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

AbstractThe 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

The Efficiency and Fidelity of Translesion Synthesis past Cisplatin and Oxaliplatin GpG Adducts by Human DNA Polymerase β

DNA polymerase beta (pol beta) is the only mammalian DNA polymerase identified to date that can catalyze extensive bypass of platinum-DNA adducts in vitro. Previous studies suggest that DNA synthesis by pol beta is distributive on primed single-stranded DNA and processive on gapped DNA. The data presented in this paper provide an analysis of translesion synthesis past cisplatin- and oxaliplatin-DNA adducts by pol beta functioning in both distributive and processive modes using primer extension and steady-state kinetic experiments. Translesion synthesis past Pt-DNA adducts was greater with gapped DNA templates than with single-stranded DNA templates. In the processive mode pol beta did not discriminate between cisplatin and oxaliplatin adducts, while in the distributive mode it displayed about 2-fold increased ability for translesion synthesis past oxaliplatin compared with cisplatin adducts. The differentiation between cisplatin and oxaliplatin adducts resulted from a K(m)-mediated increase in the efficiency of dCTP incorporation across from the 3'-G of oxaliplatin-GG adducts. Rates of misincorporation across platinated guanines determined by the steady-state kinetic assay were higher in reactions with primed single-stranded templates than with gapped DNA and a slight increase in the misincorporation of dTTP across from the 3'-G was found for oxaliplatin compared with cisplatin adducts

The Effect of DNA Structure on the Catalytic Efficiency and Fidelity of Human DNA Polymerase β on Templates with Platinum-DNA Adducts

DNA adducts formed by platinum-based anticancer drugs interfere with DNA replication. The carrier ligand of the platinum compound is likely to affect the conformation of the Pt-DNA adducts. In addition, the conformation of the adduct can also change upon binding of damaged DNA to the active site of DNA polymerase. From the crystal structures of pol beta ternary complexes it is evident that undamaged gapped and primed single-stranded (non-gapped) DNA templates exist in very different conformations when bound to pol beta. Therefore, one might expect that the constraints imposed on the damaged templates by binding to the polymerase active site should also affect the conformation of the Pt-DNA adducts and their ability to inhibit DNA replication. In support of this hypothesis we have found that the efficiency, carrier ligand specificity, site of discrimination (3'-G versus 5'-G of the Pt-GG adducts), and fidelity of translesion synthesis past Pt-DNA adducts by pol beta are strongly affected by the structure of the DNA template. Previous studies have suggested that the conformation of Pt-DNA adducts may be affected by the sequence context of the adduct. In support of this hypothesis, our data show that sequence context affects the efficiency, fidelity, and pattern of misincorporation by pol beta

A synthetic genetic polymer with an uncharged backbone chemistry based on alkyl phosphonate nucleic acids

The physicochemical properties of nucleic acids are dominated by their highly charged phosphodiester backbone chemistry. The polyelectrolyte structure decouples information content (base sequence) from bulk properties such as solubility and has been proposed as a defining trait of all informational polymers. However, this conjecture has not been tested experimentally. Here, we describe the encoded synthesis of a genetic polymer with an uncharged backbone chemistry: alkyl-phosphonate nucleic acids (phNA), in which the canonical, negatively charged phosphodiester is replaced by an uncharged P-alkylphosphonodiester backbone. Using synthetic chemistry and polymerase engineering, we describe the enzymatic, DNA-templated synthesis of P-methyl- and P-ethyl-phNAs, and the directed evolution of specific streptavidin-binding phNA aptamer ligands directly from random-sequence, mixed P-methyl- / P-ethyl-phNA repertoires. Our results establish a first example of the DNA-templated enzymatic synthesis and evolution of an uncharged genetic polymer and provide a foundational methodology for their exploration as a source of novel, functional molecules

MSH2–MSH6 stimulates DNA polymerase η, suggesting a role for A:T mutations in antibody genes

