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In a previous study we showed that replication through the N1-methyl-deoxyadenosine (1-MeA) adduct in human cells is mediated via three different Polι/Polθ, Polη, and Polζ dependent pathways. Based on biochemical studies with these Pols, in the Polι/Polθ pathway, we inferred a role for Polι in the insertion of a nucleotide (nt) opposite 1-MeA and of Polθ in extension of synthesis from the inserted nt; in the Polη pathway, we inferred that this Pol alone would replicate through 1-MeA; in the Polζ pathway, however, the Pol required for inserting a nt opposite 1-MeA had remained unidentified. In this study, we provide biochemical and genetic evidence for a role for Polλ in inserting the correct nt T opposite 1-MeA, from which Polζ would extend synthesis. The high proficiency of purified Polλ for inserting a T opposite 1-MeA implicates a role for Polλ - which normally uses W-C base pairing for DNA synthesis – in accommodating 1-MeA in a syn confirmation and forming a Hoogsteen base pair with T. The potential of Polλ to replicate through DNA lesions by Hoogsteen base pairing adds another novel aspect to Polλ's role in translesion synthesis in addition to its role as a scaffolding component of Polζ. We discuss how the action mechanisms of Polλ and Polζ could be restrained to inserting a T opposite 1-MeA and extending synthesis thereafter, respectively.

Background In stabilization of the G-quadruplex, formation of a Hoogsteen base-pair between the guanine (G) bases is essential. However, the contribution of each Hoogsteen base-pair at different positions to whole stability of the G-quadruplex has not been known. In this study, the effect of a deficiency of the Hoogsteen type hydrogen bond in the G-quadruplex stability was investigated. Spectral properties of meso-tetrakis(1-methylpyridinium-4-yl)porphyrin (TMPyP) associated with various G-quadruplexes were also examined. Methods The thermal stability of the thrombin-binding DNA aptamer 5′G1G2TTG5G6TG8TG10G11TTG14G15 G-quadruplex, in which the guanine (G) base at 1, 2, 5, 6 and 8th positions was replaced with an inosine (I) base, one at a time, was investigated by circular dichroism (CD). The absorption, CD and fluorescence decay curve for the G-quadruplex associated TMPyP were also measured. Results The transition from the G-quadruplex to a single stranded form was endothermic and induced by an increase in entropy. The order in stability was 0>8>6>2>5>1, where the numbers denote the position of the replacement and 0 represents no replacements of the G base, suggesting the significant contribution of the G1 base in the stability of the G-quadruplex. Alteration in the spectral property of TMPyP briefly followed the order in thermal stability. Conclusions Replacement of a G base with an I base resulted in destabilization of the G-quadruplex. The missing hydrogen bond at position 1 destabilized the G-quadruplex most efficiently. TMPyP binds near the I base-replaced location namely, the side of the G-quadruplex. General significance The Hoogsteen base-pairing is confirmed to be essential in stabilization of G-quadruplex. When G is replaced with I, the latter base is mobile to interact with cationic porphyrin., Graphical abstract, Highlights • Thermal stability of a quadruplex, 5´G1G2TTG5G6TG8TG9G10TTG11G12 was investigated. • Replacement of G by I base decreases melting temperature. • The stability decreased 0>8>6>2>5>1, where numbers denotes the position replaced. • Replaced I base interacts with a cationic porphyrin, TMPyP.

A new intercalating nucleic acid monomer X was obtained in high yield starting from alkylation of 4-iodophenol with (S)-(+)-2-(2,2-dimethyl-1,3-dioxolan-4-yl)ethanol under Mitsunobu conditions followed by hydrolysis with 80% aqueous acetic acid to give a diol which was coupled under Sonogashira conditions with trimethylsilylacetylene (TMSA) to achieve the TMS protected (S)-4-(4-((trimethylsilyl)ethynyl)phenoxy)butane-1,2-diol. Tetrabutylammonium flouride was used to remove the silyl protecting group to obtain (S)-4-(4-ethynylphenoxy)butane-1,2-diol which was coupled under Sonogashira conditions with 2-(9-bromo-6H-indolo[2,3-b]quinoxalin-6-yl)-N,N-dimethylethanamine to achieve (S)-4-(4-((6-(2-(dimethylamino)ethyl)-6H-indolo[2,3-b]quinoxalin-9-yl)ethynyl)phenoxy)butane-1,2-diol. This compound was tritylated with 4,4'-dimethoxytrityl chloride followed by treatment with 2-cyanoethyltetraisopropylphosphordiamidite in the presence of N,N'-diisopropyl ammonium tetrazolide to afford the corresponding phosphoramidite. This phosphoramidite was used to insert the monomer X into an oligonucleotide which was used for thermal denaturation studies of a corresponding parallel triplex.