Structures of DNA duplexes containing O6-carboxymethylguanine, a lesion associated with gastrointestinal cancer, reveal a mechanism for inducing pyrimidine transition mutations

Abstract

N-nitrosation of glycine and its derivatives generates potent alkylating agents that can lead to the formation of O6-carboxymethylguanine (O6-CMG) in DNA. O6-CMG has been identified in DNA derived from human colon tissue, and its occurrence has been linked to diets high in red and processed meats. By analogy to O6-methylguanine, O6-CMG is expected to be highly mutagenic, inducing G to A mutations during DNA replication that can increase the risk of gastrointestinal and other cancers. Two crystal structures of DNA dodecamers d(CGCG[O6-CMG]ATTCGCG) and d(CGC[O6-CMG]AATTCGCG) in complex with Hoechst33258 reveal that each can form a self-complementary duplex to retain the B-form conformation. Electron density maps clearly show that O6-CMG forms a Watson–Crick–type pair with thymine similar to the canonical A:T pair, and it forms a reversed wobble pair with cytosine. In situ structural modeling suggests that a DNA polymerase can accept the Watson–Crick–type pair of O6-CMG with thymine, but might also accept the reversed wobble pair of O6-CMG with cytosine. Thus, O6-CMG would permit the mis-incorporation of dTTP during DNA replication. Alternatively, the triphosphate that would be formed by carboxymethylation of the nucleotide triphosphate pool d[O6-CMG]TP might compete with dATP incorporation opposite thymine in a DNA template.

Bypass of Mutagenic O6-Carboxymethylguanine DNA Adducts by Human Y- and B-Family Polymerases

“…24,46,47 Despite the limited activity of Pol ι, it could be shown that the enzyme displayed a preference for incorporating dTMP over dCMP opposite O 6 -CMG, which corresponds as well with previous observations for the bypass of O 6 -alkylG adducts by Pol ι. 24,48 30,31 However, the latter is derived from a structure of the free duplex and does not account for the important influences of the enzyme. In contrast to Pol η and κ, Pol ι had no PLS activity, suggesting that its role, if any, in O 6 -CMG bypass may be for the insertion rather than extension step.…”

“…Furthermore, recent crystal structures of DNA containing O 6 -CMG suggest a chemical basis for the mispairing involving a Watson-Crick-type or high-wobble-type O 6 -CMG:T pair. 30,31 Nevertheless, to our knowledge, no data is available regarding O 6 -CMG as a substrate for TLS catalyzed by human DNA polymerases, therefore its miscoding properties are not known.…”

Nanopore Sequencing Accurately Identifies the Mutagenic DNA Lesion O6‐Carboxymethyl Guanine and Reveals Its Behavior in Replication

“…[14] O 6 -Carboxy-methylguanine (O 6 -CMG,F igure 1a)i sa ni mportant O 6 G lesion because it shows ahigh tendency to cause miscoding in DNAr eplication. [15] Indeed its appearance in vivo is proportional to the consumption of red meat and may lead to colorectal cancer. [16] Precise detection of O 6 -CMG within the genome is important to unveil the mechanisms of its formation, to determine whether certain sites are resistant to repair, [11a] or to explore its potential as at umor biomarker.…”

O6-Carboxymethylguanine in DNA forms a sequence context-dependent wobble base-pair structure with thymine

“…1) containing O 6 -CMG at residue positions that place the modified base opposite T or C in the palindromic Bform Dickerson-Drew sequence d(CGCGAATTCGCG) (Dickerson & Drew, 1981). This revealed that O 6 -CMG forms a Watson-Crick-type pair with T similar to the canonical A:T pair and forms a reversed-wobble pair with C (Zhang et al, 2013). These interaction geometries are those expected from the chemical structure of O 6 -CMG.…”

“…All of the global and local helical parameters, as well as the torsion angles and pseudo-rotation phase angles of the sugar rings, were calculated using 3DNA (Lu & Olson, 2003). Some of these are given in Supplementary Tables S1, S2 and (Zhang et al, 2013), which required the addition of Hoechst 33258 to facilitate their crystallization. In contrast, the present ODN d(CGC[O 6 -CMG]AATTTGCG) (O 6 -CMG4T) was easily crystallized: four crystal forms were obtained under different conditions, two of which were obtained in the absence of Hoechst 33258.…”