No abstract

Merbarone is a catalytic inhibitor of topoisomerase II that is in clinical trials as an anticancer agent. Despite the potential therapeutic value of this drug, the mechanism by which it blocks topoisomerase II activity has not been delineated. Therefore, to determine the mechanistic basis for the inhibitory action of merbarone, the effects of this drug on individual steps of the catalytic cycle of human topoisomerase II␣ were assessed. Concentrations of merbarone that inhibited catalytic activity >80% had no effect on either enzyme⅐DNA binding or ATP hydrolysis. In contrast, the drug was a potent inhibitor of enzyme-mediated DNA scission (in the absence or presence of ATP), and the inhibitory profiles of merbarone for DNA cleavage and relaxation were similar. These data indicate that merbarone acts primarily by blocking topoisomerase II-mediated DNA cleavage. Merbarone inhibited DNA scission in a global (rather than site-specific) fashion but did not appear to intercalate into DNA or bind in the minor groove. Since the drug competed with etoposide (a cleavage-enhancing agent that binds directly to topoisomerase II), it is proposed that merbarone exerts its inhibitory effects through interactions with the enzyme and that the drug shares an interaction domain on topoisomerase II with cleavage-enhancing agents.Topoisomerase II is the target for some of the most active anticancer drugs used in the treatment of human malignancies (1-6). Among the topoisomerase II-targeted agents currently in clinical use are etoposide, teniposide, doxorubicin, mitoxantrone, and amsacrine. These drugs kill cells in an unusual fashion. Rather than inhibiting the overall catalytic activity of the type II enzyme, they act by increasing levels of topoisomerase II-mediated DNA cleavage, thus converting this essential enzyme into a potent cellular toxin (1, 3, 5, 7-10). Hence, to distinguish their unique mechanism of action, they are referred to as topoisomerase II "poisons" (11).A second class of drugs that affect the activity of topoisomerase II also appears to have clinical potential (2, 5, 7). In contrast to poisons, these agents act by inhibiting the catalytic activity of the enzyme and display no ability to stimulate DNA cleavage. Originally, topoisomerase II "catalytic inhibitors" were defined by antibacterial compounds such as novobiocin and coumermycin (12). These coumarin-based drugs block the DNA strand passage activity of the prokaryotic type II enzyme, DNA gyrase, by interfering with the ability of the enzyme to bind its ATP cofactor (13-17).Recently, catalytic inhibitors that display high activity against eukaryotic type II topoisomerases have been described. These are typified by drugs such as merbarone (18), aclarubicin (20), fostriecin (21), staurosporine (22), and mitindomide (23), which reflect a variety of inhibitory mechanisms. Aclarubicin, for example, blocks binding of the enzyme to its DNA substrate, the initial step of the topoisomerase II catalytic cycle (24) (see Fig. 12). In contrast, ICRF-193 blocks the final s...

The yeast Mlh1-Pms1 heterodimer required for mismatch repair (MMR) binds to DNA. Here we map DNA binding to N-terminal fragments of Mlh1 and Pms1. We demonstrate that Mlh1 and Pms1 N-terminal domains (NTDs) independently bind to double-stranded and single-stranded DNA, in the absence of dimerization and with different affinities. Full-length Mlh1p alone, which can homodimerize, also binds to DNA. Substituting conserved positively charged amino acids in Mlh1 produces mutator phenotypes in a haploid yeast strain characteristic of reduced MMR. These substitutions strongly reduce DNA binding by the Mlh1 NTD and, to a lesser extent, they also reduce DNA binding by full-length Mlh1 and the Mlh1-Pms1 heterodimer. Replacement of a homologous Pms1 residue has a much smaller effect on mutation rate and does not reduce DNA binding. The results demonstrate that NTDs of yeast Mlh1 and Pms1 contain independent DNA binding sites and they suggest that the C-terminal region of Mlh1p may also contribute to DNA binding. The differential mutator effects and binding properties observed here further suggest that Mlh1 and Pms1 differ in their interactions with DNA. Finally, the results are consistent with the hypothesis that DNA binding by Mlh1 is important for MMR.

Background & Aims-Individuals with inflammatory bowel disease are at risk of developing colorectal cancer (CRC). Epidemiologic, animal, and laboratory studies suggest that 5-aminosalicylic acid (5-ASA) protects from the development of CRC by altering cell cycle progression and by inducing apoptosis. Our previous results indicate that 5-ASA improves replication fidelity in colorectal cells, an effect that is active in reducing mutations. In this study, we hypothesized that 5-ASA restrains cell cycle progression by activating checkpoint pathways in colorectal cell lines, which would prevent tumor development and improve genomic stability.

In fulfilling its biosynthetic roles in nuclear replication and in several types of repair, DNA polymerase δ (pol δ) is assisted by replication protein A (RPA), the single-stranded DNA-binding protein complex, and by the processivity clamp proliferating cell nuclear antigen (PCNA). Here we report the effects of these accessory proteins on the fidelity of DNA synthesis in vitro by yeast pol δ. We show that when RPA and PCNA are included in reactions containing pol δ, rates for single base errors are similar to those generated by pol δ alone, indicating that pol δ itself is by far the prime determinant of fidelity for single base errors. However, the rate of deleting multiple nucleotides between directly repeated sequences is reduced by ∼10-fold in the presence of either RPA or PCNA, and by ≥90-fold when both proteins are present. We suggest that PCNA and RPA suppress large deletion errors by preventing the primer terminus at a repeat from fraying and/or from relocating and annealing to a downstream repeat. Strong suppression of deletions by PCNA and RPA suggests that they may contribute to the high replication fidelity needed to stably maintain eukaryotic genomes that contain abundant repetitive sequences.