Glycosylase Recognition of Damaged DNA: Balancing Repair and Mutagenesis.

Abstract

The base excision repair (BER) pathway is responsible for repairing the majority of damaged bases that spontaneously arise in DNA. A DNA glycosylase initiates BER by catalyzing the excision of a damaged nucleobase. These enzymes face the task of finding sites of damage among the excess of undamaged nucleotides. Importantly, damaged nucleobases typically alter the basepairing from canonical Watson-Crick interactions and may facilitate base-flipping, providing a mechanism for recognizing damage. Human alkyladenine DNA glycosylase (AAG) has the remarkable ability to recognize a wide variety of alkylated and deaminated purines. Upon finding that weaker basepairing allows more efficient base excision, we showed that AAG can excise an unpaired, bulged base. Remarkably, the bulge is excised with 3-fold greater efficiency than a mismatch. This finding has important biological implications because unpaired bases are created when a polymerase slips and were previously thought to be repaired exclusively by mismatch repair (MMR). It has been shown that overexpression of AAG correlates to an increase in frameshift mutations and may interfere with MMR’s role in protecting against insertion/deletion (frameshift) mutations by providing high-fidelity repair of bulged intermediates. Biochemical experiments characterized the ability of BER to delete a bulged nucleotide, initiated by AAG excision. Production of the single nucleotide deletion product was observed in several human cancer cell extracts. The BER deletion pathway is expected to cause a bias for deletions by facilitating the repair of insertions but making deletions permanent. To determine if excision of a bulged lesion is unique to AAG, we examined the other human glycosylases. Human homolog of Endonuclease III (Nth1) was found to discriminate against excision of a bulged lesion, but nevertheless, maintained significant catalytic activity. In total, four human glycosylases have been reported to excise a bulged lesion, suggesting a common feature of glycosylase catalysis. Our results suggest that nucleotide flipping mechanism makes glycosylases vulnerable to accepting bulges as substrates. These findings explain how the overexpression of DNA glycosylases can lead to increased mutation rates, highlighting that the glycosylase’s unique ability to recognize a structurally diverse range of substrates must be carefully balanced to avoid superfluous excision leading to mutations.