Mutagenicity of a Model DNA-Peptide Cross-Link in Human Cells: Roles of Translesion Synthesis DNA Polymerases

Abstract

DNA-protein cross-links are formed upon exposure of cellular DNA to various agents, including antitumor drugs, UV light, transition metals, and reactive oxygen species. They are thought to contribute to cancer, aging, and neurodegenerative diseases. It has been proposed that DNA-protein cross-links formed in cells are subject to proteolytic degradation to the corresponding DNA-peptide cross-links (DpCs). To investigate the effects of DpCs on DNA replication, we have constructed plasmid DNA containing a 10-mer Myc peptide covalently linked to C7 of 7-deaza-dG, a hydrolytically stable mimic of N7-dG lesions. Following transfection in human embryonic kidney cells (HEK 293T), progeny plasmids were recovered and sequenced. Translesion synthesis (TLS) past DpC was 76% compared to unmodified control. The DpC induced 20% targeted G→A and G→T plus 15% semi-targeted mutations, notably at a guanine (G5) five bases 3’ to the lesion site. Proteolytic digestion of the DpC reduced the mutation frequency considerably, indicating that the covalently attached 10-mer peptide was responsible for the observed mutations. TLS efficiency and targeted mutations were reduced upon siRNA knockdown of pol η, pol κ, or pol ζ, indicating that they participate in error-prone bypass of the DpC lesion. However, the semi-targeted mutation at G5 was only reduced upon knockdown of pol ζ, suggesting its critical role in this type of mutations. Our results indicate that DpCs formed at the N7 position of guanine can induce both targeted and semi-targeted mutations in human cells and that the TLS polymerases play a critical role in their error-prone bypass.

Synthesis of Dihydroxyalkynyl and Dihydroxyalkyl Nucleotides as Building Blocks or Precursors for Introduction of Diol or Aldehyde Groups to DNA for Bioconjugations

“…We then wanted to explore capability of the diol moietyi n DNA to be converted to an aldehyde [16][17][18][19][20] and used for bioconjugations (Scheme 2). Double-stranded DNA containing the diol DNA_1U DHA and DNA_4U DHA ,a sw ell as DNA containing the aldehyde DNA_1U CHO or DNA_4U CHO werep repared by the PEX reactiono ns emi-preparative scale.…”

“…[40] On the other hand, the aldehydelinked dU CHO TP worked well in PEX but gave PCR amplification only in am ixture with natural TTP.B oth the synthesis of diollinked nucleotide buildingb locks and aldehyde-linked dNTP and DNA are shorter and more facile than the previously reported literature procedures. [16][17][18][19][20] The aldehyde-modifiedD NA prepared by either direct incorporation of an aldehyde-linked nucleotide or by incorporation of ad iol-nucleotide followed by periodate oxidativec leavage served very well for bioconjugations through hydrazone formation or reductive aminations (either using NaBH 3 CN or transfer hydrogenation using an Ir catalyst and formate). Also the diollinked nucleotides and DNA could have some potentiala pplications,f or example, in redox-labellingo fD NA through osmy-lation of diols [41] or in selection of modified DNA aptamers.…”

“…[13] Ap articularly useful reactive group is an aldehyde function, [14] which can undergo number of useful transformations. Literature examples on aldehyde-linked DNA mostly include the fluorogenic modifications of naturally occurring 5-formylcytosine or 5-formyluracil, [15] or incorporationo fv icinal diols, [16][17][18][19][20] abasic site [21] or furan [22] and their oxidative cleavage to aldehydes. [16][17][18][19][20][21][22] Reducing sugars have also been used as aldehyde equivalent for DNA metallization through the Tollens reaction.…”

“…Literature examples on aldehyde-linked DNA mostly include the fluorogenic modifications of naturally occurring 5-formylcytosine or 5-formyluracil, [15] or incorporationo fv icinal diols, [16][17][18][19][20] abasic site [21] or furan [22] and their oxidative cleavage to aldehydes. [16][17][18][19][20][21][22] Reducing sugars have also been used as aldehyde equivalent for DNA metallization through the Tollens reaction. [23] Only few examples show direct chemical [24] or enzymatic [25][26][27] incorporation of aldehyde-modified nucleotides.…”

Formation and repair of DNA-protein crosslink damage

“…For instance, glutathione DNA-peptide crosslinks and a 10-mer Myc peptide incur mutagenic consequences in human fibroblasts and HEK 293T cells, respectively, particularly generating single base substitutions [Voitkun et al, 1998; Pande et al, 2017]. Translesion synthesis polymerases are important in bypassing smaller DpC lesions, thereby alleviating the replication block [Duxin et al, 2014; Yeo et al, 2014; Wickramaratne et al, 2016; Pande et al, 2017]. It is likely that proteolytic processing of larger DPCs and DpCs enables bypass by translesion synthesis polymerases as well.…”