The C-terminal tail of the NEIL1 DNA glycosylase interacts with the human mitochondrial single-stranded DNA binding protein

Abstract

The 16.5 kb mitochondrial genome is subjected to damage from reactive oxygen species (ROS) generated in the cell during normal cellular metabolism and external sources such as ionizing radiation and ultraviolet light. ROS cause harmful damage to DNA bases that could result in mutagenesis and various diseases, if not properly repaired. The base excision repair (BER) pathway is the primary pathway involved in maintaining the integrity of mtDNA. Several enzymes that partake in BER within the nucleus have also been identified in the mitochondria. The nei-like (NEIL) DNA glycosylases initiate BER by excising oxidized pyrimidine bases and others such as the ring-opened formamidopyrimidine and the hydantoin lesions. During BER, the NEIL enzymes interact with proteins that are involved with DNA replication and transcription. In the current manuscript, we detected NEIL1 in purified mitochondrial extracts from human cells and showed that NEIL1 interacts with the human mitochondrial single-stranded DNA binding protein (mtSSB) via its C-terminal tail using protein painting, far-western analysis, and gel-filtration chromatography. Finally, we scrutinized the NEIL1-mtSSB interaction in the presence and absence of a partial-duplex DNA substrate using a combination of multi-angle light scattering (MALS) and small-angle X-ray scattering (SAXS). The data indicate that NEIL1 and homotetrameric mtSSB form a larger ternary complex in presence of DNA, however, the tetrameric form of mtSSB gets disrupted by NEIL1 in the absence of DNA as revealed by the formation of a smaller NEIL1-mtSSB_monomer complex.

Keywords: Base excision repair; Mitochondrial single-stranded DNA binding protein; NEIL1 DNA glycosylase; Protein painting; Size exclusion chromatography; Small angle X-ray scattering.