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

Abstract

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.genome damage repair | replication fork stalling | oxidized base repair at DNA replication fork S everal 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...

The C-terminal Domain (CTD) of Human DNA Glycosylase NEIL1 Is Required for Forming BERosome Repair Complex with DNA Replication Proteins at the Replicating Genome

“…This inhibition should be essential in vivo for preventing double strand break formation (5,13). To further understand the function of NEIL1 in the replicating genome, we recently provided direct experimental evidence for the "cow-catcher" role of NEIL1 in prereplicative repair of oxidized DNA bases in the template strand (5).…”

“…We have previously shown that replication protein A, a mammalian ssDNA-binding protein essential for DNA replication, inhibits DNA glycosylase activity of NEIL1/NEIL2 in vitro with the replication fork-mimicking primer-template DNA substrate (13). This inhibition should be essential in vivo for preventing double strand break formation (5,13). To further understand the function of NEIL1 in the replicating genome, we recently provided direct experimental evidence for the "cow-catcher" role of NEIL1 in prereplicative repair of oxidized DNA bases in the template strand (5).…”

“…We recently demonstrated that human DNA glycosylase NEIL1 (Nei-like protein 1) plays a critical role in prereplicative repair of oxidized lesions in S phase cells, a critical process for preventing mutations in the replicating genome (5). NEIL1 is unique among the five oxidized base-specific mammalian DNA glycosylases in the Nth or Nei family for its S phase-specific activation (1).…”

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

“…The N terminus contains the glycosylase domain of NEIL1, and the C terminus is intrinsically disordered (8,11). Whereas the C terminus is dispensable for both glycosylase and lyase activities in vitro, it interacts with many proteins in vivo and is required for efficient DNA repair activity inside the cells (12,13). NEIL1 is also unique among the three human NEIL proteins in that it is increased in an S-phase-specific manner and carries out prereplicative repair of oxidized bases in the human genome (8,12).…”

“…Whereas the C terminus is dispensable for both glycosylase and lyase activities in vitro, it interacts with many proteins in vivo and is required for efficient DNA repair activity inside the cells (12,13). NEIL1 is also unique among the three human NEIL proteins in that it is increased in an S-phase-specific manner and carries out prereplicative repair of oxidized bases in the human genome (8,12). Moreover, increasing literature has further emphasized the importance of NEIL1's cellular repair activity, as NEIL1 deficiency has led to multiple abnormalities and is associated with severe human diseases, including cancer (14)(15)(16)(17)(18)(19).…”