Nei-like 1 (NEIL1) excises 5-carboxylcytosine directly and stimulates TDG-mediated 5-formyl and 5-carboxylcytosine excision

Thymine DNA glycosylase (TDG) and Nei-like 1 (NEIL1) have both been implicated in the base excision repair step of active DNA demethylation. The robust glycosylase activity of TDG on DNA substrates containing 5-formylcytosine (5fC) or 5-carboxylcytosine (5caC) is universally accepted, but the mode of action of NEIL1 is still debated. Based on genetic experiments, it has been suggested that NEIL1 acts redundantly with TDG and excises 5fC and 5caC directly. However, this result has been disputed, and it was suggested instead that NEIL1 is recruited by the monofunctional TDG for the 2′-deoxyribose excision step. Using purified human NEIL1 and its catalytically impaired P2T and E3Q variants as controls, we detect NEIL1 activity on 5caC, but not a 5fC containing dsDNA substrate. We confirm direct NEIL1 TDG binding and NEIL1 mediated 2′-deoxyribose excision downstream of TDG glycosylase activity. NEIL1 acts not only downstream of TDG, but also enhances TDG activity on 5fC or 5caC containing DNA. NEIL1 mediated enhancement of the TDG glycosylase activity is substrate specific and does not occur for dsDNA with a T/G mismatch.