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.

Corrigendum: Identification and Characterization of Thermostable Y-Family DNA Polymerases η, ι, κ and Rev1 From a Lower Eukaryote, .

[This corrects the article DOI: 10.3389/fmolb.2021.

Identification and Characterization of Thermostable Y-Family DNA Polymerases η, ι, κ and Rev1 From a Lower Eukaryote, .

Y-family DNA polymerases (pols) consist of six phylogenetically separate subfamilies; two UmuC (polV) branches, DinB (pol IV, Dpo4, polκ), Rad30A/POLH (polη), and Rad30B/POLI (polι) and Rev1. Of these subfamilies, DinB orthologs are found in all three domains of life; eubacteria, archaea, and eukarya. UmuC orthologs are identified only in bacteria, whilst Rev1 and Rad30A/B orthologs are only detected in eukaryotes.

CroS , an ortholog of the λ Cro repressor, plays a major role in suppressing polV -dependent mutagenesis.

When subcloned into low-copy-number expression vectors, rumAB, encoding polV (RumA' B), is best characterized as a potent mutator giving rise to high levels of spontaneous mutagenesis in vivo. This is in dramatic contrast to the poorly mutable phenotype when polV is expressed from the native 88.5 kb R391, suggesting that R391 expresses cis-acting factors that suppress the expression and/or the activity of polV .

Novel Escherichia coli active site dnaE alleles with altered base and sugar selectivity.

The Escherichia coli dnaE gene encodes the α-catalytic subunit (pol IIIα) of DNA polymerase III, the cell's main replicase. Like all high-fidelity DNA polymerases, pol III possesses stringent base and sugar discrimination. The latter is mediated by a so-called "steric gate" residue in the active site of the polymerase that physically clashes with the 2'-OH of an incoming ribonucleotide.

Mysterious and fascinating: DNA polymerase ɩ remains enigmatic 20 years after its discovery.

With the publication of the first paper describing the biochemical properties of DNA polymerase iota (polɩ), the question immediately arose as to why cells harbor such a low-fidelity enzyme which often violates the Watson-Crick base pairing rules? Yet 20 years after its discovery, the cellular function of polɩ remains unknown. Here, we provide a graphical review of the unique biochemical properties of polɩ and speculate about the cellular pathways in which enigmatic polɩ may participate.

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

pol V (RumA'B) is a low-fidelity polymerase that promotes considerably higher levels of spontaneous "SOS-induced" mutagenesis than the related E. coli pol V (UmuD'C). The molecular basis for the enhanced mutagenesis was previously unknown.

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. This 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.

Ribonucleotide discrimination by translesion synthesis DNA polymerases.

The well-being of all living organisms relies on the accurate duplication of their genomes. This is usually achieved by highly elaborate replicase complexes which ensure that this task is accomplished timely and efficiently. However, cells often must resort to the help of various additional "specialized" DNA polymerases that gain access to genomic DNA when replication fork progression is hindered.

Translesion DNA polymerases in eukaryotes: what makes them tick?

Life as we know it, simply would not exist without DNA replication. All living organisms utilize a complex machinery to duplicate their genomes and the central role in this machinery belongs to replicative DNA polymerases, enzymes that are specifically designed to copy DNA. "Hassle-free" DNA duplication exists only in an ideal world, while in real life, it is constantly threatened by a myriad of diverse challenges.

Unlocking the steric gate of DNA polymerase η leads to increased genomic instability in Saccharomyces cerevisiae.

DNA polymerase η (pol η) is best characterized for its ability to perform accurate and efficient translesion DNA synthesis (TLS) through cyclobutane pyrimidine dimers (CPDs). To ensure accurate bypass the polymerase is not only required to select the correct base, but also discriminate between NTPs and dNTPs. Most DNA polymerases have a conserved "steric gate" residue which functions to prevent incorporation of NMPs during DNA synthesis.

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.

Investigating the mechanisms of ribonucleotide excision repair in Escherichia coli.

Low fidelity Escherichia coli DNA polymerase V (pol V/UmuD'2C) is best characterized for its ability to perform translesion synthesis (TLS). However, in recA730 lexA(Def) strains, the enzyme is expressed under optimal conditions allowing it to compete with the cell's replicase for access to undamaged chromosomal DNA and leads to a substantial increase in spontaneous mutagenesis. We have recently shown that a Y11A substitution in the "steric gate" residue of UmuC reduces both base and sugar selectivity of pol V, but instead of generating an increased number of spontaneous mutations, strains expressing umuC_Y11A are poorly mutable in vivo.

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.

Mechanisms employed by Escherichia coli to prevent ribonucleotide incorporation into genomic DNA by Pol V.

Escherichia coli pol V (UmuD'(2)C), 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.

Translesion DNA Synthesis.

