Role of RNase H enzymes in maintaining genome stability in Escherichia coli expressing a steric-gate mutant of pol VICE391

pol V_ICE391 (RumAʹ₂B) is a low-fidelity polymerase that promotes considerably higher levels of spontaneous “SOS-induced” mutagenesis than the related E. coli pol V (UmuDʹ₂C). The molecular basis for the enhanced mutagenesis was previously unknown. Using single molecule fluorescence microscopy to visualize pol V enzymes, we discovered that the elevated levels of mutagenesis are likely due, in part, to prolonged binding of RumB to genomic DNA leading to increased levels of DNA synthesis compared to UmuC.We have generated a steric gate pol V_ICE391 variant (pol V_ICE391_Y13A) that readily misincorporates ribonucleotides into the E. coli genome and have used the enzyme to investigate the molecular mechanisms of Ribonucleotide Excision Repair (RER) under conditions of increased ribonucleotide-induced stress. To do so, we compared the extent of spontaneous mutagenesis promoted by pol V and pol V_ICE391 to that of their respective steric gate variants. Levels of mutagenesis promoted by the steric gate variants that are lower than that of the wild-type enzyme are indicative of active RER that removes misincorporated ribonucleotides, but also misincorporated deoxyribonucleotides from the genome.Using such an approach, we confirmed that RNase HII plays a pivotal role in RER. In the absence of RNase HII, Nucleotide Excision Repair (NER) proteins help remove misincorporated ribonucleotides. However, significant RER occurs in the absence of RNase HII and NER. Most of the RNase HII and NER-independent RER occurs on the lagging strand during genome duplication. We suggest that this is most likely due to efficient RNase HI-dependent RER which recognizes the polyribonucleotide tracts generated by pol V_ICE391_Y13A. These activities are critical for the maintenance of genomic integrity when RNase HII is overwhelmed, or inactivated, as ΔrnhB or ΔrnhB ΔuvrA strains expressing pol V_ICE391_Y13A exhibit genome and plasmid instability in the absence of RNase HI.     

Mechanisms Employed by Escherichia coli to Prevent Ribonucleotide Incorporation into Genomic DNA by Pol V

Escherichia coli pol V (UmuD′₂C), the main translesion DNA polymerase, ensures continued nascent strand extension when the cellular replicase is blocked by unrepaired DNA lesions. Pol V is characterized by low sugar selectivity, which can be further reduced by a Y11A “steric-gate” substitution in UmuC that enables pol V to preferentially incorporate rNTPs over dNTPs in vitro. Despite efficient error-prone translesion synthesis catalyzed by UmuC_Y11A in vitro, strains expressing umuC_Y11A exhibit low UV mutability and UV resistance. Here, we show that these phenotypes result from the concomitant dual actions of Ribonuclease HII (RNase HII) initiating removal of rNMPs from the nascent DNA strand and nucleotide excision repair (NER) removing UV lesions from the parental strand. In the absence of either repair pathway, UV resistance and mutagenesis conferred by umuC_Y11A is significantly enhanced, suggesting that the combined actions of RNase HII and NER lead to double-strand breaks that result in reduced cell viability. We present evidence that the Y11A-specific UV phenotype is tempered by pol IV in vivo. At physiological ratios of the two polymerases, pol IV inhibits pol V–catalyzed translesion synthesis (TLS) past UV lesions and significantly reduces the number of Y11A-incorporated rNTPs by limiting the length of the pol V–dependent TLS tract generated during lesion bypass in vitro. In a recA730 lexA(Def) ΔumuDC ΔdinB strain, plasmid-encoded wild-type pol V promotes high levels of spontaneous mutagenesis. However, umuC_Y11A-dependent spontaneous mutagenesis is only ∼7% of that observed with wild-type pol V, but increases to ∼39% of wild-type levels in an isogenic ΔrnhB strain and ∼72% of wild-type levels in a ΔrnhA ΔrnhB double mutant. Our observations suggest that errant ribonucleotides incorporated by pol V can be tolerated in the E. coli genome, but at the cost of higher levels of cellular mutagenesis.     

