(A) Alternative bases that are structurally similar to standard nucleobases but have altered base-pairing specificity. The pattern of hydrogen bond acceptors and donors (indicated by arrows) is reversed for the non-standard base pair between 5-methyl isocytosine (isoC) and isoguanosine (isoG) (right) compared with a standard cytosine (C) guanosine (G) base pair (left). (B) Mismatch repair model of a template containing consecutive non-standard base pairs. The polymerase domain (purple) of a bacterial DNA polymerase catalyzes repair DNA synthesis by incorporation of nucleoside triphosphates. The 5′ nuclease domain (pink) of the polymerase excises a standard base (blue) mismatched with a non-natural base (isoG, red) in the template. DNA ligase (green) completes repair by formation of a covalent bond between isoC (green) and isoG (red).

Polymerase extension through consecutive non-standard bases. A cyanine 5 (CY5) 5′-labeled DNA primer hybridized to a complementary DNA template was extended by Taq DNA polymerase in the presence of various non-standard and standard deoxynucleoside triphosphates. Extension products were resolved by denaturing polyacrylamide gel electrophoresis and detected by fluorescence imaging. Deoxynucleoside triphosphate combinations are indicated on top of each lane. Lane 1, no triphosphate (–); lane 2, d-isoCTP (iC); lane 3, d-isoCTP + d-isoGTP (iC,iG); lane 4, d-isoCTP + d-isoGTP + dGTP (iC,iG,G); lane 5, d-isoCTP + d-isoGTP + dNTP, where N = A, C, G, T (All); lane 6, d-isoCTP + dNTP (-iG). Reaction products are diagrammed on the left and are lined up with their respective band size.

A model system demonstrating FEN-1 nuclease cleavage of a bifurcated site. DNA both 5′-labeled with fluorescein (F) and 3′-labeled with cyanine 3 (CY) was annealed to equimolar amounts of complementary hairpin-forming DNA. The 3′ ends of the hairpins either contained an isoC (iC) or no base (–) at position Y. The complementary base in the hairpin opposite position Y was isoG. The annealed substrates were treated with T.aquaticus DNA polymerase containing the 5′ FEN-1 domain in the absence of nucleoside triphosphates. Cleavage products were resolved by denaturing PAGE and detected by fluorescence imaging.

Ligation between consecutive non-standard bases. A 3′-labeled single-stranded DNA containing a 5′ phosphoryl isoguanosine (iG) was annealed to the complementary 5′ single-stranded ends of one of three DNA hairpins. The hairpins used all differed at position Y. The annealed substrates were treated with T4 DNA ligase under appropriate conditions. Ligation products including the 5′ adenylated iG probe intermediate (Ad-iG) indicated on the left of the gel were resolved by denaturing PAGE and detected by fluorescence imaging. A mock product was synthesized chemically and run along with the unligated probe as a size marker (M).

Complete repair of the mismatched bifurcated model system at a site containing consecutive non-standard DNA bases. A single-stranded DNA both 5′-labeled with fluorescein (F) and 3′-labeled with cyanine 3 (CY) was annealed to equimolar amounts of complementary hairpin-forming DNA. The 5′ thymidine (T) of the single-stranded DNA was mispaired with an isoguanosine (iG) in the hairpin. Repair reactions contained combinations of ligase, polymerase, FEN-1 nuclease and 5-methyl isocytosine triphosphate (iCTP) as indicated above the lanes. Complete repair reaction product, cleavage intermediate, 5′-phosphoryl probe and ligation intermediate adenylated probe (Ad-iG) indicated on the left of the gel were resolved by denaturing PAGE and detected by fluorescence imaging. A mock repair product was synthesized chemically and used as a size marker (M).