Abstract
DNA-protein cross-links (DPCs) are bulky DNA lesions that form both endogenously and following exposure to bis-electrophiles such as common antitumor agents. The structural and biological consequences of DPCs have not been fully elucidated due to the complexity of these adducts. The most common site of DPC formation in DNA following treatment with bis-electrophiles such as nitrogen mustards and cisplatin is the N7 position of guanine, but the resulting conjugates are hydrolytically labile and thus are not suitable for structural and biological studies. In this report, hydrolytically stable structural mimics of N7-guanine-conjugated DPCs were generated by reductive amination reactions between the Lys and Arg side chains of proteins/peptides and aldehyde groups linked to 7-deazaguanine residues in DNA. These model DPCs were subjected to in vitro replication in the presence of human translesion synthesis DNA polymerases. DPCs containing full-length proteins (11-28 kDa) or a 23-mer peptide blocked human polymerases η and κ. DPC conjugates to a 10-mer peptide were bypassed with nucleotide insertion efficiency 50-100-fold lower than for native G. Both human polymerase (hPol) κ and hPol η inserted the correct base (C) opposite the 10-mer peptide cross-link, although small amounts of T were added by hPol η. Molecular dynamics simulation of an hPol κ ternary complex containing a template-primer DNA with dCTP opposite the 10-mer peptide DPC revealed that this bulky lesion can be accommodated in the polymerase active site by aligning with the major groove of the adducted DNA within the ternary complex of polymerase and dCTP.
Highlights
DNA-protein cross-links (DPCs) are bulky DNA lesions that form both endogenously and following exposure to bis-electrophiles such as common antitumor agents
In a more recent study, we have shown that human cells deficient in nucleotide excision repair were sensitized toward nitrogen mustard toxicity and contained increased numbers of N7-guanine-conjugated DPC lesions [19]
In the resulting model crosslinks, Lys or Arg side chains of proteins and peptides are conjugated to the 7-deazaguanine residues of DNA via a two-carbon linker, creating a structure that is analogous to DPC lesions induced in cells by antitumor nitrogen mustards [3] and ethylene dibromide [26]
Summary
Synthesis and Characterization of DNA-Protein and DNAPeptide Cross-links—Model DNA-protein and DNA-peptide conjugates were prepared by reductive amination reactions between the Lys/Arg side chains of proteins/peptides and 7-deaza-7-(2-oxoethyl)-2Ј-deoxyguanosine of DNA (Fig. 1). Running start experiments were conducted using 13-mer primer P2 (5-CTA TGA FAMTGG TAC C-3) annealed to 39-mer templates T10 –T16 (5Ј-GAA AGA AGX ACA GAA GAG GGT ACC ATC ATA GAG TCA GTG-3Ј where X is unmodified dG, deaza-DHP-dG, or DNA-protein conjugate; Table 1 and Fig. 3B). In this primertemplate complex, the 3Ј-end of the primer is placed 10 nucleotides upstream from the adducted site (Ϫ10 primer; Fig. 3B). This may contribute to inefficient lesion bypass and polymerase stalling at the peptide cross-link (Figs. 6 and 7)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
