The absence of ribonuclease H1 or H2 alters the sensitivity of Saccharomyces cerevisiae to hydroxyurea, caffeine and ethyl methanesulphonate: implications for roles of RNases H in DNA replication and repair

Arudchandran, Arulvathani; Cerritelli, Susana M.; Narimatsu, Scott; Itaya, Mitsuhiro; Shin, Deug-Yong; Shimada, Yuji; Crouch, Robert J.

doi:10.1046/j.1365-2443.2000.00373.x

Cited by 121 publications

(130 citation statements)

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“…Considering the evolutionary position of these organisms, the RNase H genes of A. aeolicus and Chlamydophila perhaps represent an ancestral structure and the BC type is perhaps more primordial than the AB type. The main function of RNase H is to remove RNA primers from Okazaki fragments, process R loops to modulate replication initiation and restore DNA topology (Broccoli et al, 2004;Kogoma & Foster 1998;Arudchandran et al, 2000). Recently, a mitochondrial RNase H from the parasitic protozoon Leishmania was analysed, which is essential for the parasite's survival (Misra et al, 2005).…”

Section: Discussionmentioning

confidence: 99%

Biochemical characterization and functional complementation of ribonuclease HII and ribonuclease HIII from Chlamydophila pneumoniae AR39

et al. 2007

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Chlamydophila pneumoniae AR39 contains two different ORFs (CP0654 and CP0782) encoding ribonuclease H (RNase H) homologues, Cpn-RNase HII and Cpn-RNase HIII. Sequence alignments show that the two homologues both contain the conserved motifs of type 2 RNase H, and Cpn-RNase HII has the conserved active-site motif (DEDD) of RNase HII. Cpn-RNase HIII also contains a unique active-site motif (DEDE), common to other RNase HIIIs. Complementation assays indicated that Cpn-RNase HII can complement both Escherichia coli RNase HII and RNase HI, but Cpn-RNase HIII can only complement the latter. In vitro enzyme activity experiments showed that neither Cpn-RNase HII nor Cpn-RNase HIII is thermostable and their optimum pH values were 9.0 and 10.0, respectively. Cpn-RNase HII cleaves a 12 bp RNA-DNA substrate at multiple sites, but Cpn-RNase HIII at only one site. When a 35 bp DNA-RNA-DNA/DNA chimeric substrate was used, cleavage was only observed with Cpn-RNase HII. These results indicate that the RNase H combination of C. pneumoniae AR39 is not simple substitution of E. coli RNase H, perhaps representing a more primordial type. This is believed to be the first in vivo functional study of Chlamydophila RNase Hs and the results should cntribute to the analysis of RNase Hs of other parasite species.

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Section: Discussionmentioning

confidence: 99%

Biochemical characterization and functional complementation of ribonuclease HII and ribonuclease HIII from Chlamydophila pneumoniae AR39

et al. 2007

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“…However, evidence for this function for RNase H type 2 enzymes in vivo is lacking and, as an alternative pathway, removal of Okazaki primers is believed to be efficiently accomplished by strand displacement followed by cutting by FEN-1, thus not requiring the activity of the RNase H enzyme in the process (23). An alternative view that these enzymes are involved in removal of misincorporated ribose in DNA has been expressed previously (6,28,29). In particular, early work by Eder and Walder (28) shows that purified human RNase H1 (a type 2 enzyme) is able to incise at the site of a single ribose, as opposed to E. coli RNase H1 (a type 1 enzyme), which requires a stretch of at least 4 ribose nt for activity.…”

Section: Rnase H Enzymes and Their Proposedmentioning

confidence: 99%

“…A search of current databases identifies Ͼ100 RNase H type 2 enzymes with significant sequence homology from Rickettsia to Homo sapiens, indicating an important biological function. The phenotypic properties of RNase H(35) type 2 knockout cells of the yeast S. cerevisiae have recently been studied (23,29,30). Such knockout cells have normal growth characteristics, arguing against an essential role for the enzyme in DNA replication.…”

Section: Rnase H Enzymes and Their Proposedmentioning

confidence: 99%

Excision of misincorporated ribonucleotides in DNA by RNase H (type 2) and FEN-1 in cell-free extracts

Rydberg

Game

2002

Proc. Natl. Acad. Sci. U.S.A.

