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DNA Polymerase θ is the key actuator of the recently identified double-strand break repair pathway, theta-mediated end joining (TMEJ). It is the only known polymerase to have a 3-domain architecture containing an independently functional family A DNA polymerase tethered by a long central region to an N-terminal helicase-like domain (HLD). Full-length polymerase θ and the isolated HLD hydrolyze ATP in the presence of DNA, but no processive DNA duplex unwinding has been observed. Based on sequence and structure conservation, the HLD is classified as a member of helicase superfamily II and, more specifically, the Ski2-like family. The specific subdomain composition and organization most closely resemble that of archaeal DNA repair helicases Hel308 and Hjm. The underlying structural basis as to why the HLD is not able to processively unwind duplex DNA, despite its similarity to bona fide helicases, remains elusive. Activities of the HLD include ATP hydrolysis, protein displacement, and annealing of complementary DNA. These observations have led to speculation about the role of the HLD within the context of double-strand break repair via TMEJ, such as removal of single-stranded DNA binding proteins like RPA and RAD51 and microhomology alignment. This review summarizes the structural classification and organization of the polymerase θ HLD and its homologs and explores emerging data on its biochemical activities. We conclude with a simple, speculative model for the HLD's role in TMEJ.

Oxidative DNA damage as a result of normal cellular metabolism, inflammation, or exposure to exogenous DNA damaging agents if left unrepaired, can result in genomic instability, a precursor to cancer and other diseases. Nth-like DNA glycosylase 1 (NTHL1) is an evolutionarily conserved bifunctional DNA glycosylase that primarily removes oxidized pyrimidine lesions. NTHL1 D239Y is a germline variant identified in both heterozygous and homozygous state in the human population. Here, we have generated a knockin mouse model carrying Nthl1 D227Y (mouse homologue of D239Y) using CRISPR-cas9 genome editing technology and investigated the cellular effects of the variant in the heterozygous (Y/+) and homozygous (Y/Y) state using murine embryonic fibroblasts. We identified a significant increase in double stranded breaks, genomic instability, replication stress and impaired proliferation in both the Nthl1 D227Y heterozygous Y/+ and homozygous mutant Y/Y MEFs. Importantly, we identified that the presence of the D227Y variant interferes with repair by the WT protein, possibly by binding and shielding the lesions. The cellular phenotypes observed in D227Y mutant MEFs suggest that both the heterozygous and homozygous carriers of this NTHL1 germline mutation may be at increased risk for the development of DNA damage-associated diseases, including cancer.

Assault to DNA that leads to oxidative base damage is repaired by the base excision repair (BER) pathway with specialized enzymes called DNA glycosylases catalyzing the first step of this pathway. These glycosylases can be categorized into two families: the HhH superfamily, which includes endonuclease III (or Nth), and the Fpg/Nei family, which comprises formamidopyrimidine DNA glycosylase (or Fpg) and endonuclease VIII (or Nei). In humans there are three Nei-like (NEIL) glycosylases: NEIL1, 2, and 3. Here we present the first crystal structure of a viral ortholog of the human NEIL2/NEIL3 proteins, Mimivirus Nei2 (MvNei2), determined at 2.04 Å resolution. The C-terminal region of the MvNei2 enzyme comprises two conserved DNA binding motifs: the helix-two-turns-helix (H2TH) motif and a C-H-C-C type zinc-finger similar to that of human NEIL2. The N-terminal region of MvNei2 is most closely related to NEIL3. Like NEIL3, MvNei2 bears a valine at position 2 instead of the usual proline and it lacks two of the three conserved void-filling residues present in other members of the Fpg/Nei family. Mutational analysis of the only conserved void-filling residue methionine 72 to alanine yields an MvNei2 variant with impaired glycosylase activity. Mutation of the adjacent His73 causes the enzyme to be more productive thereby suggesting a plausible role for this residue in the DNA lesion search process.

