Interstrand DNA crosslinks (ICLs) are formed by natural products of metabolism and by chemotherapeutic reagents. Work in E. coli identified a two cycle repair scheme involving incisions on one strand on either side of the ICL (unhooking) producing a gapped intermediate with the incised oligonucleotide attached to the intact strand. The gap is filled by recombinational repair or lesion bypass synthesis. The remaining monoadduct is then removed by Nucleotide Excision Repair (NER). Despite considerable effort, our understanding of each step in mammalian cells is still quite limited. In part this reflects the variety of crosslinking compounds, each with distinct structural features, used by different investigators. Also, multiple repair pathways are involved, variably operative during the cell cycle. G1 phase repair requires functions from NER, although the mechanism of recognition has not been determined. Repair can be initiated by encounters with the transcriptional apparatus, or a replication fork. In the case of the latter, the reconstruction of a replication fork, stalled or broken by collision with an ICL, adds to the complexity of the repair process. The enzymology of unhooking, the identity of the lesion bypass polymerases required to fill the first repair gap, and the functions involved in the second repair cycle are all subjects of active inquiry. Here we will review current understanding of each step in ICL repair in mammalian cells.

The bacteriorhodopsin gene has been identified in a 5.3-kilobase restriction endonuclease fragment isolated from Halobacterium halobium DNA, using a cloned cDNA fragment as the probe. Of the 1229 nucleotides whose sequence was determined in the genomic fragment, 786 correspond to the structural gene of bacteriorhodopsin, 360 are upstream from the initiator methionine codon, and 83 are downstream from the COOH terminus. The bacteriorhodopsin gene codes for a precursor sequence of 13 amino acids at the NH2 terminus, 248 amino acids that are present in the mature protein, and an additional aspartic acid at the COOH terminus. This determination of the DNA sequence for an archaebacterial gene reveals that the standard genetic code is used; however, there is a marked preference for either G or C in the third codon position. The gene does not contain any intervening sequences and no prokaryotic promoter can be identified in the region immediately upstream from the structural gene. The bacteriorhodopsin mRNA contains at the 5' terminus only three nucleotides beyond the initiating AUG codon and this terminus can form a hairpin structure. Immediately downstream from this structure there is a sequence complementary to the 3' terminus of H. halobium 16S rRNA.Bacteriorhodopsin, the only protein in the purple membrane of Halobacterium halobium, catalyzes the light-dependent translocation of protons and thus generates a transmembrane electrochemical gradient (1). The protein consists of a single polypeptide chain of 248 amino acids (2) and contains one molecule of retinaldehyde per protein linked as a Schiff's base to the a-amino group of a lysine residue (1, 3). The amino acid sequence ofthe protein is known (2, 4), and a three dimensional model has been developed (5) that is compatible with this sequence and the diffraction data (6, 7). According to this model the polypeptide chain traverses the membrane seven times in the form of a-helical rods.In view of these and other studies (1)

Triple helix forming oligonucleotides (TFOs) recognize and bind sequences in duplex DNA and have received considerable attention because of their potential for targeting specific genomic sites. TFOs can deliver DNA reactive reagents to specific sequences in purified chromosomal DNA (ref. 4) and nuclei. However, chromosome targeting in viable cells has not been demonstrated, and in vitro experiments indicate that chromatin structure is incompatible with triplex formation. We have prepared modified TFOs, linked to the DNA-crosslinking reagent psoralen, directed at a site in the Hprt gene. We show that stable Hprt-deficient clones can be recovered following introduction of the TFOs into viable cells and photoactivation of the psoralen. Analysis of 282 clones indicated that 85% contained mutations in the triplex target region. We observed mainly deletions and some insertions. These data indicate that appropriately constructed TFOs can find chromosomal targets, and suggest that the chromatin structure in the target region is more dynamic than predicted by the in vitro experiments.

