinity for single-stranded DNA.

Homologous recombination enhancement conferred by the Z-DNA motif d(TG)30 is abrogated by simian virus 40 T antigen binding to adjacent DNA sequences.

The Z-DNA motif polydeoxythymidylic-guanylic [d(TG)].polydeoxyadenylic-cytidylic acid [d(AC)], present throughout eucaryotic genomes, is capable of readily forming left-handed Z-DNA in vitro and has been shown to promote homologous recombination. The effects of simian virus 40 T-antigen-dependent substrate replication upon the stimulation of recombination conferred by the Z-DNA motif d(TG)30 were analyzed. Presence of d(TG)30 adjacent to a T-antigen-binding site I can stimulate homologous recombination between nonreplicating plasmids, providing that T antigen is absent, in both simian CV-1 cells and human EJ cells (W. P. Wahls, L. J. Wallace, and P. D. Moore, Mol. Cell. Biol. 10:785-793). It has also been shown elsewhere that the presence of d(TG)n not adjacent to the T-antigen-binding site can stimulate homologous recombination in simian virus 40 molecules replicating in the presence of T antigen (P. Bullock, J. Miller, and M. Botchan, Mol. Cell. Biol. 6:3948-3953, 1986). However, it is demonstrated here that d(TG)30 nine base pairs distant from a T-antigen-binding site bound with T antigen does not stimulate recombination between either replicating or nonreplicating substrates in somatic cells. The bound T antigen either prevents the d(TG)30 sequence from acquiring a recombinogenic configuration (such as left-handed Z-DNA), or it prevents the interaction of recombinase proteins with the sequence by stearic hindrance.

Homologous recombination catalyzed by human cell extracts.

Two plasmids containing noncomplementing and nonreverting deletions in a bacterial phosphotransferase gene conferring resistance to neomycin (Neor) were incubated with human cell extracts, and the mixtures were used to transform recombination-deficient (recA-) Escherichia coli cells. We were able to obtain Neor colonies at a frequency of 2 X 10(-3). This frequency was 100 to 1,000 times higher than that obtained with no extracts. The removal of riboadenosine 5'-triphosphate, Mg2+, or deoxynucleoside triphosphates from the reaction mixture severely reduced the yield of Neor colonies. Examination of plasmid DNA from the Neor colonies revealed that they resulted from gene conversion and reciprocal recombination. On the basis of these results, we conclude that mammalian somatic cells in culture have the enzymatic machinery to catalyze homologous recombination in vitro.

The Z-DNA motif d(TG)30 promotes reception of information during gene conversion events while stimulating homologous recombination in human cells in culture.

Tracts of the alternating dinucleotide polydeoxythymidylic-guanylic [d(TG)].polydeoxyadenylic-cytidylic acid [d(AC)], present throughout the human genome, are capable of readily forming left-handed Z-DNA in vitro. We have analyzed the effects of the Z-DNA motif d(TG)30 upon homologous recombination between two nonreplicating plasmid substrates cotransfected into human cells in culture. In this study, the sequence d(TG)30 is shown to stimulate homologous recombination up to 20-fold. Enhancement is specific to the Z-DNA motif; a control DNA fragment of similar size does not alter the recombination frequency. The stimulation of recombination is observed at a distance (237 to 1,269 base pairs away from the Z-DNA motif) and involves both gene conversion and reciprocal exchange events. Maximum stimulation is observed when the sequence is present in both substrates, but it is capable of stimulating when present in only one substrate. Analysis of recombination products indicates that the Z-DNA motif increases the frequency and alters the distribution of multiple, unselected recombination events. Specifically designed crosses indicate that the substrate containing the Z-DNA motif preferentially acts as the recipient of genetic information during gene conversion events. Models describing how left-handed Z-DNA sequences might promote the initiation of homologous recombination are presented.

Interactions between DNA polymerase and aminofluorene adducts that affect the recognition and possibly the mutagenicity of the lesions.

