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

Abstract

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.

Keywords: DNA polymerase I; Flap endonuclease; Mismatch repair; Nucleotide excision repair; Ribonuclease H; Ribonucleotide excision repair.

Fig. 1. A model for Ribonucleotide Excision Repair

DNA replication catalyzed by an error-prone DNA polymerase might lead to occasional misinsertion of rNMPs along with erroneous bases (1). Type 2 RNase H recognizes a nucleotide with an extra 2'-OH group (2) and cleaves the phosphodiester bond at the RNA-DNA junction 5' to the rNMP (3). A DNA polymerase initiates strand-displacement DNA synthesis at the nick and the displaced single rNMP, or 5'-RNA-terminated DNA fragment is excised by the 5' nuclease activity either intrinsic to the polymerase or associated with a separate protein (4). When a DNA fragment in the vicinity of rNMP contains mispaired bases, its replacement by RER reduces the mutagenic consequences of DNA synthesis by an error-prone DNA polymerase. The DNA template left after completion of excision and re-synthesis steps contains a nick with 3'-hydroxyl and 5'-phosphate ends that is readily sealed by DNA ligase, thus completing the RER pathway (5). Mispaired bases outside the re-synthesis patch of RER can be replaced by the MMR. It has been proposed that in eukaryotes, a transient nick created during RER can serve as a signal that directs MMR to the mispaired base on the nascent strand.

Fig. 2. Processing of a nicked DNA substrate with a single ribonucleotide (U) in the 5' terminus of the nick by the combined action of a DNA polymerase and nuclease

A. Sequence of the oligonucleotides used to generate the nicked DNA substrate (also shown schematically to the right of each gel panel as “a”), consisting of the 49-mer DNA template (brown) with an abasic site (X) annealed with a 17-mer primer (red) and a 32-mer downstream blocker (blue). The template in which either primer, or blocker, is P³²-labeled (indicated by the star, *), together with the purified recombinant human DNA polymerase (pol β, pol η, or pol ι) and Fen1 protein was used in in vitro reactions B. P³²-labeling of the 17-mer primer (red *) allows for visualization of the primer extension products. The processivity of the strand displacement synthesis, decreasing in the order pol β> pol η> or pol τ□ can be judged by the intensity of the intermediate bands such as the 19-mer oligonucleotide, whose structure (“b”) is schematically shown to the right of the gel. The ~27 nt-long bands correspond to replication blockage by the abasic site on the DNA template (c), and the 49-mer band corresponds to the full-sized product generated by translesion replication to the end of the template (d). C. P³²- labeling of the 32-mer blocker (blue *) allows one to visualize products of the 5'→3' exo-(green ▼), and flap endonucleolytic (orange ▼) cleavage.

Fig. 3. Enzymatic properties of pol I on a nicked DNA substrate containing an abasic site (X) located in the template strand and a 5'-terminal mono-ribonucleotide in the downstream blocking oligonucleotide

