Miscoding and DNA Polymerase Stalling by Methoxyamine-Adducted Abasic Sites

Apurinic/apyrimidinic (AP) sites appear in DNA spontaneously and as intermediates of base excision DNA repair. AP sites are noninstructive lesions: they strongly block DNA polymerases, and if bypassed, the nature of the incorporated dNMP is mostly guided by the interactions within the polymerase-DNA active site. Many DNA polymerases follow the A-rule, preferentially incorporating dAMP opposite to natural AP sites. Methoxyamine (MX), a small molecule, efficiently reacts with the aldehyde moiety of natural AP sites, thereby preventing their cleavage by APEX1, the major human AP endonuclease. MX is currently regarded as a possible sensitizer of cancer cells toward DNA-damaging drugs. To evaluate the mutagenic potential of MX, we have studied the utilization of various dNTPs by five DNA polymerases of different families encountering MX-AP adducts in the template in comparison with the natural aldehydic AP site. The Klenow fragment of Escherichia coli DNA polymerase I strictly followed the A-rule with both natural AP and MX-adducted AP sites. Phage RB69 DNA polymerase, a close relative of human DNA polymerases delta and epsilon, efficiently incorporated both dAMP and dGMP. DNA polymerase beta mostly incorporated dAMP and dCMP, preferring dCMP opposite to the natural AP site and dAMP opposite to the MX-AP site, while DNA polymerase lambda was selective for dGMP, apparently via the primer misalignment mechanism. Finally, translesion DNA polymerase. also followed the A-rule for MX-AP and additionally incorporated dCMP opposite to a natural AP site. Overall, the MX-AP site, despite structural differences, was similar to the natural AP site in terms of the dNMP misincorporation preference but was bypassed less efficiently by all polymerases except for Pol kappa.

Automated summary

AP sites in DNA are noninstructive lesions that block DNA polymerases. Methoxyamine is considered as a potential sensitizer of cancer cells to DNA-damaging drugs, but may lead to base misincorporation in certain DNA polymerases.