Adducts of 2-aminofluorene on the C-8 position of guanine block DNA synthesis and lead to mutation. N-acetylated adducts adopt the syn conformation such that in DNA the guanine is displaced from the helix by the fluorene ring while unacetylated adducts prefer the anti conformation allowing normal base pairing of the guanine with cytosine. In vitro synthesis by both E. coli DNA polymerase I and T4 DNA polymerase terminates predominantly one nucleotide before acetylated adducts but has an increased tendency to terminate opposite the unacetylated adducts apparently reflecting the preferred conformations of the two species of reacted nucleoside. In complete contrast AMV reverse transcriptase correctly inserts cytosine opposite the acetylated adducts but prefers to terminate one nucleotide before the unacetylated adducts. We interpret these results as indicating that the specific properties of a replicating polymerase can influence the conformation of a reacted nucleoside thus altering its recognition and possibly its mutagenic activity.

Radiation-sensitive pyrimidine auxotrophs of Ustilago maydis. I. Isolation and characterization of mutants.

The relationship between UV sensitivity and pyrimidine auxotrophy has been examined. Fourteen pyrimidine-requiring mutants have been classified on the basis of genetic complementation and utilization of biosynthetic intermediates and have been assigned to at least four loci. All the mutants studied were sensitive to UV, although the degree of sensitivity varied both between loci and amongst alleles at the same locus. A double mutant strain carrying pyrimidine mutants at two loci was only as sensitive to UV as the more sensitive of the singles. This suggests that both mutants are deficient in the same repair mechanism. Suppressor mutations which restored endogenous pyrimidine biosynthesis were isolated. These suppressors restored UV resistance in the presence of the original mutation. The results indicate that the UV sensitivity of these mutants is a direct result of pyrimidine auxotrophy and not to any secondary properties of the mutants.

Radiation-sensitive pyrimidine auxotrophs of Ustilago maydis. II. A study of repair mechanisms and UV recovery in pyr I.

Two mutants at the pyr I locus have been used to study the radiation sensitivity of pyrimidine auxotrophs of U. maydis. The mutant pry I-I has a reduced level of thymidine nucleotides, and this is a likely basis of the sensitivity. This strain is able to excise pyrimidine dimers from its DNA and is cross-sensitive to gamma-rays and nitrosoguanidine (NG) as well as to UV. A diploid heteroallelic at the pyr I locus was UV-sensitive but not deficient in UV-induced mitotic recombination. The results suggest that the UV sensitivity may be due to the failure of a repair DNA polymerase to fill post-excision single-strand gaps in the DNA. The mutant pyr I-I exhibits the property of UV recovery, and this is shown to be dependent on the presence of dimers in the DNA. A mechanism for UV recovery is proposed in which a repair system, possibly involving recombination, is induced by the UV irradiation.

Homologous recombination in a Chinese hamster X-ray-sensitive mutant.

We have tested the mutant Chinese hamster cell line xrs-5, which is sensitive to ionizing radiation, for the ability to carry out homologous recombination. In an in vivo assay to detect recombination between two transfected plasmids carrying non-complementing mutants in the neomycin resistance gene, xrs-5 showed a 6-fold reduction in recombination frequency when compared to the parental cell line K1. Extracts prepared from nuclei of the mutant were also tested for their ability to catalyze homologous recombination between the same two plasmids in vitro. Extracts from xrs-5 were found to mediate recombination in this assay at frequencies not significantly different from those obtained with extracts from the parental cell line.

Induction of homologous recombination in Saccharomyces cerevisiae.

We have investigated the effects of UV irradiation of Saccharomyces cerevisiae in order to distinguish whether UV-induced recombination results from the induction of enzymes required for homologous recombination, or the production of substrate sites for recombination containing regions of DNA damage. We utilized split-dose experiments to investigate the induction of proteins required for survival, gene conversion, and mutation in a diploid strain of S. cerevisiae. We demonstrate that inducing doses of UV irradiation followed by a 6 h period of incubation render the cells resistant to challenge doses of UV irradiation. The effects of inducing and challenge doses of UV irradiation upon interchromosomal gene conversion and mutation are strictly additive. Using the yeast URA3 gene cloned in non-replicating single- and double-stranded plasmid vectors that integrate into chromosomal genes upon transformation, we show that UV irradiation of haploid yeast cells and homologous plasmid DNA sequences each stimulate homologous recombination approximately two-fold, and that these effects are additive. Non-specific DNA damage has little effect on the stimulation of homologous recombination, as shown by studies in which UV-irradiated heterologous DNA was included in transformation/recombination experiments. We further demonstrate that the effect of competing single- and double-stranded heterologous DNA sequences differs in UV-irradiated and unirradiated cells, suggesting an induction of recombinational machinery in UV-irradiated S. cerevisiae cells.

