Misincorporated ribonucleotides in DNA will cause DNA backbone distortion and may be targeted by DNA repair enzymes. Using double-stranded oligonucleotide probes containing a single ribose, we demonstrate a robust activity in human, yeast, and Escherichia coli cell-free extracts that nicks 5 of the ribose. The human and yeast extracts also make a subsequent cut 3 of the ribonucleotide releasing a ribonucleotide monophosphate. The resulting 1-nt gap is an ideal substrate for polymerase and ligase to complete a proposed repair sequence that effectively replaces the ribose with deoxyribose. T here is presently a nearly total lack of information about repair of deoxyribose modifications in DNA. Such modifications can be caused by external agents, such as oxidizing agents and ionizing radiation (1-3), and can also occur naturally by misincorporation of ribonucleotides into DNA during DNA replication (4). The presence of ribose in DNA is a hindrance to formation of normal B form DNA as evidenced by the structure of RNA͞DNA hybrid molecules (5), and consequently a single ribose in DNA will result in a local DNA backbone distortion (6). Other bulky modified sugars are also likely to cause backbone distortions, and it can be hypothesized that they pose a hindrance for DNA polymerases and can be mutagenic.Progressive DNA and RNA polymerases are similar in structure and belong to the same class of proteins (4), probably with a common evolutionary origin (7). The specificity toward deoxyribonucleoside triphosphates (dNTPs) or ribonucleoside triphosphates (rNTPs) has been found to be determined by subtle differences at the active site (4). Gao et al. (8) could largely eliminate the discrimination between the rNTPs and dNTPs by introducing a single amino acid change in a reverse transcriptase, and similar observations in mutant polymerases have recently been made by several investigators (7, 9-13). On the basis of such observations, it has been suggested that the discrimination against ribonucleotides by DNA polymerases is largely accomplished by a ''steric gate'' that will not give enough space for the 2Ј hydroxyl group present in rNTPs (4). However, the discrimination against rNTPs is not 100%, and detectable incorporation of rNTPs has been found in vitro by using purified DNA polymerases with a wide variety of discrimination factors ranging from a few thousand-fold (7, 11) to several million-fold (13). However, it is presently not known to what extent ribonucleotides are misincorporated into DNA during normal in vivo DNA replication. The intranuclear milieu contains both ribonucleotides and deoxyribonucleotides, with the ribonucleotide concentration generally higher than the deoxyribonucleotide concentration (14). The deoxyribonucleotides are produced from the ribonucleotide pool by the enzyme ribonucleotide diphosphate reductase. This enzyme can be inhibited in eukaryotic cells by hydroxyurea (HU), which blocks DNA replication when given to cell cultures in sufficient concentration.Gao and Goff (15) mutagenized a viral p...

dUTP pyrophosphatase (dUTPase; EC 3.6.1.23) catalyses the hydrolysis of dUTP to dUMP and PPi and thereby prevents the incorporation of uracil into DNA during replication. Although it is widely believed that dUTPase is essential for cell viability because of this role, direct evidence supporting this assumption has not been presented for any eukaryotic system. We have analysed the role of dUTPase (DUT1) in the life cycle of yeast. Using gene disruption and tetrad analysis, we find that DUT1 is necessary for the viability of S. cerevisiae; however, under certain conditions dut1 null mutants survive if supplied with exogenous thymidylate (dTMP). Analyses with isogenic uracil‐DNA‐glycosylase (UNG1) deficient or proficient strains indicate that in the absence of dUTPase, cell death results from the incorporation of uracil into DNA and the attempted repair of this damage by UNG1‐mediated excision repair. However, in dut1 ung1 double mutants, starvation for dTMP causes dividing cells to arrest and die in all phases of the cell cycle. This latter effect suggests that the extensive stable substitution of uracil for thymine in DNA leads to a general failure in macromolecular synthesis. These results are in general agreement with previous models in thymine‐less death that implicate dUTP metabolism. They also suggest an alternative approach for chemotherapeutic drug design.

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Mammalian Bre1 complexes (BRE1A/B (RNF20/40) in humans and Bre1a/b (Rnf20/40) in mice) function similarly to their yeast homolog Bre1 as ubiquitin ligases in monoubiquitination of histone H2B. This ubiquitination facilitates methylation of histone H3 at K4 and K79, and accounts for the roles of Bre1 and its homologs in transcriptional regulation. Recent studies by others suggested that Bre1 acts as a tumor suppressor, augmenting expression of select tumor suppressor genes and suppressing select oncogenes. In this study we present an additional mechanism of tumor suppression by Bre1 through maintenance of genomic stability. We track the evolution of genomic instability in Bre1-deficient cells from replication-associated double-strand breaks (DSBs) to specific genomic rearrangements that explain a rapid increase in DNA content and trigger breakage-fusion-bridge cycles. We show that aberrant RNA-DNA structures (R-loops) constitute a significant source of DSBs in Bre1-deficient cells. Combined with a previously reported defect in homologous recombination, generation of R-loops is a likely initiator of replication stress and genomic instability in Bre1-deficient cells. We propose that genomic instability triggered by Bre1 deficiency may be an important early step that precedes acquisition of an invasive phenotype, as we find decreased levels of BRE1A/B and dimethylated H3K79 in testicular seminoma and in the premalignant lesion in situ carcinoma.