Ribonucleotides as nucleotide excision repair substrates

The incorporation of ribonucleotides in DNA has attracted considerable notice in recent years, since the pool of ribonucleotides can exceed that of the deoxyribonucleotides by at least 10–20-fold, and single ribonucleotide incorporation by DNA polymerases appears to be a common event. Moreover ribonucleotides are potentially mutagenic and lead to genome instability. As a consequence, errantly incorporated ribonucleotides are rapidly repaired in a process dependent upon RNase H enzymes. On the other hand, global genomic nucleotide excision repair (NER) in prokaryotes and eukaryotes removes damage caused by covalent modifications that typically distort and destabilize DNA through the production of lesions derived from bulky chemical carcinogens, such as polycyclic aromatic hydrocarbon metabolites, or via crosslinking. However, a recent study challenges this lesion-recognition paradigm. The work of Vaisman et al. (2013) [34] reveals that even a single ribonucleotide embedded in a deoxyribonucleotide duplex is recognized by the bacterial NER machinery in vitro. In their report, the authors show that spontaneous mutagenesis promoted by a steric-gate pol V mutant increases in uvrA, uvrB, or uvrC strains lacking rnhB (encoding RNase HII) and to a greater extent in an NER-deficient strain lacking both RNase HI and RNase HII. Using purified UvrA, UvrB, and UvrC proteins in in vitro assays they show that despite causing little distortion, a single ribonucleotide embedded in a DNA duplex is recognized and doubly-incised by the NER complex. We present the hypothesis to explain the recognition and/or verification of this small lesion, that the critical 2′-OH of the ribonucleotide – with its unique electrostatic and hydrogen bonding properties – may act as a signal through interactions with amino acid residues of the prokaryotic NER complex that are not possible with DNA. Such a mechanism might also be relevant if it were demonstrated that the eukaryotic NER machinery likewise incises an embedded ribonucleotide in DNA.     

Redundancy in ribonucleotide excision repair: Competition,compensation, and cooperation

The survival of all living organisms is determined by their ability to reproduce, which in turn depends on accurate duplication of chromosomal DNA. In order to ensure the integrity of genome duplication, DNA polymerases are equipped with stringent mechanisms by which they select and insert correctly paired nucleotides with a deoxyribose sugar ring. However, this process is never 100% accurate. To fix occasional mistakes, cells have evolved highly sophisticated and often redundant mechanisms. A good example is mismatch repair (MMR), which corrects the majority of mispaired bases and which has been extensively studied for many years. On the contrary, pathways leading to the replacement of nucleotides with an incorrect sugar that is embedded in chromosomal DNA have only recently attracted significant attention. This review describes progress made during the last few years in understanding such pathways in both prokaryotes and eukaryotes. Genetic studies in Escherichia coli and Saccharomyces cerevisiae demonstrated that MMR has the capacity to replace errant ribonucleotides, but only when the base is mispaired. In contrast, the major evolutionarily conserved ribonucleotide repair pathway initiated by the ribonuclease activity of type 2 Rnase H has broad specificity. In yeast, this pathway also requires the concerted action of Fen1 and pol δ, while in bacteria it can be successfully completed by DNA polymerase I. Besides these main players, all organisms contain alternative enzymes able to accomplish the same tasks, although with differing efficiency and fidelity. Studies in bacteria have very recently demonstrated that isolated rNMPs can be removed from genomic DNA by error-free nucleotide excision repair (NER), while studies in yeast suggest the involvement of topoisomerase 1 in alternative mutagenic ribonucleotide processing. This review summarizes the most recent progress in understanding the ribonucleotide repair mechanisms in prokaryotes and eukaryotes.     

Cleavage of a DNA-RNA-DNA/DNA chimeric substrate containing a single ribonucleotide at the DNA-RNA junction with prokaryotic RNases HII

We have analyzed the cleavage specificities of various prokaryotic Type 2 ribonucleases H (RNases H) on chimeric DNA-RNA-DNA/DNA substrates containing one to four ribonucleotides. RNases HII from Bacillus subtilis and Thermococcus kodakaraensis cleaved all of these substrates to produce a DNA segment with a 5'-monoribonucleotide. Consequently, these enzymes cleaved even the chimeric substrate containing a single ribonucleotide at the DNA-RNA junction (5'-side of the single ribonucleotide). In contrast, Escherichia coli RNase HI and B. subtilis RNase HIII did not cleave the chimeric substrate containing a single ribonucleotide. These results suggest that bacterial and archaeal RNases HII are involved in excision of a single ribonucleotide misincorporated into DNA.     