Bloom syndrome and Werner syndrome are genome instability disorders, which result from mutations in two different genes encoding helicases. Both enzymes are members of the RecQ family of helicases, have a 3 3 5 polarity, and require a 3 single strand tail. In addition to their activity in unwinding duplex substrates, recent studies show that the two enzymes are able to unwind G2 and G4 tetraplexes, prompting speculation that failure to resolve these structures in Bloom syndrome and Werner syndrome cells may contribute to genome instability. The triple helix is another alternate DNA structure that can be formed by sequences that are widely distributed throughout the human genome. Here we show that purified Bloom and Werner helicases can unwind a DNA triple helix. The reactions are dependent on nucleoside triphosphate hydrolysis and require a free 3 tail attached to the third strand. The two enzymes unwound triplexes without requirement for a duplex extension that would form a fork at the junction of the tail and the triplex. In contrast, a duplex formed by the third strand and a complement to the triplex region was a poor substrate for both enzymes. However, the same duplex was readily unwound when a noncomplementary 5 tail was added to form a forked structure. It seems likely that structural features of the triplex mimic those of a fork and thus support efficient unwinding by the two helicases.Despite the obvious importance of genetic stability, the mammalian genome has an abundance of sequences that are potentially destabilizing. Elements that can form non-duplex structures, such as DNA triple helices (1, 2), G quartets (3-5), hairpins (6, 7), and cruciforms (8), all have the capacity to interfere with transcription and replication. Moreover, many studies have described the role of these elements in DNA rearrangements such as deletions, sister chromatid exchanges, homologous and illegitimate recombination events, etc. (reviewed in Refs. 9 and 10). With such an array of provocative sequence elements, it seems likely that cells would have developed the capacity for controlling the potential of these sequences for genome destabilization.Some insight into the nature of the enzymology involved in the maintenance of genetic integrity has come from the study of two "genome instability" disorders, Bloom and Werner syndrome. Patients with Werner syndrome (WS) 1 are characterized by premature aging (11) and a high incidence of certain cancers. They have elevated frequencies of spontaneous deletion mutations in the HPRT gene (12) and show a variety of karyotypic abnormalities including inversions, translocations, and chromosomal losses (13,14). The mutant gene in WS has been identified as a member of the RecQ helicase family, and the protein is a 3Ј-5Ј-helicase (15, 16) and a 3Ј-5Ј-exonuclease (17-19). A role in replication is implied by the demonstration that the WRN helicase interacts with and is stimulated by the human replication protein A (RPA) (20, 21). The protein has been recovered from cells in a replication comp...

Successful gene-targeting reagents must be functional under physiological conditions and must bind chromosomal target sequences embedded in chromatin. Triple helix-forming oligonucleotides (TFOs) recognize and bind specific sequences via the major groove of duplex DNA and may have potential for gene targeting in vivo. We have constructed chemically modified, psoralenlinked TFOs that mediate site-specific mutagenesis of a chromosomal gene in living cells. Here we show that targeting efficiency is sensitive to the biology of the cell, specifically, cell cycle status. Targeted mutagenesis was variable across the cycle with the greatest activity in S phase. This was the result of differential TFO binding as measured by cross-link formation. Targeted cross-linking was low in quiescent cells but substantially enhanced in S phase cells with adducts in ϳ20 -30% of target sequences. 75-80% of adducts were repaired faithfully, whereas the remaining adducts were converted into mutations (>5% mutation frequency). Clones with mutations could be recovered by direct screening of colonies chosen at random. These results demonstrate high frequency target binding and target mutagenesis by TFOs in living cells. Successful protocols for TFOmediated manipulation of chromosomal sequences are likely to reflect a combination of appropriate oligonucleotide chemistry and manipulation of the cell biology.Effective gene-targeting reagents would have a broad application as probes of chromatin structure and in a variety of genomic manipulations, e.g. gene knock-out, targeted gene conversion and/or recombination, and gene therapy. Successful constructs must be functional in the nuclear environment of living cells. Furthermore, their intended targets must be accessible despite the packaging and condensation of nuclear DNA by chromosomal proteins. One targeting strategy that has been of interest for many years is based on triple-helix forming oligonucleotides (TFOs). 1A DNA triple helix can form when a TFO lies in the major groove of intact duplex DNA (1-5). The most stable structures assemble on polypurine:polypyrimidine sequences with hydrogen bonds formed between the bases in the third strand and those in the purine strand of the duplex. The purine or pyrimidine motif third strands may be involved in triplex formation depending on the target sequence, and a binding code for the design of the third strands has been defined (6). Although conventional oligonucleotides have features that limit their activity under physiological conditions, there is a variety of chemical modifications that ameliorate these limitations (7). Thus, for pyrimidine TFOs, the replacement of cytosine by 5-methylcytosine and the use of 2Ј-O-methyl (2Ј-OMe)-modified sugars permit stable triplex formation at physiological pH in vitro (8 -13). Other derivatives contribute to the bioactivity of TFOs of both motifs as shown by us and others in recent publications (14 -18).Although chemical modifications can enhance TFO affinity and triplex stability, they obviously cannot add...