Evidence for the formation of hybrid DNA during mitotic recombination in Chinese hamster cells.

Direct evidence is provided for the formation of hybrid DNA during mitotic recombination in CHO cells. The cells were labeled for one round of replication in medium containing BUdR, so that the density of the DNA was heavy light (HL) and then returned to light medium. Further DNA synthesis, during either repair or chromosome replication, can only result in HL or fully light (LL) DNA; however, the formation of hybrid DNA as part of the process of recombinational repair will produce some fully heavy (HH) DNA. A small fraction of DNA containing regions of HH DNA has been detected on neutral CsC1 gradients, and the amount of this DNA is increased by treatment of the cells with mitomycin C. Increasing doses of mitomycin C produce smimlar increases in both the amount of HH DNA and the frequency of sister chromatid exchanges measured cytologically. This correlation provides evidence that the HH DNA is hybrid DNA, formed as an intermediate in recombinational repair.

Transformation and recombination in rad mutants of Saccharomyces cerevisiae.

Disruption/deletion mutations in genes of the RAD52 epistasis group of Saccharomyces cerevisiae were examined for their effects on recombination between single- and double-stranded circular DNA substrates and chromosomal genes in a transformation assay. In rad50 mutants there was a small reduction in recombination with single-stranded DNA at the leu2-3, 112 allele; in addition there was an almost complete elimination of recombination at trp1-1 for both single- and double-stranded DNA. Reintroduction of a wild-type RAD50 gene on a replicating plasmid carrying CEN4 restored recombinational competence at trp1-1, indicating that rad50 is defective in gene replacement of this allele. In rad52 mutants a reduction of 30%-50% in recombination involving either single- or double-stranded circular DNA was observed in each experiment when compared to the wild type. This reduction of recombination in rad52 mutants was similar for recombination at the ura3-52 mutant locus where only integration events have been observed, and at the trp1-1 mutant locus, where recombination occurs predominantly by gene replacement. Neither the rad54 nor the rad57 mutations had a significant effect on recombination with single- or double-stranded DNA substrates.

Relative frequencies of homologous recombination between plasmids introduced into DNA repair-deficient and other mammalian somatic cell lines.

Twelve mammalian somatic cell lines, some of them DNA damage-sensitive mutants paired with their respective wild-type parental lines, were assayed for their ability to catalyze extrachromosomal, intermolecular homologous recombination between pSV2neo plasmid recombination substrates. All of the somatic cell lines analyzed are capable of catalyzing homologous recombination; however, there is a wide range of efficiencies with which they do so. Five human cell lines display a fourfold range of recombination frequencies, and six hamster cell lines vary almost 20-fold. Linearizing one of the recombination substrates stimulates recombination in all but one of the cell lines. Two of the three paired mutant cell lines display a threefold reduction in their ability to catalyze homologous recombination when compared to their respective parental cell lines, indicating that the mutations that render them sensitive to DNA damaging agents might also play a role in homologous recombination.

Molecular recombination and the repair of DNA double-strand breaks in CHO cells.

Molecular recombination and the repair of DNA double-strand breaks (DSB) have been examined in the G-0 and S phase of the cell cycle using a temperature-sensitive CHO cell line to test i) if there are cell cycle restrictions on the repair of DSB's' ii) the extent to which molecular recombination can be induced between either sister chromatids or homologous chromosomes and iii) whether repair of DSB's involves recombination (3). Mitomycin C (1-2 micrograms/ml) or ionizing radiation (50 krad) followed by incubation resulted in molecular recombination (hybrid DNA) in S phase cells. Approximately 0.03 to 0.10% of the molecules (number average molecular weight: 5.6 x 10(6) Daltons after shearing) had hybrid regions for more than 75% of their length. However, no recombination was detected in G-0 cells. Since the repair of DSB was observed in both stages with more than 50% of the breaks repaired in 5 hours, it appears that DSB repair in G-0 cells does not involve recombination between homologous chromosomes. The possibility is not excluded that repair in G-0 cells involves only small regions (less than 4 x 10(6) Daltons).

Antiparallel, intramolecular triplex DNA stimulates homologous recombination in human cells.