Rotational and translational positions determine the structural and dynamic impact of a single ribonucleotide incorporated in the nucleosome

Ribonucleotides misincorporated by replicative DNA polymerases are by far the most common DNA lesion. The presence of ribonucleotides in DNA is associated with genome instability, causing replication stress, chromosome fragility, gross chromosomal rearrangements, and other mutagenic events. Furthermore, nucleosome and chromatin assembly as well as nucleosome positioning are affected by the presence of ribonucleotides. Notably, nucleosome formation is significantly reduced by a single ribonucleotide. Single ribonucleotides are primarily removed from DNA by the ribonucleotide excision repair (RER) pathway via the RNase H2 enzyme, which incises the DNA backbone on the 5′-side of the ribonucleotide. While the structural implications of a single ribonucleotide in free duplex DNA have been well studied, how a single ribonucleotide embedded in nucleosomal DNA impacts nucleosome structure and dynamics, and the possible consequent impact on RER, have not been explored. We have carried out 3.5 μs molecular dynamics simulations of a single ribonucleotide incorporated at various translational and rotational positions in a nucleosome core particle. We find that the presence of the 2′−OH group on the ribose impacts the local conformation and dynamics of both the ribonucleotide and nearby DNA nucleotides as well as their interactions with histones; the nature of these disturbances depends on the rotational and translational setting, including whether the ribose faces toward or away from the histones. The ribonucleotide’s preferred C3′-endo pucker is stabilized by interactions with the histones, and furthermore the ribonucleotide can cause dynamic local duplex disturbance involving an abnormal C3′-endo population of the adjacent deoxyribose pucker, minor groove opening, ruptured Watson-Crick pairing, and duplex unwinding that are governed by translation-dependent histone-nucleotide interactions. Possible effects of these disturbances on RER are considered.     

Formation and Repair of Mismatches Containing Ribonucleotides and Oxidized Bases at Repeated DNA Sequences

The cellular pool of ribonucleotide triphosphates (rNTPs) is higher than that of deoxyribonucleotide triphosphates. To ensure genome stability, DNA polymerases must discriminate against rNTPs and incorporated ribonucleotides must be removed by ribonucleotide excision repair (RER). We investigated DNA polymerase β (POL β) capacity to incorporate ribonucleotides into trinucleotide repeated DNA sequences and the efficiency of base excision repair (BER) and RER enzymes (OGG1, MUTYH, and RNase H2) when presented with an incorrect sugar and an oxidized base. POL β incorporated rAMP and rCMP opposite 7,8-dihydro-8-oxoguanine (8-oxodG) and extended both mispairs. In addition, POL β was able to insert and elongate an oxidized rGMP when paired with dA. We show that RNase H2 always preserves the capacity to remove a single ribonucleotide when paired to an oxidized base or to incise an oxidized ribonucleotide in a DNA duplex. In contrast, BER activity is affected by the presence of a ribonucleotide opposite an 8-oxodG. In particular, MUTYH activity on 8-oxodG:rA mispairs is fully inhibited, although its binding capacity is retained. This results in the reduction of RNase H2 incision capability of this substrate. Thus complex mispairs formed by an oxidized base and a ribonucleotide can compromise BER and RER in repeated sequences.     

