TY - JOUR

T1 - Abasic site-peptide cross-links are blocking lesions repaired by AP endonucleases

AU - Yudkina, Anna V

AU - Bulgakov, Nikita A

AU - Kim, Daria V

AU - Baranova, Svetlana V

AU - Ishchenko, Alexander A

AU - Saparbaev, Murat K

AU - Koval, Vladimir V

AU - Zharkov, Dmitry O

N1 - FUNDING: Russian Science Foundation [21-74-00061 to A.V.Y., all biochemical experiments]; Fondation ARC [PJA2021060003796 to A.A.I.]; Russian Ministry of Higher Education and Science [121031300056-8 to D.O.Z.]; D.V.K. is supported by a graduate student fellowship from the Russian Foundation for Basic Research [20-34-90092]. Funding for open access charge: SB RAS ICBFM intramural program. © The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.

PY - 2023/7/7

Y1 - 2023/7/7

N2 - Apurinic/apyrimidinic (AP) sites are abundant DNA lesions arising from spontaneous hydrolysis of the N-glycosidic bond and as base excision repair (BER) intermediates. AP sites and their derivatives readily trap DNA-bound proteins, resulting in DNA-protein cross-links. Those are subject to proteolysis but the fate of the resulting AP-peptide cross-links (APPXLs) is unclear. Here, we report two in vitro models of APPXLs synthesized by cross-linking of DNA glycosylases Fpg and OGG1 to DNA followed by trypsinolysis. The reaction with Fpg produces a 10-mer peptide cross-linked through its N-terminus, while OGG1 yields a 23-mer peptide attached through an internal lysine. Both adducts strongly blocked Klenow fragment, phage RB69 polymerase, Saccharolobus solfataricus Dpo4, and African swine fever virus PolX. In the residual lesion bypass, mostly dAMP and dGMP were incorporated by Klenow and RB69 polymerases, while Dpo4 and PolX used primer/template misalignment. Of AP endonucleases involved in BER, Escherichia coli endonuclease IV and its yeast homolog Apn1p efficiently hydrolyzed both adducts. In contrast, E. coli exonuclease III and human APE1 showed little activity on APPXL substrates. Our data suggest that APPXLs produced by proteolysis of AP site-trapped proteins may be removed by the BER pathway, at least in bacterial and yeast cells.

AB - Apurinic/apyrimidinic (AP) sites are abundant DNA lesions arising from spontaneous hydrolysis of the N-glycosidic bond and as base excision repair (BER) intermediates. AP sites and their derivatives readily trap DNA-bound proteins, resulting in DNA-protein cross-links. Those are subject to proteolysis but the fate of the resulting AP-peptide cross-links (APPXLs) is unclear. Here, we report two in vitro models of APPXLs synthesized by cross-linking of DNA glycosylases Fpg and OGG1 to DNA followed by trypsinolysis. The reaction with Fpg produces a 10-mer peptide cross-linked through its N-terminus, while OGG1 yields a 23-mer peptide attached through an internal lysine. Both adducts strongly blocked Klenow fragment, phage RB69 polymerase, Saccharolobus solfataricus Dpo4, and African swine fever virus PolX. In the residual lesion bypass, mostly dAMP and dGMP were incorporated by Klenow and RB69 polymerases, while Dpo4 and PolX used primer/template misalignment. Of AP endonucleases involved in BER, Escherichia coli endonuclease IV and its yeast homolog Apn1p efficiently hydrolyzed both adducts. In contrast, E. coli exonuclease III and human APE1 showed little activity on APPXL substrates. Our data suggest that APPXLs produced by proteolysis of AP site-trapped proteins may be removed by the BER pathway, at least in bacterial and yeast cells.

KW - African Swine Fever Virus/metabolism

KW - Animals

KW - DNA Damage

KW - DNA Repair

KW - DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism

KW - DNA/metabolism

KW - Endonucleases/metabolism

KW - Escherichia coli/genetics

KW - Humans

KW - Peptides

KW - Saccharomyces cerevisiae/metabolism

KW - Swine

UR - https://www.scopus.com/record/display.uri?eid=2-s2.0-85164936476&origin=inward&txGid=ec2c6afdda5feb0a724adc396b257108

UR - https://www.mendeley.com/catalogue/75da7beb-ac04-3cca-ac9f-84bb18ab0c6d/

U2 - 10.1093/nar/gkad423

DO - 10.1093/nar/gkad423

M3 - Article

C2 - 37216593

VL - 51

SP - 6321

EP - 6336

JO - Nucleic Acids Research

JF - Nucleic Acids Research

SN - 0305-1048

IS - 12

M1 - gkad423

ER -