Prereplicative repair of oxidized bases in the human genome is mediated by NEIL1 DNA glycosylase together with replication proteins

Base oxidation by endogenous and environmentally induced reactive oxygen species preferentially occurs in replicating single-stranded templates in mammalian genomes, warranting prereplicative repair of the mutagenic base lesions. It is not clear how such lesions (which, unlike bulky adducts, do not block replication) are recognized for repair. Furthermore, strand breaks caused by base excision from ssDNA by DNA glycosylases, including Nei-like (NEIL) 1, would generate double-strand breaks during replication, which are not experimentally observed. NEIL1, whose deficiency causes a mutator phenotype and is activated during the S phase, is present in the DNA replication complex isolated from human cells, with enhanced association with DNA in S-phase cells and colocalization with replication foci containing DNA replication proteins. Furthermore, NEIL1 binds to 5-hydroxyuracil, the oxidative deamination product of C, in replication protein A-coated ssDNA template and inhibits DNA synthesis by DNA polymerase δ. We postulate that, upon encountering an oxidized base during replication, NEIL1 initiates prereplicative repair by acting as a “cowcatcher” and preventing nascent chain growth. Regression of the stalled replication fork, possibly mediated by annealing helicases, then allows lesion repair in the reannealed duplex. This model is supported by our observations that NEIL1, whose deficiency slows nascent chain growth in oxidatively stressed cells, is stimulated by replication proteins in vitro. Furthermore, deficiency of the closely related NEIL2 alone does not affect chain elongation, but combined NEIL1/2 deficiency further inhibits DNA replication. These results support a mechanism of NEIL1-mediated prereplicative repair of oxidized bases in the replicating strand, with NEIL2 providing a backup function.Several dozen oxidatively modified, and mostly mutagenic, bases are induced in the genomes of aerobic organisms by endogenous and environmentally induced reactive oxygen species (ROS) (1, 2). For example, 5-hydroxyuracil (5-OHU), a predominant lesion generated by oxidative deamination of C, is mutagenic because of its mispairing with A (3). The bases in the single-stranded (ss) replicating DNA template are particularly prone to oxidation (4); the lack of their repair before replication could fix the mutations. The bulky base adducts if formed in the template strand would block replication and trigger DNA damage-response signaling. In contrast, oxidized bases with minor modifications, which are continuously formed in much higher abundance than the bulky adducts, would mostly allow replication. This raises the question of how these bases are marked for repair before replication to avoid mutagenic consequences. Oxidized base repair in mammalian genomes occurs primarily via the base excision repair (BER) pathway which is initiated with lesion base excision mediated by one of five major DNA glycosylases belonging to the Nth or Nei families, with distinct structural features and reaction mechanisms (1). Nei-like (NEIL) 1 and NEIL2 DNA glycosylases (5, 6) of the Nei family (which also contains the less characterized NEIL3; ref. 7) are distinct from NTH1 and OGG1 of the Nth family because the NEILs can excise damaged bases from ssDNA substrates (8). Furthermore, NEIL1 is activated during the S phase (5). Our earlier studies also showed that NEIL1 functionally interacts with many DNA replication proteins including sliding clamp proliferating cell nuclear antigen (PCNA), flap endonuclease 1 (FEN-1), and Werner RecQ helicase (WRN) via its disordered C-terminal segment (9–12). Importantly, mammalian ssDNA-binding replication protein A (RPA), essential for DNA replication and most other DNA transactions, inhibits NEIL1 or NEIL2 activity with primer-template DNA substrates mimicking the replication fork, presumably to prevent double-strand break formation (13). Although they collectively implicate NEIL1 in the repair of replicating DNA, those observations did not provide direct evidence for NEIL1’s role in prereplicative repair, nor did they address whether NEIL1 is unique for this function. In this report, we document that NEIL1 binds to the lesion base in an RPA-coated ssDNA template in vitro, without excising the lesion and cleaving the DNA strand, and blocks primer elongation by the replicative DNA polymerase δ (Polδ). This strongly suggests that the replication complex at the lesion site is stalled in vivo in the presence of NEIL1, which provides the signal for repair of lesions in the template strand before replication.