Analysis of Ribonucleotide Removal from DNA by Human Nucleotide Excision Repair

Ribonucleotides are incorporated into the genome during DNA replication. The enzyme RNase H2 plays a critical role in targeting the removal of these ribonucleotides from DNA, and defects in RNase H2 activity are associated with both genomic instability and the human autoimmune/inflammatory disorder Aicardi-Goutières syndrome. Whether additional general DNA repair mechanisms contribute to ribonucleotide removal from DNA in human cells is not known. Because of its ability to act on a wide variety of substrates, we examined a potential role for canonical nucleotide excision repair in the removal of ribonucleotides from DNA. However, using highly sensitive dual incision/excision assays, we find that ribonucleotides are not efficiently targeted by the human nucleotide excision repair system in vitro or in cultured human cells. These results suggest that nucleotide excision repair is unlikely to play a major role in the cellular response to ribonucleotide incorporation in genomic DNA in human cells.     

Involvement of Ser94 in RNase HIII from Chlamydophila pneumoniae in the recognition of a single ribonucleotide misincorporated into double-stranded DNA

We recently provided the first report that RNase HIII can cleave a DNA-rN₁-DNA/DNA substrate (rN₁, one ribonucleotide) in vitro. In the present study, mutagenesis analyses and molecular dynamics (MD) simulations were performed on RNase HIII from Chlamydophila pneumoniae AR39 (CpRNase HIII). Our results elucidate the mechanism of ribonucleotide recognition employed by CpRNase HIII, indicating that the G95/K96/G97 motif of CpRNase HIII represents the main surface interacting with single ribonucleotides, in a manner similar to that of the GR(K)G motif of RNase HIIs. However, CpRNase HIII lacks the specific tyrosine required for RNase HII to recognize single ribonucleotides in double-stranded DNA (dsDNA). Interestingly, MD shows that Ser94 of CpRNase HIII forms a stable hydrogen bond with the deoxyribonucleotide at the (5')RNA–DNA(3') junction, moving this nucleotide away from the chimeric ribonucleotide. This movement appears to deform the nucleic acid backbone at the RNA–DNA junction and allows the ribonucleotide to interact with the GKG motif. Based on the inferences drawn from MD simulations, biochemical results indicated that Ser94 was necessary for catalytic activity on the DNA-rN₁-DNA/DNA substrate; mutant S94V could bind this substrate but exhibited no cleavage. Mismatches opposite the single ribonucleotide misincorporated in dsDNA inhibited cleavage by CpRNase HIII to varying degrees but did not interfere with CpRNase/substrate binding. Further MD results implied that mismatches impair the interaction between Ser94 and the deoxyribonucleotide at the RNA–DNA junction. Consequently, recognition of the misincorporated ribonucleotide was disturbed. Our results may help elucidate the distinct substrate-recognition properties of different RNase Hs.     

Accessibility of DNA polymerases to repair synthesis during nucleotide excision repair in yeast cell-free extracts

Nucleotide excision repair (NER) removes a variety of DNA lesions. Using a yeast cell-free repair system, we have analyzed the repair synthesis step of NER. NER was proficient in yeast mutant cell-free extracts lacking DNA polymerases (Pol) β, ζ or η. Base excision repair was also proficient without Polβ. Repair synthesis of NER was not affected by thermal inactivation of the temperature-sensitive mutant Polα (pol1-17), but was reduced after thermal inactivation of the temperature-sensitive mutant Polδ (pol3-1) or Pol (pol2-18). Residual repair synthesis was observed in pol3-1 and pol2-18 mutant extracts, suggesting a repair deficiency rather than a complete repair defect. Deficient NER in pol3-1 and pol2-18 mutant extracts was specifically complemented by purified yeast Polδ and Pol, respectively. Deleting the polymerase catalytic domain of Pol (pol2-16) also led to a deficient repair synthesis during NER, which was complemented by purified yeast Pol, but not by purified yeast Polη. These results suggest that efficient repair synthesis of yeast NER requires both Polδ and Pol in vitro, and that the low fidelity Polη is not accessible to repair synthesis during NER.     

