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DNA polymerases η and κ bypass N²-guanine-O⁶-alkylguanine DNA alkyltransferase cross-linked DNA-peptides.

ABSTRACT: DNA-protein cross-links are formed when proteins become covalently trapped with DNA in the presence of exogenous or endogenous alkylating agents. If left unrepaired, they inhibit transcription as well as DNA unwinding during replication and may result in genome instability or even cell death. The DNA repair protein O⁶-alkylguanine DNA-alkyltransferase (AGT) is known to form DNA cross-links in the presence of the carcinogen 1,2-dibromoethane, resulting in G:C to T:A transversions and other mutations in both bacterial and mammalian cells. We hypothesized that AGT-DNA cross-links would be processed by nuclear proteases to yield peptides small enough to be bypassed by translesion (TLS) polymerases. Here, a 15-mer and a 36-mer peptide from the active site of AGT were cross-linked to the N2 position of guanine via conjugate addition of a thiol containing a peptide dehydroalanine moiety. Bypass studies with DNA polymerases (pols) η and κ indicated that both can accurately bypass the cross-linked DNA peptides. The specificity constant (k_cat/K_m) for steady-state incorporation of the correct nucleotide dCTP increased by 6-fold with human (h) pol κ and 3-fold with hpol η, with hpol η preferentially inserting nucleotides in the order dC > dG > dA > dT. LC-MS/MS analysis of the extension product also revealed error-free bypass of the cross-linked 15-mer peptide by hpol η. We conclude that a bulky 15-mer AGT peptide cross-linked to the N2 position of guanine can retard polymerization, but that overall fidelity is not compromised because only correct bases are inserted and extended.

SUBMITTER: Ghodke PP

PROVIDER: S-EPMC8463853 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:1,1,2,2-cis-diamminedichloroplatinum (II) (cisplatin) is a chemotherapeutic agent widely used in the clinic to treat various cancers. The antitumor activity of cisplatin is generally attributed to its ability to form intrastrand and interstrand DNA-DNA cross-links via sequential platination of two nucleophilic sites within the DNA duplex. However, cisplatin also induces DNA- protein lesions (DPCs) that may contribute to its biological effects due to their ability to block DNA replication and transcription. We previously reported that over 250 nuclear proteins including high mobility group proteins, histone proteins, and elongation factors formed DPCs in human HT1080 cells treated with cisplatin (Ming et al. Chem. Res. Toxicol. 2017, 30, 980-995). Interestingly, cisplatin-induced DNA-protein conjugates were reversed upon heating, by an unknown mechanism. In the present work, DNA repair protein O6-alkylguanine DNA alkyltransferase (AGT) was used as a model to investigate the molecular details of cisplatin-mediated DNA-protein cross-linking and to establish the mechanism of their reversal. We found that AGT is readily cross-linked to DNA in the presence of cisplatin. HPLC-ESI+-MS/MS sequencing of tryptic peptides originating from dG-Pt-AGT complexes revealed that the cross-linking occurred at six sites within this protein including Glu110, Lys125, Cys145, His146, Arg147, and Cys150. Cisplatin-induced Lys-Gua cross-links (1,1-cis-diammine-2-(5-amino-5-carboxypentyl)amino-2-(2'-deoxyguanosine-7-yl)-platinum(II) (dG-Pt-Lys) were detected by HPLC-ESI+-MS/MS of total digests of modified protein in comparison with the corresponding authentic standard. Upon heating, dG-Pt-AGT complexes were subject to platination migration from protein to DNA, forming cis-[Pt(NH3)2{d(GpG)}] cross-links which were detected by HPLC-ESI+-MS/MS. Our results provide a new insight into the mechanism of cisplatin-mediated DNA-protein cross-linking and their dynamic equilibrium with the corresponding DNA-DNA lesions.

Project description:Overexpression of human DNA polymerase kappa (hpol ?) in glioblastoma is associated with shorter survival time and resistance to the alkylating agent temozolomide (TMZ), making it an attractive target for the development of small-molecule inhibitors. We previously reported on the development and characterization of indole barbituric acid-derived (IBA) inhibitors of translesion DNA synthesis polymerases (TLS pols). We have now identified a potent and selective inhibitor of hpol ? based on the indole-aminoguanidine (IAG) chemical scaffold. The most promising IAG analogue, IAG-10, exhibited greater inhibitory action against hpol ? than any other human Y-family member, as well as pols from the A-, B-, and X-families. Inhibition of hpol ? by IAG analogues appears to proceed through a mechanism that is distinct from inhibition of hpol ? based on changes in DNA binding affinity and nucleotide insertion kinetics. By way of comparison, both IAG and IBA analogues inhibited binary complex formation by hpol ? and ternary complex formation by hpol ?. Decreasing the concentration of enzyme and DNA in the reaction mixture lowered the IC50 value of IAG-10 to submicromolar values, consistent with inhibition of binary complex formation for hpol ?. Chemical footprinting experiments revealed that IAG-10 binds to a cleft between the finger, little finger, and N-clasp domains on hpol ? and that this likely disrupts the interaction between the N-clasp and the TLS pol core. In cell culture, IAG-10 potentiated the antiproliferative activity and DNA damaging effects of TMZ in hpol ?-proficient cells but not in hpol ?-deficient cells, indicative of a target-dependent effect. Mutagenic replication across alkylation damage increased in hpol ?-proficient cells treated with IAG-10, while no change in mutation frequency was observed for hpol ?-deficient cells. In summary, we developed a potent and selective small-molecule inhibitor of hpol ? that takes advantage of structural features unique to this TLS enzyme to potentiate TMZ, a standard-of-care drug used in the treatment of malignant brain tumors. Furthermore, the IAG scaffold represents a new chemical space for the exploration of TLS pol inhibitors, which could prove useful as a strategy for improving patient response to genotoxic drugs.

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DNA polymerases η and κ bypass N²-guanine-O⁶-alkylguanine DNA alkyltransferase cross-linked DNA-peptides.

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DNA polymerases η and κ bypass N2-guanine-O6-alkylguanine DNA alkyltransferase cross-linked DNA-peptides.

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DNA polymerases η and κ bypass N²-guanine-O⁶-alkylguanine DNA alkyltransferase cross-linked DNA-peptides.