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

Abstract

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...

Ribonuclease H2 mutations induce a cGAS/STING‐dependent innate immune response

“…Although the exact in vivo substrates of these nucleases remain to be identified, they are thought to act on RNA:DNA hybrids such as those arising during nuclear DNA replication and R‐loop formation (Cerritelli & Crouch, 2009). Unlike RNase H1, RNase H2 also cleaves and initiates the removal of single ribonucleotides embedded in DNA (Eder et al , 1993; Rydberg & Game, 2002), a process known as ribonucleotide excision repair (RER) (Sparks et al , 2012). RNase H2 is essential for mammalian genome stability, with complete loss of RNase H2 resulting in embryonic lethality (Reijns et al , 2012; Hiller et al , 2012).…”

lmo1273, a novel gene involved in Listeria monocytogenes virulence

“…However, only RNase HII is capable of removing single RNA from DNA-RNA/DNA-DNA hybrids, such as those produced when RNA is misincorporated into DNA (Haruki et al, 2002;Rydberg & Game, 2002). A recent extensive phylogenetic analysis of 353 prokaryotic genomes revealed that the RNase H group has evolved in such a way that genes encoding RNase HI and RNase HIII are inherited in a mutually exclusive manner, which may be attributed to the functional redundancy of the two enzymes.…”

Roles of DNA helicases and Exo1 in the avoidance of mutations induced by Top1-mediated cleavage at ribonucleotides in DNA

“…In the case of rad27D, which affects the processing of RNA primers in Okazaki fragments, the increase in slippage mutations is expected based on the fact that there are more rNMPs remaining in the DNA of mutant cells. 4 Accordingly, the double mutant rnh202D rad27D displayed a synergistic increase in slippage mutations, indicative of overlap in function in removing rNMPs from DNA. The pol3-01 mutant gave an increase in mutation slippage in an RNaseH2-proficient strain, confirming the role of the Pol3 editing function in mutation prevention.…”

“…Mutation rates for the (AG) 4 slippage reporter and Can r were determined by the Lea and Coulson median method as previously described. 1,2,18 For the (AG) 4 slippage reporter, single colonies were inoculated into 5 mL YPD and grown for 2 d at 30 C, washed and resuspended in 1 mL H 2 O.…”

“…1,2,18 For the (AG) 4 slippage reporter, single colonies were inoculated into 5 mL YPD and grown for 2 d at 30 C, washed and resuspended in 1 mL H 2 O. Cells were plated onto SC-lysine medium to select for slippage mutants and onto SC medium for total cell number.…”