Differential Activities of DNA Polymerases in Processing Ribonucleotides during DNA Synthesis in Archaea

Consistent with the fact that ribonucleotides (rNTPs) are in excess over deoxyribonucleotides (dNTPs) in vivo, recent findings indicate that replicative DNA polymerases (DNA Pols) are able to insert ribonucleotides (rNMPs) during DNA synthesis, raising crucial questions about the fidelity of DNA replication in both Bacteria and Eukarya. Here, we report that the level of rNTPs is 20-fold higher than that of dNTPs in Pyrococcus abyssi cells. Using dNTP and rNTP concentrations present in vivo, we recorded rNMP incorporation in a template-specific manner during in vitro synthesis, with the family-D DNA Pol (PolD) having the highest propensity compared with the family-B DNA Pol and the p41/p46 complex. We also showed that ribonucleotides accumulate at a relatively high frequency in the genome of wild-type Thermococcales cells, and this frequency significantly increases upon deletion of RNase HII, the major enzyme responsible for the removal of RNA from DNA. Because ribonucleotides remain in genomic DNA, we then analyzed the effects on polymerization activities by the three DNA Pols. Depending on the identity of the base and the sequence context, all three DNA Pols bypass rNMP-containing DNA templates with variable efficiency and nucleotide (mis)incorporation ability. Unexpectedly, we found that PolD correctly base-paired a single ribonucleotide opposite rNMP-containing DNA templates. An evolutionary scenario is discussed concerning rNMP incorporation into DNA and genome stability.     

Proofreading of ribonucleotides inserted into DNA by yeast DNA polymerase ɛ

We have investigated the ability of the 3′ exonuclease activity of Saccharomyces cerevisiae DNA polymerase ? (Pol ?) to proofread newly inserted ribonucleotides (rNMPs). During DNA synthesis in vitro, Pol ? proofreads ribonucleotides with apparent efficiencies that vary from none at some locations to more than 90% at others, with rA and rU being more efficiently proofread than rC and rG. Previous studies show that failure to repair ribonucleotides in the genome of rnh201Δ strains that lack RNase H2 activity elevates the rate of short deletions in tandem repeat sequences. Here we show that this rate is increased by 2–4-fold in pol2-4 rnh201Δ strains that are also defective in Pol ? proofreading. In comparison, defective proofreading in these same strains increases the rate of base substitutions by more than 100-fold. Collectively, the results indicate that although proofreading of an ‘incorrect’ sugar is less efficient than is proofreading of an incorrect base, Pol ? does proofread newly inserted rNMPs to enhance genome stability.     

Alternative Excision Repair Pathways

Alternative excision repair (AER) is a category of excision repair initiated by a single nick, made by an endonuclease, near the site of DNA damage, and followed by excision of the damaged DNA, repair synthesis, and ligation. The ultraviolet (UV) damage endonuclease in fungi and bacteria introduces a nick immediately 5′ to various types of UV damage and initiates its excision repair that is independent of nucleotide excision repair (NER). Endo IV-type apurinic/apyrimidinic (AP) endonucleases from Escherichia coli and yeast and human Exo III-type AP endonuclease APEX1 introduce a nick directly and immediately 5′ to various types of oxidative base damage besides the AP site, initiating excision repair. Another endonuclease, endonuclease V from bacteria to humans, binds deaminated bases and cleaves the phosphodiester bond located 1 nucleotide 3′ of the base, leading to excision repair. A single-strand break in DNA is one of the most frequent types of DNA damage within cells and is repaired efficiently. AER makes use of such repair capability of single-strand breaks, removes DNA damage, and has an important role in complementing BER and NER.NER and base excision repair (BER) are the major excision repair pathways present in almost all organisms. In NER, dual incisions are introduced, the damaged DNA between the incised sites is then removed, and DNA synthesis fills the single-stranded gap, followed by ligation. In BER, an AP site, formed by depurination or created by a base damage-specific DNA glycosylase, is recognized by an AP endonuclease that introduces a nick immediately 5′ to the AP site, followed by repair synthesis, removal of the AP site, and final ligation. Besides these two fundamental excision repair systems, investigators have found another category of excision repair—AER—an example of which is the excision repair of UV damage, initiated by an endonuclease called UV damage endonuclease (UVDE). UVDE introduces a single nick immediately 5′ to various types of UV lesions as well as other types of base damage, and this nick leads to the removal of the lesions by an AER process designated as UVDE-mediated excision repair (UVER or UVDR). Genetic analysis in Schizosaccharomyces pombe indicates that UVER provides cells with an extremely rapid removal of UV lesions, which is important for cells exposed to UV in their growing phase.Endo IV–type AP endonucleases from Escherichia coli and budding yeast and the Exo III–type human AP endonuclease APEX1 are able to introduce a nick at various types of oxidative base damage and initiate a form of excision repair that has been designated as nucleotide incision repair (NIR). Endonuclease V (ENDOV) from bacteria to humans recognizes deaminated bases, introduces a nick 1 nucleotide 3′ of the base, and leads to excision repair initiated by the nick. These endonucleases introduce a single nick near the DNA-damage site, leaving 3′-OH termini, and initiate repair of both the DNA damage and the nick. The mechanisms of AER may be similar to those of single-strand break (SSB) repair or BER except for the initial nicking process. However, how DNA damage is recognized determines the repair process within the cell. This article discusses the mechanisms and functional roles of AER. We begin with AER of UV damage, because genetic analysis has shown functional differences between this AER and NER in S. pombe.     

