Computational Evaluation of Nucleotide Insertion Opposite Expanded and Widened DNA by the Translesion Synthesis Polymerase Dpo4

Abstract

Expanded (x) and widened (y) deoxyribose nucleic acids (DNA) have an extra benzene ring incorporated either horizontally (xDNA) or vertically (yDNA) between a natural pyrimidine base and the deoxyribose, or between the 5- and 6-membered rings of a natural purine. Far-reaching applications for (x,y)DNA include nucleic acid probes and extending the natural genetic code. Since modified nucleobases must encode information that can be passed to the next generation in order to be a useful extension of the genetic code, the ability of translesion (bypass) polymerases to replicate modified bases is an active area of research. The common model bypass polymerase DNA polymerase IV (Dpo4) has been previously shown to successfully replicate and extend past a single modified nucleobase on a template DNA strand. In the current study, molecular dynamics (MD) simulations are used to evaluate the accommodation of expanded/widened nucleobases in the Dpo4 active site, providing the first structural information on the replication of (x,y)DNA. Our results indicate that the Dpo4 catalytic (palm) domain is not significantly impacted by the (x,y)DNA bases. Instead, the template strand is displaced to accommodate the increased C1'-C1' base-pair distance. The structural insights unveiled in the present work not only increase our fundamental understanding of Dpo4 replication, but also reveal the process by which Dpo4 replicates (x,y)DNA, and thereby will contribute to the optimization of high fidelity and efficient polymerases for the replication of modified nucleobases.

Keywords: DNA replication; Dpo4; bypass polymerase; expanded DNA; molecular dynamics; translesion synthesis; widened DNA; xDNA; yDNA.

Figure 1

Chemical structures of the DNA, xDNA and yDNA bases considered in the current study.

Figure 2

The finger, palm, thumb, and little finger domains of Dpo4, as well as the tether region. The positions of potentially important residues in each domain are indicated with spheres.

Figure 3

(a) Hydrogen-bonding interactions with the dNTP (blue) and primer terminus (purple), with the reaction distance shown in green; (b) Active site coordination of the catalytic (blue) and binding (red) Mg²⁺ ions.

Figure 4

Geometry of the dN:dNTP hydrogen-bonding interaction during Dpo4 replication of a DNA, xDNA or yDNA base. Average distance and angle for each hydrogen-bonding interaction are given, as well as hydrogen-bonding occupancies, which are based on a distance cutoff of <3.4 Å and an angle cutoff of >120°.

Figure 5

Overlays of representative MD structures of the (x,y)DNA base pairs with representative MD structures of the (a) dG:dCTP (black) or (b) dT:dATP (white) pair in the Dpo4 ternary complexes.

Figure 6

Alterations in the Dpo4 conformation to accommodate an (x,y)DNA base in the template strand. (a) Overlay of the representative MD structures of the Dpo4 ternary complex for the replication of dxA (green) and dG (black). Overlays for all systems are provided in the Supplementary Material (Figure S8); (b) Possible hydrogen-bonding arrangements within the Ser40···Ser34···d(x,y)N chain.

Figure 7

Overlay of the Cα protein backbone in representative MD structures of the Dpo4 ternary complex for the replication of: (a) dyT (blue); or (b) dxC (yellow), and dT (white). See Figure S10 for all systems.

Figure 8

Examples of size-extended anti-purine:anti-purine mismatches.

Figure 9

Damaged DNA bases with extended ring systems that lack Watson–Crick hydrogen bonding.