172

167

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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...

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“…3 reveals that a rNMP in a DNA template strand slows synthesis by a polymerase that participates in leading strand DNA replication (21). This may be related to the finding that deletion of RNase H(35) increases the sensitivity of S. cerevisiae to hydroxyurea (32), an inhibitor of replication that reduces dNTP pools and alters the dNTP∶rNTP ratio.…”

mentioning

confidence: 95%

Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases

McElhinny

Watts

Kumar

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

375

582

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Measurements of nucleoside triphosphate levels in Saccharomyces cerevisiae reveal that the four rNTPs are in 36-to 190-fold molar excess over their corresponding dNTPs. During DNA synthesis in vitro using the physiological nucleoside triphosphate concentrations, yeast DNA polymerase ε, which is implicated in leading strand replication, incorporates one rNMP for every 1,250 dNMPs. Pol δ and Pol α, which conduct lagging strand replication, incorporate one rNMP for every 5,000 or 625 dNMPs, respectively. Discrimination against rNMP incorporation varies widely, in some cases by more than 100-fold, depending on the identity of the base and the template sequence context in which it is located. Given estimates of the amount of replication catalyzed by Pols α, δ, and ε, the results are consistent with the possibility that more than 10,000 rNMPs may be incorporated into the nuclear genome during each round of replication in yeast. Thus, rNMPs may be the most common noncanonical nucleotides introduced into the eukaryotic genome. Potential beneficial and negative consequences of abundant ribonucleotide incorporation into DNA are discussed, including the possibility that unrepaired rNMPs in DNA could be problematic because yeast DNA polymerase ε has difficulty bypassing a single rNMP present within a DNA template.DNA replication | nucleotide precursors | nucleotide selectivity T he integrity of the eukaryotic genome is ensured in part by the chemical nature of the storage medium-DNA. Compared to RNA, DNA is inherently more resistant to strand cleavage due to the absence of a reactive 2′ hydroxyl on the ribose ring. The active sites of most DNA polymerases are evolved to efficiently exclude ribonucleoside triphosphates (rNTPs) from being incorporated during DNA synthesis (reviewed in (1)). However, rNTP exclusion is not absolute. Early studies (reviewed in (1, 2)) revealed that DNA polymerases do incorporate rNMPs during DNA synthesis. Kinetic studies (3-13) have further demonstrated that selectivity for insertion of dNMPs into DNA rather than rNMPs varies from 10-fold to >10 6 -fold, depending on the DNA polymerase and the dNTP/rNTP pair examined. rNMP incorporation during DNA synthesis is potentially made more probable by the fact that the concentrations of rNTPs in vivo are higher than are the concentrations of dNTPs (e.g., see refs. 2, 14 and results of this study). Thus some rNMPs are likely to be stably incorporated into DNA during replication, and possibly during DNA repair, e.g., nonhomologous end joining (NHEJ) of double strand breaks in DNA (9, 15). This possibility is supported by biochemical studies implicating RNase H2 and FEN1 in the repair of single ribonucleotides in DNA (16,17). It is therefore of interest to know just how frequently rNMPs are incorporated into DNA by the DNA polymerases that synthesize the most DNA in a eukaryotic cell, namely DNA polymerases α, δ, and ε. Here we investigate this by first measuring the rNTP and dNTP concentrations in budding yeast. We then use these concentrations in DNA sy...

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The absence of ribonuclease H1 or H2 alters the sensitivity of Saccharomyces cerevisiae to hydroxyurea, caffeine and ethyl methanesulphonate: implications for roles of RNases H in DNA replication and repair

Abstract: Background: RNA of RNA-DNA hybrids can be degraded by ribonucleases H present in all organisms including the eukaryote Saccharomyces cerevisiae. Determination of the number and roles of the RNases H in eukaryotes is quite feasible in S. cerevisiae.

Cited by 121 publications

References 49 publications

Biochemical characterization and functional complementation of ribonuclease HII and ribonuclease HIII from Chlamydophila pneumoniae AR39

Biochemical characterization and functional complementation of ribonuclease HII and ribonuclease HIII from Chlamydophila pneumoniae AR39

Excision of misincorporated ribonucleotides in DNA by RNase H (type 2) and FEN-1 in cell-free extracts

Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases

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