Selective inhibition of sequence-specific protein-DNA interactions by incorporation of 6-thioguanine: cleavage by restriction endonucleases.

Incorporation of the antileukemic agent 6-thioguanine (TG) into cellular DNA has been demonstrated to be a major determinant of its cytotoxicity. We have previously shown that complete replacement of G by TG within one DNA strand of the SV40 origin of replication can completely inhibit sequence-specific binding of the viral replication protein T antigen. The aim of the present study was to determine the effect of more selective TG substitutions on DNA-protein interactions, by utilizing the simpler base recognition sequence motifs of restriction endonucleases. In the first part of our study, we replaced G with TG in one or two of four possible sites within the duplex hexameric recognition sequence of BamHI (5'-G decreases GATCC-3'), by enzymatic extension of primed oligonucleotides. This extension was stalled, but not completely inhibited, at locations where insertion of consecutive TG moieties was required. Both strands of molecules containing a single substitution were cleaved by BamHI at reduced rates, with the substituted strand inhibited to a greater degree. In molecules containing two substitutions, neither strand was cut by BamHI. In contrast, we found that scission of these same mono- and disubstituted substrates by the less stringent isoschizomer MboI (5'-N decreases GATCN-3') was inhibited only slightly. In the second part of our study, we investigated the effect of analog substitution on scission by the type II-S enzymes AlwI and FokI, in order to separately determine the effects of restriction site modification versus scission site modification. We found that the reactivity of these enzymes was completely abolished by TG substitution within the recognition site, whereas substitution at the scission site had no effect. Our results demonstrate that infrequent TG substitutions within symmetric DNA sequences can inhibit sequence-specific interactions in an asymmetric fashion. In addition, although previous reports have shown that TG forms a relatively weak base pair with cytosine, it appears that the inhibition of restriction endonuclease-mediated cleavage resulting from TG incorporation is a function of the sequence requirements of the protein and not a general consequence of disrupted base-pairing at the recognition locus. These data support the idea that the cytotoxic consequences of TG incorporation may be due to inhibition of sequence-specific protein-DNA interactions.

Effects of incorporation of 6-thioguanine into SV40 DNA.

The antileukemic agent 6-thioguanine (TG) is thought to inhibit DNA synthesis as a result of its incorporation into DNA. In the present study we have examined the nature of this inhibition, using replication of SV40 viral DNA as a model system. Addition of TG to SV40-infected CV1P cells from 22 to 24 hr post infection causes a dose-dependent inhibition of viral DNA synthesis. This inhibition plateaus between 250 and 2500 microM TG, resulting in a maximum decrease of viral DNA synthesis of about 50%. Pulse-chase experiments showed no detectable slowing of elongation of nascent DNA chains, whereas measurement of the conversion of incorporated 3H-dThd into supercoiled viral DNA suggested that elongation might be slightly inhibited, but by no more than 20%. Since inhibition of elongation could not account for the total depression of DNA synthesis, we hypothesized that inhibition of initiation of DNA replication takes place. This hypothesis was tested by radioactively labeling newly synthesized viral DNA and then assessing the ability of these molecules to reenter the replicating pool by density labeling with bromodeoxyuridine. The fraction of TG-containing molecules able to re-initiate replication was decreased 15%, compared to control. This effect, which was dependent on the concentration of TG added to the medium, was closely correlated to the extent of TG incorporation into the viral genome. We concluded that a portion of SV40 viral DNA synthesis inhibited by TG is due to an effect on initiation, and hypothesized that this effect may be caused by the substitution of TG for guanine in critical recognition sequences at the origin of replication. We proceeded to test this hypothesis by constructing SV40 origin sequences containing TG and then measuring their ability to bind T-antigen in vitro. The necessary deoxynucleoside triphosphate, TdGTP, was obtained by chemical phosphorylation of thiodeoxyguanosine. In order to selectively place TG within the desired region, a plasmid containing the T-antigen binding sequences was linearized so as to place these sequences at one end of the molecule, and then digested briefly with exonuclease III. The excised strand was resynthesized by use of the Klenow fragment of DNA polymerase I along with various nucleotide mixtures. Although resynthesis with mixtures containing TdGTP in place of dGTP was impeded somewhat, it was possible to achieve complete resynthesis with this analog.(ABSTRACT TRUNCATED AT 400 WORDS)

Dissimilar actions of 6-mercaptopurine and 6-thioguanine in Chinese hamster ovary cells.

The actions of 6-thioguanine (TG) and 6-mercaptopurine (MP) were compared in Chinese hamster ovary (CHO) cells. Several differences were noted between these two agents. TG caused a greater maximal loss of clonogenicity, leaving about one log fewer survivors than did MP, although the cells killed by MP appeared to succumb much more rapidly than those killed by TG. MP-treated populations experienced a G1 or G1/S arrest which was quickly reversed upon drug removal, while TG-treated cells were arrested in late S/G2, after some delay. Although TG induced a gross chromosome deformation [unilateral chromatid damage, as described earlier in Maybaum and Mandel, Cancer Res. 43, 3852 (1983)] MP caused little or no such deformation. Addition of 4-amino-5-imidazolecarboxamide (AIC) to MP treatments antagonized MP-induced loss of clonogenicity, while AIC caused a dose-dependent potentiation of TG-induced loss of clonogenicity. The interaction between TG and AIC does not seem to represent an increase in either purine starvation or incorporation of TG into DNA, suggesting that a third mechanism is involved. We suggest that this additional mechanism may possibly be related to the induction of differentiation by TG that has been reported in other systems.