Anti-ALKBH2 (C-terminal) polyclonal antibody

ALKBH2 is a direct DNA repair dioxygenase guarding the mammalian genome against N-1-methyladenine, N-3-methylcytosine and 1,N-6-ethenoadenine damage. A prerequisite for repair is to identify these lesions in the genome. Here we present crystal structures of human ALKBH2 bound to different duplex DNAs. Together with computational and biochemical analyses, our results suggest that DNA interrogation by ALKBH2 has two previously unknown features: (i) ALKBH2 probes base-pair stability and detects base pairs with reduced stability, and (ii) ALKBH2 does not have nor need a damage-checking site, which is critical for preventing spurious base cleavage for several glycosylases. The demethylation mechanism of ALKBH2 insures that only cognate lesions are oxidized and reversed to normal bases, and that a flipped, non-substrate base remains intact in the active site. Overall, the combination of duplex interrogation and oxidation chemistry allows ALKBH2 to detect and process diverse lesions efficiently and correctly.

1,N-6-ethenoadenine (epsilon A) is a mutagenic lesion and biomarker observed in numerous cancerous tissues. Two pathways are responsible for its repair: base excision repair (BER) and direct reversal repair (DRR). Alkyladenine DNA glycosylase (AAG) is the primary enzyme that excises epsilon A in BER, generating stable intermediates that are processed by downstream enzymes. For DRR, the Fe(II)/alpha-ketoglutarate-dependent ALKBH2 enzyme repairs epsilon A by direct conversion of epsilon A to A. While the molecular mechanism of each enzyme is well understood on unpackaged duplex DNA, less is known about their actions on packaged DNA. The nucleosome core particle (NCP) forms the minimal packaging unit of DNA in eukaryotic organisms and is composed of 145-147 base pairs wrapped around a core of eight histone proteins. In this work, we investigated the activity of AAG and ALKBH2 on epsilon A lesions globally distributed at positions throughout a strongly positioned NCP. Overall, we examined the repair of epsilon A at 23 unique locations in packaged DNA. We observed a strong correlation between rotational positioning of epsilon A and AAG activity but not ALKBH2 activity. ALKBH2 was more effective than AAG at repairing occluded epsilon A lesions, but only AAG was capable of full repair of any epsilon A in the NCP. However, notable exceptions to these trends were observed, highlighting the complexity of the NCP as a substrate for DNA repair. Modeling of binding of the repair enzymes to NCPs revealed that some of these observations can be explained by steric interference caused by DNA packaging. Specifically, interactions between ALKBH2 and the histone proteins obstruct binding to DNA, which leads to diminished activity. Taken together, these results support in vivo observations of alkylation damage profiles and contribute to our understanding of mutational hotspots.