A. The sequence of the oligonucleotides used to generate the various DNA substrates is essentially the same as indicated in Figure 1A. The structures of the DNA substrates are schematically presented on the top of each gel image (brown – template, red – primer, blue - blocker). B. The products of pol I-catalyzed strand displacement synthesis and 3'→5' exonucleolytic proofreading are detected using DNA substrates with the P³²-labeled 17-mer primer (red star, *). C. 5'-end P³²-labeling of the 32-mer blocker (blue star, *) is used to visualize the 5' nuclease activity of pol I. D. Labeling of the blocker at the 3' termini by addition of a single P³²-ATP (and converting the blocker into the 33-mer) allows for the detection of repair track synthesis. E. Schematic representation of DNA templates with arrows pointing to the cleavage sites on the DNA made by the 3'→5' exo- (purple Ξ in a), 5'→3' exo- (green ▼ in b²), and FLAP-endonuclease (orange ▼in b¹) activities of pol I. Lanes 1, 4, & 7 in each gel panel represent control reactions incubated in the absence of pol I. Pausing of strand-displacement DNA synthesis after incorporation of 1 or 2 nucleotides (indicated by the red arrows, lanes 2 & 3 on the panel B) is accompanied by the release of the mono- or di-nucleotides catalyzed by the 5'→3'exonucleolytic activity of pol I (indicated by the green arrows, lanes 2 & 3, panel C, and illustrated on the scheme b², panel E). Blocking DNA synthesis with an abasic site (27-mer on the panel B) is accompanied by the emergence of the 3'- end labeled 21- and 22-mers (panel D). These bands are mainly formed as a result of 5'→3'exonucleolytic cleavage by pol I, although a low level contribution of FLAP-endonuclease cannot be excluded. The attempts of pol I to replicate past the lesion is largely overcome by the 3'→5' exonuclease proofreading (scheme a, panel E) as indicated by the emergence of the mono- or di-nucleotides (lanes 2 & 3, panel B), although a small amount of full-sized translesion replication products (49-mer) can also be seen. Futile cycling (bypass synthesis/3'→5' proofreading) causes release of a significant portion of the downstream blocker which is degraded by the 5' exo- and/or endonuclease activity of pol I, which is manifested by the 3'-end labeled 10-17-mer bands (panel D and scheme b², gray arrows). Introduction of one (lanes 4-6, Y=A) or two (lanes 7-9, X & Y=A) mismatched base pairs facilitates strand displacement and as a consequence, increases the processivity of DNA synthesis (manifested by the significant reduction of band intensities at the +1 and +2 positions, lanes 5, 6, 8, 9, panel B). For the template with the single mismatch, this results in a shift in the size of the pol I cleavage products from a 2-mer to 3-mer (indicated by the green arrows, lanes 5 & 6, panel C). Correspondingly, length of the 3'-end labeled blocker reduces to 27-28 nucleotides (panel D). At the same time, products of the FLAP-endonuclease activity also become more visible as 9-11 nucleotide long oligonucleotides in panel C (also illustrated on the scheme b¹, panel E). Products of the FLAP-endonuclease are much more prevalent with a DNA template containing two mispaired bases (lanes 8 & 9, panels C & D).

Fig. 4. Pathways for keeping genomic DNA free from errant ribonucleotides: from competition to cooperation

While copying the parental DNA strand (shown in yellow), a low fidelity DNA polymerase, such as E. coli pol V, besides the correct nucleotides (shown in pink), often incorporates nucleotides with wrong base (shown in red) and/or wrong sugar moiety (correctly paired ribonucleotides are indicated in light blue, mispaired rNMPs are indicated in dark blue). These mistakes can be corrected by multiple repair pathways with distinct but overlapping specificities. The cartoon illustrates interplay between those pathways schematically presented as colored boxes connected with the corresponding target sites by a set of arrows. The first responder to the errantly incorporated rNMPs is type 2 RNase H. This enzyme initiates the primary and most efficient pathway of rNMP repair (RER², blue). Type 2 RNase H preferentially cleaves DNA templates with a single ribonucleotide, although it is also able to incise templates containing multiple rNTPs. In contrast, type 1 RNase H initiates repair (RER¹, orange) that operates only on a tract of at least four sequential ribonucleotides within the DNA strand. Ribonucleotides that for some reason escape RER, can be excised by means of NER (green), most likely activated later than the RNase H-dependent pathway, or when it is impaired. All three pathways, RER², RER¹, and NER, are specifically triggered by the rNMPs, either correctly paired or mispaired, but concomitantly they can remove mispaired dNMPs positioned within the repair patch (indicated by the dashed lines). In contrast, coupled to replication MMR (crimson) is able to replace nucleotides with wrong sugar only when the base is also wrong. In this sense MMR cannot be considered as truly rNMP-targeted repair. Similar to NER, Top1-initiated excision of rNMPs (violet) is activated later than RER and MMR. Moreover, it seems that this pathway operates only when RNase H is impaired. In contrast to other mechanisms of rNMP repair, the Top1-initiated pathway is highly mutagenic and to date has been detected only in yeast. It remains to be determined whether additional, as yet unidentified pathways of ribonucleotide repair (shown in grey and by question mark) exist in other species.