Probing the Conformational Restraints of DNA Damage Recognition with β-L-Nucleotides

Abstract

The DNA building blocks 2'-deoxynucleotides are enantiomeric, with their natural β-D-configuration dictated by the sugar moiety. Their synthetic β-L-enantiomers (βLdNs) can be used to obtain L-DNA, which, when fully substituted, is resistant to nucleases and is finding use in many biosensing and nanotechnology applications. However, much less is known about the enzymatic recognition and processing of individual βLdNs embedded in D-DNA. Here, we address the template properties of βLdNs for several DNA polymerases and the ability of base excision repair enzymes to remove these modifications from DNA. The Klenow fragment was fully blocked by βLdNs, whereas DNA polymerase κ bypassed them in an error-free manner. Phage RB69 DNA polymerase and DNA polymerase β treated βLdNs as non-instructive but the latter enzyme shifted towards error-free incorporation on a gapped DNA substrate. DNA glycosylases and AP endonucleases did not process βLdNs. DNA glycosylases sensitive to the base opposite their cognate lesions also did not recognize βLdNs as a correct pairing partner. Nevertheless, when placed in a reporter plasmid, pyrimidine βLdNs were resistant to repair in human cells, whereas purine βLdNs appear to be partly repaired. Overall, βLdNs are unique modifications that are mostly non-instructive but have dual non-instructive/instructive properties in special cases.

Keywords: AP endonucleases; DNA glycosylases; DNA polymerases; DNA repair; translesion synthesis; β-L-nucleotides.

Figure 1

(a) Structures of normal β-D- (top) and mirror-image β-L-deoxynucleotides (bottom). (b) Structure of normal DNA (top, 5′-TTC-3′/5′-GAA-3′, PDB ID 355D [12]) and a hypothetical structure of 5′-T[βLdT]C-3′/5′-GAA-3′ maintaining Watson–Crick bonding in DNA (bottom; see Section 4 for the description of model building). Dashes indicate hydrogen bonds.

Figure 2

Primer extension by RBpol and Pol κ across βLdNs on a primer–template substrate. (a) Scheme of the substrate. FAM, fluorescent label; β, modified nucleotide. (b) Nucleotide incorporation by RBpol. (c) Nucleotide incorporation by Pol κ. +1 …+3, number of nucleotides added.

Figure 3

Primer extension by Pol β across βLdNs on primer–template(pt) and gapped (gap) substrates. (a) Scheme of the substrates. FAM, fluorescent label; β, modified nucleotide; p, 5′-terminal phosphate. (b–e) Insertion of dNMPs by Pol β opposite to βLdA (b), βLdG (c), βLdT (d) and βLdC (e). +1 …+2, number of nucleotides added.

Figure 4

Cleavage of substrates containing βLdNs by DNA glycosylases MutY (a), MBD4 (b), Fpg (c) and OGG1 (d). S, oligonucleotide substrate; P, cleavage product. βA, βLdA; βC, βLdC; βG, βLdG; βT, βLdT.

Figure 6

Transcriptional mutagenesis and repair induced by βLdNs in HeLa cells. (a) Scheme of the processes induced by a lesion in the eGFP reporter gene. (b) Relative EGFP expression normalized for the fluorescence of the control G-construct (n = 3, mean ± SD shown). Differences between constructs: p < 0.05 (*); p < 0.01 (**); p < 0.005 (***); two-tailed Student’s t-test with Bonferroni correction applied. The brightness of the bars is proportional to the EGFP expression level.

Figure 5

Cleavage of substrates containing βLdNs by AP endonucleases. (a) Cleavage of βLdA:T, βLdT:A, βLdG:C and βLdC:G substrates by human APE1 and E. coli Nfo and Xth. (b) Cleavage of βLdA:T by Xth under different conditions. S, substrate, P, cleavage product.