Triple helix forming oligonucleotides (TFOs) that bind chromosomal targets in living cells may become tools for genome manipulation, including gene knockout, conversion, or recombination. However, triplex formation by DNA third strands, particularly those in the pyrimidine motif, requires nonphysiological pH and Mg(2+) concentration, and this limits their development as gene-targeting reagents. Recent advances in oligonucleotide chemistry promise to solve these problems. For this study TFOs containing 2'-O-methoxy (2'-OMe) and 2'-O-(2-aminoethyl) (2'-AE) ribose substitutions in varying proportion have been constructed. The TFOs were linked to psoralen and designed to target and mutagenize a site in the hamster HPRT gene. T(m) analyses showed that triplexes formed by these TFOs were more stable than the underlying duplex, regardless of 2'-OMe/2'-AE ratio. However, TFOs with 2'-AE residues were more stable in physiological pH than those with only 2'-OMe sugars, as a simple function of 2'-AE content. In contrast, gene knockout assays revealed a threshold requirement--TFOs with three or four 2'-AE residues were at least 10-fold more active than the TFO with two 2'-AE residues. The HPRT knockout frequencies with the most active TFOs were 300-400-fold above the background, whereas there was no activity against the APRT gene, a monitor of nonspecific mutagenesis.

The soybean genome hosts a family of several hundred, relatively homogeneous copies of a large, copia͞Ty1-like retroelement designated SIRE-1. A copy of this element has been recovered from a Glycine max genomic library. DNA sequence analysis of two SIRE-1 subclones revealed that SIRE-1 contains a long, uninterrupted, ORF between the 3 end of the pol ORF and the 3 long terminal repeat (LTR), a region that harbors the env gene in retroviral genomes. Conceptual translation of this second ORF produces a 70-kDa protein. Computer analyses of the amino acid sequence predicted patterns of transmembrane domains, ␣-helices, and coiled coils strikingly similar to those found in mammalian retroviral envelope proteins. In addition, a 65-residue, proline-rich domain is characterized by a strong amino acid compositional bias virtually identical to that of the 60-amino acid, proline-rich neutralization domain of the feline leukemia virus surface protein. The assignment of SIRE-1 to the copia͞ Ty1 family was confirmed by comparison of the conceptual translation of its reverse transcriptase-like domain with those of other retroelements. This finding suggests the presence of a proretrovirus in a plant genome and is the strongest evidence to date for the existence of a retrovirus-like genome closely related to copia͞Ty1 retrotransposons.

The binding of Escherichia coli Gal repressor to linear DNA fragments containing two binding sites (OE and OI) within the gal operon was analyzed in vitro with quantitative footprint and mobility-shift techniques. In vivo analysis of the regulation of the gal operon [Haber, R., & Adhya, S. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 9683-9687] has suggested the role of a regulatory "looped complex" mediated by the association of Gal repressor dimers bound at OE and OI. The binding of Gal repressor to a single site can be described by a model in which monomer and dimer are in equilibrium and only the dimer binds to DNA. At pH 7.0, 25 mM KCl, and 20 degrees C, the binding and dimerization free energies are comparable, suggesting that the equilibrium governing the formation of dimers may be important to regulation. The two intrinsic binding constants, delta GI and delta GE, and a constant describing cooperativity, delta GIE, were determined by footprint titration analysis as a function of pH, [KCl], and temperature. Only at 4 and 0 degrees C was delta GIE negative, signifying cooperative binding. These results are thought to be due to a weak dimer to tetramer association interface. delta GE and delta GI had maximal values between pH 6 and pH 7. The dependence of these constants on [KCl] corresponded to the displacement of approximately 2 ion equiv. The temperature dependence could be described by a change in the heat capacity, delta Cp, of -2.3 kcal mol-1 deg-1. Mobility-shift titration experiments conducted at 20 and 0 degrees C yielded values for delta GIE that were consistent with those resolved from the footprint analysis. Unique values of delta GIE were determined by analysis of mobility-shift titrations of Gal repressor with wild-type operator subject to the constraint that delta GE = delta GI: a procedure that eliminates the need to simultaneously analyze wild-type titrations with titrations of OE- and OI- operators.