7,8-Dihydro-8-oxoguanine (8-oxoG) is the major oxidative product of guanine and the most prevalent base lesion observed in DNA molecules. Because 8-oxoG has the capability to form a Hoogsteen pair with adenine (8-oxoG:A) in addition to a normal Watson–Crick pair with cytosine (8-oxoG:C), this lesion can lead to a G:C → T:A transversion after replication. However, 8-oxoG is recognized and excised by the 8-oxoguanine DNA glycosylase (Ogg) of the base excision repair pathway. Members of the Ogg1 family usually display a strong preference for a C opposite the lesion. In contrast, the atypical Ogg1 from Clostridium actetobutylicum (CacOgg) can excise 8-oxoG when paired with either one of the four bases, albeit with a preference for C and A. Here we describe the first high-resolution crystal structures of CacOgg in complex with duplex DNA containing the 8-oxoG lesion paired to cytosine and to adenine. A structural comparison with human OGG1 provides a rationale for the lack of opposite base specificity displayed by the bacterial Ogg.

The repair of free-radical oxidative DNA damage is carried out by lesion-specific DNA glycosylases as the first step of the highly conserved base excision repair (BER) pathway. In humans, three orthologs of the prototypical endonuclease VIII (Nei), the Nei-like NEIL1-3 enzymes are involved in the repair of oxidized DNA lesions. In recent years, several genome and cancer single-nucleotide polymorphic variants of the NEIL1 glycosylase have been identified. In this study we characterized four variants of human NEIL1: S82C, G83D, P208S, and ΔE28, and tested their ability to excise pyrimidine-derived lesions such as thymine glycol (Tg), 5-hydroxyuracil (5-OHU), and dihydrouracil (DHU) and the purine-derived guanidinohydantoin (Gh), spiroiminodihydantoin 1 (Sp1), and methylated 2,6-diamino-4-hydroxy-5-formamidopyrimidine (MeFapyG). The P208S variant has near wild-type activity on all substrates tested. The S82C and ΔE28 variants exhibit decreased Tg excision compared to wild-type. G83D displays little to no activity with any of the substrates tested, with the exception of Gh and Sp1. Human NEIL1 is known to undergo editing whereby the lysine at position 242 is recoded into an arginine. The non-edited form of NEIL1 is more efficient at cleaving Tg than the R242 form, but the G83D variant does not cleave Tg regardless of the edited status of NEIL1. The corresponding G86D variant in Mimivirus Nei1 similarly lacks glycosylase activity. A structure of a G86D-DNA complex reveals a rearrangement in the β4/5 loop comprising Leu84, the highly-conserved void-filling residue, thereby providing a structural rationale for the decreased glycosylase activity of the glycine to aspartate variant.

Formamidopyrimidine-DNA glycosylase (Fpg; MutM) is a DNA repair enzyme widely distributed in bacteria. Fpg recognizes and excises oxidatively modified purines, 4,6-diamino-5-formamidopyrimidine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine and 8-oxoguanine (8-oxoG), with similar excision kinetics. It exhibits some lesser activity toward 8-oxoadenine. Fpg enzymes are also present in some plant and fungal species. The eukaryotic Fpg homologs exhibit little or no activity on DNA containing 8-oxoG, but they recognize and process its oxidation products, guanidinohydantoin (Gh) and spiroiminohydantoin (Sp). To date, several structures of bacterial Fpg enzymes unliganded or in complex with DNA containing a damaged base have been published but there is no structure of a eukaryotic Fpg. Here we describe the first crystal structure of a plant Fpg, Arabidopsis thaliana (AthFpg), unliganded and bound to DNA containing an abasic site analog, tetrahydrofuran (THF). Although AthFpg shares a common architecture with other Fpg glycosylases, it harbors a zincless finger, previously described in a subset of Nei enzymes, such as human NEIL1 and Mimivirus Nei1. Importantly the "αF-β9/10 loop" capping 8-oxoG in the active site of bacterial Fpg is very short in AthFpg. Deletion of a segment encompassing residues 213-229 in Escherichia coli Fpg (EcoFpg) and corresponding to the "αF-β9/10 loop" does not affect the recognition and removal of oxidatively damaged DNA base lesions, with the exception of 8-oxoG. Although the exact role of the loop remains to be further explored, it is now clear that this protein segment is specific to the processing of 8-oxoG.