Human DNA Glycosylase NEIL1's Interactions with Downstream Repair Proteins Is Critical for Efficient Repair of Oxidized DNA Base Damage and Enhanced Cell Survival

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.

Keywords: DNA glycosylase; NEIL1; base excision repair; common interaction domain; disordered structure; oxidative base damage and repair; protein-protein interaction; reactive oxygen species.

Figure 1

Schematic representation of NEIL1-initiated base excision repair (BER) sub-pathways in mammalian cells. Excision of the base lesion and subsequent abasic (AP) lyase activity of NEIL1 causing βδ-elimination generates a 1-nt gap at single-strand break with 3' and 5' phosphate (P) ends. The 3'P is removed by polynucleotide kinase 3' phosphatase (PNKP) in the next step to produce 3'OH which serves as primer for gap filling synthesis; this may involve incorporation of 1 nt (SN-BER) by Polβ, or of 2–8 nts (LP-BER) by Polδor Polβ in collaboration with FEN-1. Other details are given in the text.

Figure 2

NEIL1’s common interaction domain (CID)-containing C-terminus is dispensable for DNA glycosylase activity in vitro. Recombinant wild-type (WT) and truncated (N311) mutant of NEIL1 (A; Coomassie-stained gel in B) show similar DNA glycosylase/AP lyase activity with a 5-OHU-containing 5'-³²P-labeled 51-mer oligonucleotide duplex substrate to produce 25 nt oligo (C). Lanes 2 and 3: 10 and 50 fmol WT NEIL1; lanes 4 and 5: 10 and 50 fmol N311 mutant.

Figure 3

NEIL1’s C-terminus provides CID for its partner proteins. Far Western analysis with purified proteins (A), and FLAG co-IP analysis in HEK293 cell extracts expressing FLAG-WT NEIL1 or the FLAG-N311 mutant (B) show the requirement of the CID for NEIL1’s interactions with downstream SN-BER proteins. (C) PLA analysis confirms in-cell association of SN-BER proteins in the nucleus with ectopic WT NEIL1 but not the N311 mutant. Other details are in the Experimental Section.

Figure 4

NEIL1’s CID is required for efficient repair of oxidized DNA lesions. (A) Repair of the 5-OHU-containing plasmid was monitored by the incorporation of ³²P-dTMP and analysis of a 32 nt Nt.BstNB1 restriction repaired fragment after denaturing gel electrophoresis [18,38]. (B) In vitro reconstitution of NEIL1-intitiated SN-BER with purified proteins (10 and 50 fmol WT NEIL1 [lanes 2 and 3] or N311mutant [lanes 4 and 5] and 50 fmol each of PNKP, Polβ, LigIIIα and XRCC1). 5'-³²P-labeled 32 and 20-mer oligos were used as size markers (lane 6). The histogram shows quantitation of repair. (C-E) FLAG-N311 IP, with reduced DNA glycosylase activity compared to FLAG-WT NEIL1 IP (D), was extremely deficient in overall repair (E). The DNA glycosylase activity was measured with 5-OHU-duplex oligo and complete repair with the plasmid substrate. FLAG levels in stably expressing cells were compared by Western analysis (C).

Figure 5

The lack of NEIL1’s CID sensitizes HEK293 cells to oxidative stress. (A) Western analysis of NEIL1 levels after its depletion by 3'-UTR specific siRNA in HEK293 cells and simultaneous expression of FLAG-WT NEIL1 or FLAG-N311 mutant polypeptide. (B) Survival of HEK293 cells transfected with control siRNA or siRNA for NEIL1, and simultaneous co-transfection of FLAG-WT NEIL1 or FLAG-N311 mutant expression plasmids. The details are in the Experimental Section.