Synthesis of Site-Specific DNA–Protein Conjugates and Their Effects on DNA Replication

Abstract

DNA–protein cross-links (DPCs) are bulky, helix-distorting DNA lesions that form in the genome upon exposure to common antitumor drugs, environmental/occupational toxins, ionizing radiation, and endogenous free-radical-generating systems. As a result of their considerable size and their pronounced effects on DNA–protein interactions, DPCs can interfere with DNA replication, transcription, and repair, potentially leading to mutagenesis, genotoxicity, and cytotoxicity. However, the biological consequences of these ubiquitous lesions are not fully understood due to the difficulty of generating DNA substrates containing structurally defined, site-specific DPCs. In the present study, site-specific cross-links between the two biomolecules were generated by copper-catalyzed [3 + 2] Huisgen cycloaddition (click reaction) between an alkyne group from 5-(octa-1,7-diynyl)-uracil in DNA and an azide group within engineered proteins/polypeptides. The resulting DPC substrates were subjected to in vitro primer extension in the presence of human lesion bypass DNA polymerases η, κ, ν, and ι. We found that DPC lesions to the green fluorescent protein and a 23-mer peptide completely blocked DNA replication, while the cross-link to a 10-mer peptide was bypassed. These results indicate that the polymerases cannot read through the larger DPC lesions and further suggest that proteolytic degradation may be required to remove the replication block imposed by bulky DPC adducts.

Error-prone Translesion Synthesis Past DNA-Peptide Cross-links Conjugated to the Major Groove of DNA via C5 of Thymidine

“…A biotinylated 23-mer primer (biotin-5Ј-(T) 10 GGG GGA AGG AUT C-3Ј) and 13-mer primer (5Ј-GGG GGA AGG ATT C-3Ј) were purchased from Integrated DNA Technologies (Coralville, IA). All oligodeoxynucleotides were purified by semi-preparative HPLC, desalted by Illustra NAP-5 columns, characterized by HPLC-ESI Ϫ -MS, and quantified by UV spectrophotometry as described previously (37).…”

“…To assess the purity of the isolated conjugates, an aliquot of the purified sample was radiolabeled with [␥- 32 P]ATP and resolved on a 20% (w/v) denaturing polyacrylamide gel containing 7 M urea, followed by visualization using a Typhoon FLA 7000 phosphorimager (GE Healthcare). A separate aliquot was subjected to alkaline phosphatase/phosphodiesterase digestion, and the resulting nucleoside-peptide conjugates were characterized by nano-LC-nanospray-MS/MS (37).…”

“…We have recently developed an efficient methodology for generating site-specific DNA-protein and DNA-peptide conjugates using 1,3-dipolar cycloaddition between azide-containing proteins/peptides and alkyne-functionalized oligodeoxynucleotides in the presence of copper (37). This approach generates covalent conjugates between the C5 position of thymine in DNA and azide-functionalized polypeptides (Scheme 1).…”

“…The resulting DNA-peptide conjugates (Scheme 1) were isolated by denaturing PAGE (37), and their purity was confirmed by sequencing PAGE. Only DNA templates that were at least 99% pure were employed for primer extension assays.…”

Bypass of DNA-Protein Cross-links Conjugated to the 7-Deazaguanine Position of DNA by Translesion Synthesis Polymerases

“…Two peptides of increasing size, a 10-mer peptide derived from c-Myc protein (EQKLISEEDL) and a 23-mer derived from tetanus toxoid (N 3 (CH 2 ) 3 CO-PDAQLVPGINGKAIHLVNNESSE) were used. The same two peptides were used in our earlier publication investigating replication bypass of DPCs conjugated to the C5 position of thymidine using Cu(I)-catalyzed azide-alkyne cycloaddition (29). In addition, histone H2A (14 kDa), histone H4 (11.5 kDa), and AlkB (22.9 kDa) were chosen for these experiments based on the documented ability of histones and DNA repair proteins to engage in the formation of DNA-protein cross-links (3,4,8,10).…”