Abstract
N1-methyl adenine (1-MeA) is formed in DNA by reaction with alkylating agents and naturally occurring methyl halides. The 1-MeA lesion impairs Watson-Crick base pairing and blocks normal DNA replication. Here we identify the translesion synthesis (TLS) DNA polymerases (Pols) required for replicating through 1-MeA in human cells and show that TLS through this lesion is mediated via three different pathways in which Pols ι and θ function in one pathway and Pols η and ζ, respectively, function in the other two pathways. Our biochemical studies indicate that in the Polι/Polθ pathway, Polι would carry out nucleotide insertion opposite 1-MeA from which Polθ would extend synthesis. In the Polη pathway, this Pol alone would function at both the nucleotide insertion and extension steps of TLS, and in the third pathway, Polζ would extend from the nucleotide inserted opposite 1-MeA by an as yet unidentified Pol. Whereas by pushing 1-MeA into the syn conformation and by forming Hoogsteen base pair with the T residue, Polι would carry out TLS opposite 1-MeA, the ability of Polη to replicate through 1-MeA suggests that despite its need for Watson-Crick hydrogen bonding, Polη can stabilize the adduct in its active site. Remarkably, even though Pols η and ι are quite error-prone at inserting nucleotides opposite 1-MeA, TLS opposite this lesion in human cells occurs in a highly error-free fashion. This suggests that the in vivo fidelity of TLS Pols is regulated by factors such as post-translational modifications, protein-protein interactions, and possibly others.
Highlights
N1-methyladenine (1-MeA)3 is formed in DNA by reaction with Sn2 methylating agents such as methyl methanesulfonate and naturally occurring methyl halides [1,2,3]
translesion synthesis (TLS) Pols Required for Replicating through the 1-MeA Lesion—To identify the TLS Pols required for replicating through the 1-MeA lesion, we determined the effects of siRNA depletions of TLS Pols individually and in combinations on TLS frequency opposite this lesion carried on the leading strand template of the SV40-based plasmid
A Major Role of TLS in the Replicative Bypass of 1-MeA— Previously, we have determined the extent, genetic control, and mutagenicity of TLS opposite three DNA lesions, cis-syn TT dimer, [] TT photoproduct, and thymine glycol. These studies indicated that in nucleotide excision repair-defective XPA human fibroblasts, TLS opposite a cis-syn TT dimer or a [] TT photoproduct occurs with a frequency of ϳ40%, whereas TLS opposite these lesions in nucleotide excision repair-proficient fibroblasts occurs with a frequency of ϳ20%
Summary
Construction of Plasmid Vectors Containing 1-MeA—The 16-mer oligonucleotides containing an N1-methyl deoxyadenosine (Fig. 1A) were purchased from Trilink Biotechnologies (Santa Cruz, CA). Plasmid DNA obtained from blue colonies was analyzed to determine the mutation frequency, and the mutational changes were incorporated during TLS For details of these methods, see Yoon et al [11]. For nucleotide incorporation assays with Pol, 50 M dATP, dTTP, dCTP, or dGTP (Roche Applied Science) was used, and for examining synthesis through the 1-MeA lesion all 4 dNTPs (50 M each) were used. Primer extension by human Pol (1 nM) or yeast Pol (1 nM) was assayed in the presence of 10 M each dATP, dGTP, dTTP, and dCTP (Roche Applied Science) on DNA containing an A/T or 1-MeA/T primer terminal base pair; the standard DNA polymerase reaction (5 l) contained 25 mM Tris1⁄7HCl (pH 7.5), 5 mM MgCl2, 1 mM dithiothreitol, 100 g/ml BSA, 10% glycerol,. Reactions containing human Pol were carried out at 37 °C and yeast Pol at 30 °C for 10 min
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