All living organisms are continually exposed to agents that damage their DNA, which threatens the integrity of their genome. As a consequence, cells are equipped with a plethora of DNA repair enzymes to remove the damaged DNA. Unfortunately, situations nevertheless arise where lesions persist, and these lesions block the progression of the cell's replicase.

Escherichia coli UmuC active site mutants: effects on translesion DNA synthesis, mutagenesis and cell survival.

Escherichia coli polymerase V (pol V/UmuD(2)'C) is a low-fidelity DNA polymerase that has recently been shown to avidly incorporate ribonucleotides (rNTPs) into undamaged DNA. The fidelity and sugar selectivity of pol V can be modified by missense mutations around the "steric gate" of UmuC. Here, we analyze the ability of three steric gate mutants of UmuC to facilitate translesion DNA synthesis (TLS) of a cyclobutane pyrimidine dimer (CPD) in vitro, and to promote UV-induced mutagenesis and cell survival in vivo.

A short adaptive path from DNA to RNA polymerases.

DNA polymerase substrate specificity is fundamental to genome integrity and to polymerase applications in biotechnology. In the current paradigm, active site geometry is the main site of specificity control. Here, we describe the discovery of a distinct specificity checkpoint located over 25 Å from the active site in the polymerase thumb subdomain.

Critical amino acids in Escherichia coli UmuC responsible for sugar discrimination and base-substitution fidelity.

The active form of Escherichia coli DNA polymerase V responsible for damage-induced mutagenesis is a multiprotein complex (UmuD'(2)C-RecA-ATP), called pol V Mut. Optimal activity of pol V Mut in vitro is observed on an SSB-coated single-stranded circular DNA template in the presence of the β/γ complex and a transactivated RecA nucleoprotein filament, RecA*. Remarkably, under these conditions, wild-type pol V Mut efficiently incorporates ribonucleotides into DNA.

Simple and efficient purification of Escherichia coli DNA polymerase V: cofactor requirements for optimal activity and processivity in vitro.

Most damage induced mutagenesis in Escherichia coli is dependent upon the UmuD'(2)C protein complex, which comprises DNA polymerase V (pol V). Biochemical characterization of pol V has been hindered by the fact that the enzyme is notoriously difficult to purify, largely because overproduced UmuC is insoluble. Here, we report a simple and efficient protocol for the rapid purification of milligram quantities of pol V from just 4 L of bacterial culture.

Molecular breeding of polymerases for resistance to environmental inhibitors.

Potent inhibitors limit the use of PCR assays in a wide spectrum of specimens. Here, we describe the engineering of polymerases with a broad resistance to complex environmental inhibitors using molecular breeding of eight different polymerase orthologues from the genus Thermus and directed evolution by CSR in the presence of inhibitors. Selecting for resistance to the inhibitory effects of Neomylodon bone powder, we isolated 2D9, a chimeric polymerase comprising sequence elements derived from DNA polymerases from Thermus aquaticus, Thermus oshimai, Thermus thermophilus and Thermus brockianus.

Uracil residues dependent on the deaminase AID in immunoglobulin gene variable and switch regions.

Activation-induced deaminase (AID) initiates diversity of immunoglobulin genes through deamination of cytosine to uracil. Two opposing models have been proposed for the deamination of DNA or RNA by AID. Although most data support DNA deamination, there is no physical evidence of uracil residues in immunoglobulin genes.

Molecular breeding of polymerases for amplification of ancient DNA.

In the absence of repair, lesions accumulate in DNA. Thus, DNA persisting in specimens of paleontological, archaeological or forensic interest is inevitably damaged. We describe a strategy for the recovery of genetic information from damaged DNA.

Characterization of polVR391: a Y-family polymerase encoded by rumA'B from the IncJ conjugative transposon, R391.

Although best characterized for their ability to traverse a variety of DNA lesions, Y-family DNA polymerases can also give rise to elevated spontaneous mutation rates if they are allowed to replicate undamaged DNA. One such enzyme that promotes high levels of spontaneous mutagenesis in Escherichia coli is polV(R391), a polV-like Y-family polymerase encoded by rumA'B from the IncJ conjugative transposon R391. When expressed in a DeltaumuDC lexA(Def) recA730 strain, polV(R391) promotes higher levels of spontaneous mutagenesis than the related MucA'B (polR1) or UmuD'C (polV) polymerases respectively.

Normal hypermutation in antibody genes from congenic mice defective for DNA polymerase iota.

Several low fidelity DNA polymerases participate in generating mutations in immunoglobulin genes. Polymerase eta is clearly involved in the process by causing substitutions of A:T base pairs, whereas polymerase iota has a controversial role. Although the frequency of mutations was decreased in the BL2 cell line deficient for polymerase iota, hypermutation was normal in the 129 strain of mice, which has a natural nonsense mutation in the Poli gene.