Abstract
NEIL1 is unique among the oxidatively damaged base repair-initiating DNA glycosylases in the human genome due to its S phase-specific activation and ability to excise substrate base lesions from single-stranded DNA. We recently characterized NEIL1’s specific binding to downstream canonical repair and non-canonical accessory proteins, all of which involve NEIL1’s disordered C-terminal segment as the common interaction domain (CID). This domain is dispensable for NEIL1’s base excision and abasic (AP) lyase activities, but is required for its interactions with other repair proteins. Here, we show that truncated NEIL1 lacking the CID is markedly deficient in initiating in vitro repair of 5-hydroxyuracil (an oxidative deamination product of C) in a plasmid substrate compared to the wild-type NEIL1, thus suggesting a critical role of CID in the coordination of overall repair. Furthermore, while NEIL1 downregulation significantly sensitized human embryonic kidney (HEK) 293 cells to reactive oxygen species (ROS), ectopic wild-type NEIL1, but not the truncated mutant, restored resistance to ROS. These results demonstrate that cell survival and NEIL1-dependent repair of oxidative DNA base damage require interactions among repair proteins, which could be explored as a cancer therapeutic target in order to increase the efficiency of chemo/radiation treatment.
Highlights
Reactive oxygen species (ROS), generated endogenously during cellular respiration or induced after exposure to various exogenous agents/stress, inflict oxidative damage on macromolecules, including DNA, and are implicated in various human pathologies, including aging, age-related neurodegenerative diseases, arthritis and cancer [1,2,3,4,5,6,7]
For several years our laboratory has focused on characterizing NEIL1’s interactions with downstream repair proteins, and identified its pairwise binding to XRCC1, polymerase β (Polβ), LigIII [12], flap endonuclease 1 (FEN-1) [26], PCNA [21], replication protein A (RPA) [22], Werner helicase (WRN) [20] and hnRNP-U [18]
base excision repair (BER) is essential for survival of aerobic organisms in order to repair both endogenously produced and exogenously inflicted genomic damage, including ROS-induced, cytotoxic and mutagenic oxidized DNA bases [40]
Summary
Reactive oxygen species (ROS), generated endogenously during cellular respiration or induced after exposure to various exogenous agents/stress, inflict oxidative damage on macromolecules, including DNA, and are implicated in various human pathologies, including aging, age-related neurodegenerative diseases, arthritis and cancer [1,2,3,4,5,6,7]. Gap filling can involve 1-nt incorporation by DNA polymerase β (Polβ) in the short-patch repair ( named single nucleotide incorporation repair, SN-BER) sub-pathway, or displacement synthesis of 2-8 nts by either. We have previously shown that NEIL1 directly interacts with downstream conventional repair as well as non-canonical accessory proteins (Table 1) via its CID near the C-terminus [12,18,20,21,22,26] This region is predicted to have an intrinsically disordered conformation, based on PONDR modeling [8,27], consistent with its required deletion to obtain a crystallizable form of the protein [28]. We demonstrate the requirement of these interactions for optimum repair of oxidatively damaged bases, resulting in enhanced cell survival
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