Persistently stalled replication forks inhibit nucleotide excision repair in trans by sequestering Replication protein A

Rev3, the catalytic subunit of DNA polymerase ζ, is essential for translesion synthesis of cytotoxic DNA photolesions, whereas the Rev1 protein plays a noncatalytic role in translesion synthesis. Here, we reveal that mammalian Rev3^−/− and Rev1^−/− cell lines additionally display a nucleotide excision repair (NER) defect, specifically during S phase. This defect is correlated with the normal recruitment but protracted persistence at DNA damage sites of factors involved in an early stage of NER, while repair synthesis is affected. Remarkably, the NER defect becomes apparent only at 2 h post-irradiation indicating that Rev3 affects repair synthesis only indirectly, rather than performing an enzymatic role in NER. We provide evidence that the NER defect is caused by scarceness of Replication protein A (Rpa) available to NER, resulting from its sequestration at stalled replication forks. Also the induction of replicative stress using hydroxyurea precludes the accumulation of Rpa at photolesion sites, both in Rev3^−/− and in wild-type cells. These data support a model in which the limited Rpa pool coordinates replicative stress and NER, resulting in increased cytotoxicity of ultraviolet light when replicative stress exceeds a threshold.     

Involvement of Ser94 in RNase HIII from Chlamydophila pneumoniae in the recognition of a single ribonucleotide misincorporated into double-stranded DNA

We recently provided the first report that RNase HIII can cleave a DNA-rN(1)-DNA/DNA substrate (rN(1), one ribonucleotide) in vitro. In the present study, mutagenesis analyses and molecular dynamics (MD) simulations were performed on RNase HIII from Chlamydophila pneumoniae AR39 (CpRNase HIII). Our results elucidate the mechanism of ribonucleotide recognition employed by CpRNase HIII, indicating that the G95/K96/G97 motif of CpRNase HIII represents the main surface interacting with single ribonucleotides, in a manner similar to that of the GR(K)G motif of RNase HIIs. However, CpRNase HIII lacks the specific tyrosine required for RNase HII to recognize single ribonucleotides in double-stranded DNA (dsDNA). Interestingly, MD shows that Ser94 of CpRNase HIII forms a stable hydrogen bond with the deoxyribonucleotide at the (5')RNA-DNA(3') junction, moving this nucleotide away from the chimeric ribonucleotide. This movement appears to deform the nucleic acid backbone at the RNA-DNA junction and allows the ribonucleotide to interact with the GKG motif. Based on the inferences drawn from MD simulations, biochemical results indicated that Ser94 was necessary for catalytic activity on the DNA-rN(1)-DNA/DNA substrate; mutant S94V could bind this substrate but exhibited no cleavage. Mismatches opposite the single ribonucleotide misincorporated in dsDNA inhibited cleavage by CpRNase HIII to varying degrees but did not interfere with CpRNase/substrate binding. Further MD results implied that mismatches impair the interaction between Ser94 and the deoxyribonucleotide at the RNA-DNA junction. Consequently, recognition of the misincorporated ribonucleotide was disturbed. Our results may help elucidate the distinct substrate-recognition properties of different RNase Hs.