The DNA motif 5'-AAGGGAGAAXGGGTATAGGGYAAGAGGGAA-3' (named XY32) is an H-palindrome and has been shown to undergo a superhelix-induced, pH-dependent structural transition to H-form (pyrimidine purineo pyrimidine triplex) DNA when X = Y = A (AA32) or X = Y = G (GG32), but when X = A and Y = G (AG32) or X = G and Y = A (GA32), the transition is much more difficult [Mirkin, S. (1987) Nature (London) 330, 495-497]. Furthermore, AA32, GG32, and GA32 triplexes have the proper sequence structure to potentially form pyrimidineopurineopurine (*H-form) triplexes, but AG32 does not [Beal, P. A. & Dervan, P. B. (1992) Nucleic Acids Res. 20, 2773-2776]. Using an in vivo plasmid-plasmid recombination assay system in cultured human cells, we have found that AA32, GA32, and GG32 stimulate homologous recombination between plasmids 3- to 5-fold when both recombination substrates contain these triplex-forming sequences, whereas AG32, which differs from the others by only 1 or 2 bp, does not significantly affect the frequency of recombination. Double-strand breaks, which destroy supercoiling, nullify the stimulation. Therefore, stimulation of homologous recombination between plasmids containing these sequences correlates with their triplex-forming potential. Crosses in which the triplex-forming sequence is inserted into only one substrate exhibit an intermediate stimulation, suggesting that the inserts are acting alone as intramolecular triplexes.

Recombination hotspot activity of hypervariable minisatellite DNA requires minisatellite DNA binding proteins.

Hypervariable minisatellite DNA repeats are found at tens of thousands of loci in the mammalian genome. These sequences stimulate homologous recombination in mammalian cells [Cell 60:95-103]. To test the hypothesis that protein-DNA interaction is required for hotspot function in vivo, we determined whether a second protein binding nearby could abolish hotspot activity. Intermolecular recombination between pairs of plasmid substrates was measured in the presence or absence of the cis-acting recombination hotspot and in the presence or absence of the second trans-acting DNA binding protein. Minisatellite DNA had hotspot activity in two cell lines, but lacked hotspot activity in two closely related cell lines expressing a site-specific helicase that bound to DNA adjacent to the hotspot. Suppression of hotspot function occurred for both replicating and non-replicating recombination substrates. These results indicate that hotspot activity in vivo requires site occupancy by minisatellite DNA binding proteins.

Latex allergy and the podiatric surgeon.

Latex allergy in recent years has become a more visible problem in the medical community. There are certain populations found to be at greater risk for this problem. Those with myelodysplasia, congenital urinary anomalies, and a history of a significant number of prior surgeries are particularly at risk, followed less commonly by health care workers and the general population. A detailed patient history is the most reliable predictor of latex sensitivity. There are various immunological and serological screening tests on the market, which are not always reliable predictors of allergy or readily available to the physician. This article reviews the current literature on latex allergy and provides insight into populations at risk, the type of allergic response seen, predictors of sensitivity, management of patients with latex allergy, and measures to prevent it.

The influence of DNA binding protein on the substrate affinities of DNA polymerase from Ustilago maydis: one polymerase implicated in both DNA replication and repair.

The DNA polymerase of Ustilago maydis is stimulated by a DNA binding protein from the same organism. Analysis of this stimulation shows that there is an increase in affinity for both substrates of the reaction. The apparent Km for deoxynucleoside triphosphates is decreased 3 fold, and that for denatured DNA by 4 fold. In both cases the maximum velocity (Vmax) is increased 1.2 to 1.4 fold. It is suggested that the variability in the affinity of the enzyme for deoxynucleoside triphosphates mediated by the binding protein may provide the basis for the UV sensitivity of pyrimidine auxotrophs in this organism.

Hypervariable minisatellite DNA is a hotspot for homologous recombination in human cells.

Hypervariable minisatellite DNA sequences are short tandemly repeated sequences that are present throughout the human genome and are implicated to enhance recombination. We have constructed a consensus hypervariable minisatellite sequence and analyzed its effect on homologous recombination in human cells in culture. The consensus sequence d(AGAGGTGGGCAGGTGG)6.5 is shown to stimulate homologous recombination up to 13.5-fold. The stimulation occurs at a distance and in both directions but does show a quantitative directionality. Stimulation occurs in a codominant manner, and the sequence is inherited equally in the products. Enhancement is maintained, but at a reduced level, when double-strand breaks are introduced into the substrates. Multiple unselected recombination events are promoted, and preferential stimulation of reciprocal exchange events is demonstrated.