Activation-induced cytidine deaminase deaminates cytosine to uracil (dU) in DNA, which leads to mutations at C:G basepairs in immunoglobulin genes during somatic hypermutation. The mechanism that generates mutations at A:T basepairs, however, remains unclear. It appears to require the MSH2–MSH6 mismatch repair heterodimer and DNA polymerase (pol) η, as mutations of A:T are decreased in mice and humans lacking these proteins. Here, we demonstrate that these proteins interact physically and functionally. First, we show that MSH2–MSH6 binds to a U:G mismatch but not to other DNA intermediates produced during base excision repair of dUs, including an abasic site and a deoxyribose phosphate group. Second, MSH2 binds to pol η in solution, and endogenous MSH2 associates with the pol in cell extracts. Third, MSH2–MSH6 stimulates the catalytic activity of pol η in vitro. These observations suggest that the interaction between MSH2–MSH6 and DNA pol η stimulates synthesis of mutations at bases located downstream of the initial dU lesion, including A:T pairs

Strand specificity of ribonucleotide excision repair in Escherichia coli

In Escherichia coli, replication of both strands of genomic DNA is carried out by a single replicase—DNA polymerase III holoenzyme (pol III HE). However, in certain genetic backgrounds, the low-fidelity TLS polymerase, DNA polymerase V (pol V) gains access to undamaged genomic DNA where it promotes elevated levels of spontaneous mutagenesis preferentially on the lagging strand. We employed active site mutants of pol III (pol IIIα_S759N) and pol V (pol V_Y11A) to analyze ribonucleotide incorporation and removal from the E. coli chromosome on a genome-wide scale under conditions of normal replication, as well as SOS induction. Using a variety of methods tuned to the specific properties of these polymerases (analysis of lacI mutational spectra, lacZ reversion assay, HydEn-seq, alkaline gel electrophoresis), we present evidence that repair of ribonucleotides from both DNA strands in E. coli is unequal. While RNase HII plays a primary role in leading-strand Ribonucleotide Excision Repair (RER), the lagging strand is subject to other repair systems (RNase HI and under conditions of SOS activation also Nucleotide Excision Repair). Importantly, we suggest that RNase HI activity can also influence the repair of single ribonucleotides incorporated by the replicase pol III HE into the lagging strand

Removal of Misincorporated Ribonucleotides from Prokaryotic Genomes: An Unexpected Role for Nucleotide Excision Repair

Stringent steric exclusion mechanisms limit the misincorporation of ribonucleotides by high-fidelity DNA polymerases into genomic DNA. In contrast, low-fidelity Escherichia coli DNA polymerase V (pol V) has relatively poor sugar discrimination and frequently misincorporates ribonucleotides. Substitution of a steric gate tyrosine residue with alanine (umuC_Y11A) reduces sugar selectivity further and allows pol V to readily misincorporate ribonucleotides as easily as deoxynucleotides, whilst leaving its poor base-substitution fidelity essentially unchanged. However, the mutability of cells expressing the steric gate pol V mutant is very low due to efficient repair mechanisms that are triggered by the misincorporated rNMPs. Comparison of the mutation frequency between strains expressing wild-type and mutant pol V therefore allows us to identify pathways specifically directed at ribonucleotide excision repair (RER). We previously demonstrated that rNMPs incorporated by umuC_Y11A are efficiently removed from DNA in a repair pathway initiated by RNase HII. Using the same approach, we show here that mismatch repair and base excision repair play minimal back-up roles in RER in vivo. In contrast, in the absence of functional RNase HII, umuC_Y11A-dependent mutagenesis increases significantly in ΔuvrA, uvrB5 and ΔuvrC strains, suggesting that rNMPs misincorporated into DNA are actively repaired by nucleotide excision repair (NER) in vivo. Participation of NER in RER was confirmed by reconstituting ribonucleotide-dependent NER in vitro. We show that UvrABC nuclease-catalyzed incisions are readily made on DNA templates containing one, two, or five rNMPs and that the reactions are stimulated by the presence of mispaired bases. Similar to NER of DNA lesions, excision of rNMPs proceeds through dual incisions made at the 8th phosphodiester bond 5′ and 4th-5th phosphodiester bonds 3′ of the ribonucleotide. Ribonucleotides misinserted into DNA can therefore be added to the broad list of helix-distorting modifications that are substrates for NER

DNA Polymerases ? and ?

No description supplie