TY - JOUR

T1 - Miscoding and DNA Polymerase Stalling by Methoxyamine-Adducted Abasic Sites

AU - Yudkina, Anna V.

AU - Zharkov, Dmitry O.

N1 - Funding Information: This research was supported by Russian Science Foundation (grant 19-74-20069). A.V.Y. is the recipient of Russian Presidential Fellowship for Young Scientists (SP-174.2021.4). MALDI experiments were done at the ICBFM Core Facility of Mass Spectrometric Analysis (Novosibirsk, Russia). Partial salary support from the Ministry of Science and Higher Education of the Russian Federation (121031300056-8) is recognized. Publisher Copyright: © 2022 American Chemical Society

PY - 2022/2/21

Y1 - 2022/2/21

N2 - 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 δ and ϵ, efficiently incorporated both dAMP and dGMP. DNA polymerase β mostly incorporated dAMP and dCMP, preferring dCMP opposite to the natural AP site and dAMP opposite to the MX-AP site, while DNA polymerase λ was selective for dGMP, apparently via the primer misalignment mechanism. Finally, translesion DNA polymerase κ 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 κ.

AB - 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 δ and ϵ, efficiently incorporated both dAMP and dGMP. DNA polymerase β mostly incorporated dAMP and dCMP, preferring dCMP opposite to the natural AP site and dAMP opposite to the MX-AP site, while DNA polymerase λ was selective for dGMP, apparently via the primer misalignment mechanism. Finally, translesion DNA polymerase κ 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 κ.

UR - http://www.scopus.com/inward/record.url?scp=85124009710&partnerID=8YFLogxK

U2 - 10.1021/acs.chemrestox.1c00359

DO - 10.1021/acs.chemrestox.1c00359

M3 - Article

C2 - 35089032

AN - SCOPUS:85124009710

VL - 35

SP - 303

EP - 314

JO - Chemical Research in Toxicology

JF - Chemical Research in Toxicology

SN - 0893-228X

IS - 2

ER -