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.

Scheme 1. Generation of Site-Specific DNA–Protein Conjugates 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 Modified Green Fluorescent Protein (6×His-eGFP)

The azide group was introduced by enzymatic prenylation of eGFP protein containing a C-terminal CVIA sequence with protein farnesyltransferase (PFTase) using an azide-containing farnesyl diphosphate substrate analogue.

Scheme 2. Synthesis of Site-Specific DNA–Peptide Cross-Links by Copper-Catalyzed Azide–Alkyne Cycloaddition Reaction

Synthetic 10-mer and 23-mer peptides were prepared via solid phase peptide synthesis and appended with an N-terminal 4-azidobutanoic acid group.

Figure 1

Generation of site-specific DNA–protein cross-links (DPCs) by Cu-catalyzed azide–alkyne cycloaddition. (A) SDS-PAGE analysis of DPCs generated by using 6×His-eGFP-N₃ protein and ³²P-end-labeled DNA 23-mer (5′-AGG GTT TTC CCA G C8-alkyne-dUC ACG ACG TT-3′, where C8-alkyne-dU is 5-(octa-1,7-diynyl)-uracil). Lane 1: Alkyne containing DNA. Lane 2: Reaction mixture following cycloaddition between C8-alkyne-dU-containing DNA and 6×His-eGFP-N₃ protein. Lane 3: Proteinase K digested reaction from lane 2. (B) The same reaction as in panel A conducted with unlabeled DNA and separated by 12% SDS-PAGE. Proteins were visualized via SimplyBlue staining. Lane M: protein marker. Lane 1: 6×His-eGFP-N₃. Lane 2: reaction mixture following Cu-catalyzed cycloaddition between 6×His-eGFP-N₃ protein and alkyne containing DNA. Lane 3: Reaction mixture following cycloaddition conducted in the absence of Cu. (C) The yields of cycloaddition-induced DPCs increase with increased protein:DNA molar ratios. The reaction was conducted as in panel B, but the molar ratio of DNA:6×His-eGFP-N₃ was varied between 1:1 and 1:6. (D) Denaturing PAGE analysis of DNA–peptide conjugates generated using 10-mer peptide (N₃(CH₂)₃COEQKLISEEDLNH₂) and alkyne containing DNA 23-mer. Lane 1: C8-alkyne-dU containing 23-mer alone. Lane 2: Reaction mixture following Cu-catalyzed cycloaddition between C8-alkyne-dU-containing DNA 23-mer and peptide-N₃. Lane 3: The same reaction as in Lane 2 conducted in the absence of Cu. Lane 4: Proteinase K digested reaction from Lane 2.

Figure 2

Mass spectrometry characterization of DNA–peptide and DNA–protein conjugates. (A) NanoLC-nanospray-MS/MS characterization of DNA–peptide conjugates generated using 10-mer peptide (N₃(CH₂)₃COEQKLISEEDLNH₂) and C8-alkyne-dU-containing DNA 20-mer. Following gel purification as shown in Figure 1, the DNA component of the cross-link was digested with phosphodiesterases and alkaline phosphatase, and the resulting peptide-nucleoside conjugate (m/z 823.40, doubly charged) was sequenced by nanoLC-nanospray-MS/MS on an Orbitrap Velos mass spectrometer. (B) NanoLC-nanospray-MS/MS spectrum of eGFP tryptic peptide, CVIA, cross-linked to 5-(octa-1,7-diynyl)-2′-deoxyuridine monophosphate. DPCs were generated by Cu-catalyzed cycloaddition between 6×His-eGFP-N₃ and C8-alkyne-dU containing DNA 20-mer, and DPCs were isolated by 12% SDS-PAGE as shown in Figure 1. DNA component of the DPCs was digested with phosphodiesterase I, and the resulting protein–nucleotide conjugate (m/z 532.77, doubly charged) was subjected to tryptic digestion followed by MS/MS analysis on an Orbitrap Velos mass spectrometer.

Scheme 3. Sequences of DNA Oligomers Used for Conjugation Reactions with Proteins and Peptides (A) and DNA Substrates Employed in Standing Start (B) and Running Start Primer Extension Experiments (C)

Figure 3

Extension of ³²P-labeled primers containing unmodified dT or DNA–protein and DNA–peptide conjugates of increased size adduct by human lesion bypass polymerases hPol κ (A–C) and hPol η (D–F) under standing start conditions. 13-Mer primers were annealed with 18-mer templates containing unmodified dT or covalent cross-links to 6×His-eGFP, 23-mer peptide, or 10-mer peptide (Scheme 3B). The resulting primer–template complexes (40 nM) were incubated in the presence of hPol κ (400 nM) or hPol η (160 nM). The polymerase reactions were started by the addition of the four dNTPs (500 μM) and quenched at the indicated time points. The quenched samples were separated by 20% (w/v) denaturing polyacrylamide gel electrophoresis and visualized by phosphorimaging analysis.

Figure 4

Extension of ³²P-labeled primers containing unmodified dT or DNA–protein and DNA–peptide conjugates of increased size adduct by human lesion bypass polymerases hPol κ (A–C) and hPol η (D–F) under running start conditions. The ³²P-end-labeled 9-mer primers were annealed to the 18-mer templates containing unmodified dT, GFP, 23-mer peptide, or 10-mer peptide (Scheme 3C). The resulting primer–template complexes (40 nM) were incubated at 37 °C in the presence of hPol κ (400 nM) and hPol η (160 nM). Reactions were started by the addition of all four dNTPs (500 μM) and quenched at indicated time points. The extension products were resolved by 20% (w/v) denaturing PAGE and visualized by phosphorimaging analysis.