Arabidopsis thaliana PrimPol is a primase and lesion bypass DNA polymerase with the biochemical characteristics to cope with DNA damage in the nucleus, mitochondria, and chloroplast

PrimPol is a novel Primase–Polymerase that synthesizes RNA and DNA primers de novo and extents from these primers as a DNA polymerase. Animal PrimPol is involved in nuclear and mitochondrial DNA replication by virtue of its translesion DNA synthesis (TLS) and repriming activities. Here we report that the plant model Arabidopsis thaliana encodes a functional PrimPol (AtPrimPol). AtPrimPol is a low fidelity and a TLS polymerase capable to bypass DNA lesions, like thymine glycol and abasic sites, by incorporating directly across these lesions or by skipping them. AtPrimPol is also an efficient primase that preferentially recognizes the single-stranded 3′-GTCG-5′ DNA sequence, where the 3′-G is cryptic. AtPrimPol is the first DNA polymerase that localizes in three cellular compartments: nucleus, mitochondria, and chloroplast. In vitro, AtPrimPol synthesizes primers that are extended by the plant organellar DNA polymerases and this reaction is regulated by organellar single-stranded binding proteins. Given the constant exposure of plants to endogenous and exogenous DNA-damaging agents and the enzymatic capabilities of lesion bypass and re-priming of AtPrimPol, we postulate a predominant role of this enzyme in avoiding replication fork collapse in all three plant genomes, both as a primase and as a TLS polymerase.

Alleviation of C⋅C Mismatches in DNA by the Escherichia coli Fpg Protein

DNA polymerase III mis-insertion may, where not corrected by its 3′→ 5′ exonuclease or the mismatch repair (MMR) function, result in all possible non-cognate base pairs in DNA generating base substitutions. The most thermodynamically unstable base pair, the cytosine (C)⋅C mismatch, destabilizes adjacent base pairs, is resistant to correction by MMR in Escherichia coli, and its repair mechanism remains elusive. We present here in vitro evidence that C⋅C mismatch can be processed by base excision repair initiated by the E. coli formamidopyrimidine-DNA glycosylase (Fpg) protein. The kcat for C⋅C is, however, 2.5 to 10 times lower than for its primary substrate 8-oxoguanine (oxo8G)⋅C, but approaches those for 5,6-dihydrothymine (dHT)⋅C and thymine glycol (Tg)⋅C. The KM values are all in the same range, which indicates efficient recognition of C⋅C mismatches in DNA. Fpg activity was also exhibited for the thymine (T)⋅T mismatch and for N4- and/or 5-methylated C opposite C or T, Fpg activity being enabled on a broad spectrum of DNA lesions and mismatches by the flexibility of the active site loop. We hypothesize that Fpg plays a role in resolving C⋅C in particular, but also other pyrimidine⋅pyrimidine mismatches, which increases survival at the cost of some mutagenesis.

Lesion Recognition and Cleavage of Damage-Containing Quadruplexes and Bulged Structures by DNA Glycosylases

Human telomeres as well as more than 40% of human genes near the promoter regions have been found to contain the sequence that may form a G-quadruplex structure. Other non-canonical DNA structures comprising bulges, hairpins, or bubbles may have a functionally important role during transcription, replication, or recombination. The guanine-rich regions of DNA are hotspots of oxidation that forms 7,8-dihydro-8-oxoguanine, thymine glycol, and abasic sites: the lesions that are handled by the base excision repair pathway. Nonetheless, the features of DNA repair processes in non-canonical DNA structures are still poorly understood. Therefore, in this work, a comparative analysis of the efficiency of the removal of a damaged nucleotide from various G-quadruplexes and bulged structures was performed using endonuclease VIII-like 1 (NEIL1), human 8-oxoguanine-DNA glycosylase (OGG1), endonuclease III (NTH1), and prokaryotic formamidopyrimidine-DNA glycosylase (Fpg), and endonuclease VIII (Nei). All the tested enzymes were able to cleave damage-containing bulged DNA structures, indicating their important role in the repair process when single-stranded DNA and intermediate non–B-form structures such as bubbles and bulges are formed. Nevertheless, our results suggest that the ability to cleave damaged quadruplexes is an intrinsic feature of members of the H2tH structural family, suggesting that these enzymes can participate in the modulation of processes controlled by the formation of quadruplex structures in genomic DNA.

Plant organellar DNA polymerases bypass thymine glycol using two conserved lysine residues

Plant organelles cope with endogenous DNA damaging agents, byproducts of respiration and photosynthesis, and exogenous agents like ultraviolet light. Plant organellar DNA polymerases (DNAPs) are not phylogenetically related to yeast and metazoan DNAPs and they harbor three insertions not present in any other DNAPs. Plant organellar DNAPs from Arabidopsis thaliana (AtPolIA and AtPolIB) are translesion synthesis (TLS) DNAPs able to bypass abasic sites, a lesion that poses a strong block to replicative polymerases. Besides abasic sites, reactive oxidative species and ionizing radiation react with thymine resulting in thymine glycol (Tg), a DNA adduct that is also a strong block to replication. Here, we report that AtPolIA and AtPolIB bypass Tg by inserting an adenine opposite the lesion and efficiently extend from a Tg-A base pair. The TLS ability of AtPolIB is mapped to two conserved lysine residues: K593 and K866. Residue K593 is situated in insertion 1 and K866 is in insertion 3. With basis on the location of both insertions on a structural model of AtPolIIB, we hypothesize that the two positively charged residues interact to form a clamp around the primer-template. In contrast with nuclear and bacterial replication, where lesion bypass involves an interplay between TLS and replicative DNA polymerases, we postulate that plant organellar DNAPs evolved to exert replicative and TLS activities.

The Biochemical Role of the Human NEIL1 and NEIL3 DNA Glycosylases on Model DNA Replication Forks

Endonuclease VIII-like (NEIL) 1 and 3 proteins eliminate oxidative DNA base damage and psoralen DNA interstrand crosslinks through initiation of base excision repair. Current evidence points to a DNA replication associated repair function of NEIL1 and NEIL3, correlating with induced expression of the proteins in S/G2 phases of the cell cycle. However previous attempts to express and purify recombinant human NEIL3 in an active form have been challenging. In this study, both human NEIL1 and NEIL3 have been expressed and purified from E. coli, and the DNA glycosylase activity of these two proteins confirmed using single- and double-stranded DNA oligonucleotide substrates containing the oxidative bases, 5-hydroxyuracil, 8-oxoguanine and thymine glycol. To determine the biochemical role that NEIL1 and NEIL3 play during DNA replication, model replication fork substrates were designed containing the oxidized bases at one of three specific sites relative to the fork. Results indicate that whilst specificity for 5- hydroxyuracil and thymine glycol was observed, NEIL1 acts preferentially on double-stranded DNA, including the damage upstream to the replication fork, whereas NEIL3 preferentially excises oxidized bases from single stranded DNA and within open fork structures. Thus, NEIL1 and NEIL3 act in concert to remove oxidized bases from the replication fork.

NTH1 Is a New Target for Ubiquitylation-Dependent Regulation by TRIM26 Required for the Cellular Response to Oxidative Stress

ABSTRACT Endonuclease III-like protein 1 (NTH1) is a DNA glycosylase required for the repair of oxidized bases, such as thymine glycol, within the base excision repair pathway. We examined regulation of NTH1 protein by the ubiquitin proteasome pathway and identified the E3 ubiquitin ligase tripartite motif 26 (TRIM26) as the major enzyme targeting NTH1 for polyubiquitylation. We demonstrate that TRIM26 catalyzes ubiquitylation of NTH1 predominantly on lysine 67 present within the N terminus of the protein in vitro . In addition, the stability of a ubiquitylation-deficient protein mutant of NTH1 (lysine to arginine) at this specific residue was significantly increased in comparison to the wild-type protein when transiently expressed in cultured cells. We also demonstrate that cellular NTH1 protein is induced in response to oxidative stress following hydrogen peroxide treatment of cells and that accumulation of NTH1 on chromatin is exacerbated in the absence of TRIM26 through small interfering RNA (siRNA) depletion. Stabilization of NTH1 following TRIM26 siRNA also causes significant acceleration in the kinetics of DNA damage repair and cellular resistance to oxidative stress, which can be recapitulated by moderate overexpression of NTH1. This demonstrates the importance of TRIM26 in regulating the cellular levels of NTH1, particularly under conditions of oxidative stress.

Tautomerization-dependent recognition and excision of oxidation damage in base-excision DNA repair

NEIL1 (Nei-like 1) is a DNA repair glycosylase guarding the mammalian genome against oxidized DNA bases. As the first enzymes in the base-excision repair pathway, glycosylases must recognize the cognate substrates and catalyze their excision. Here we present crystal structures of human NEIL1 bound to a range of duplex DNA. Together with computational and biochemical analyses, our results suggest that NEIL1 promotes tautomerization of thymine glycol (Tg)—a preferred substrate—for optimal binding in its active site. Moreover, this tautomerization event also facilitates NEIL1-catalyzed Tg excision. To our knowledge, the present example represents the first documented case of enzyme-promoted tautomerization for efficient substrate recognition and catalysis in an enzyme-catalyzed reaction.