Crystal Structure of an i-Motif from the HRAS Oncogene Promoter

Abstract

An i-motif is a non-canonical DNA structure implicated in gene regulation and linked to cancers. The C-rich strand of the HRAS oncogene, 5'-CGCCCGTGCCCTGCGCCCGCAACCCGA-3' (herein referred to as iHRAS), forms an i-motif in vitro but its exact structure was unknown. HRAS is a member of the RAS proto-oncogene family. About 19 % of US cancer patients carry mutations in RAS genes. We solved the structure of iHRAS at 1.77 Å resolution. The structure reveals that iHRAS folds into a double hairpin. The two double hairpins associate in an antiparallel fashion, forming an i-motif dimer capped by two loops on each end and linked by a connecting region. Six C-C⁺ base pairs form each i-motif core, and the core regions are extended by a G-G base pair and a cytosine stacking. Extensive canonical and non-canonical base pairing and stacking stabilizes the connecting region and loops. The iHRAS structure is the first atomic resolution structure of an i-motif from a human oncogene. This structure sheds light on i-motifs folding and function in the cell.

Keywords: Dimers; Gene Regulation; Non-Canonical Base Pairing; X-Ray Crystallography; i-Motifs.

Figure 1.

Structure of the dimeric iHRAS iM. A) Cartoon representation with purines, pyrimidines, and sugars shown as filled rings. Chains A and B are colored green and blue, respectively. B) iHRAS structure surrounded by the electron density at I/σ = 1.0. C) iHRAS schematics with nucleotide numbering. Nucleotides are colored by base: thymines are blue, guanines are green, cytosines are brown, adenines are magenta, and the 5Br-C located at position 4 is colored dark brown. Nucleotides from chain B are marked with an apostrophe (’).

Figure 2.

Illustration of non-canonical base pairs observed in the structure of iHRAS.

Figure 3.

Base pairing and stacking interactions in iHRAS. A) The homopurine pair, G6-G19’, stacks onto the cytosine pair, C5-C18’. This arrangement is further stabilized by stacking with C20’. B) A four-adenine π-stack is formed by A21’-A22’-A22*-A21*. C) The G2’-C14 Watson-Crick pair and the C1’-G15 Hoogsteen pair interact in the connecting region. D) T12-T12’ homopyrimidine pair stacks onto the G2-C14’ and the G2’-C14 base pairs. E) G13-G13’ stacks onto T12-T12’. Note, interactions listed in A-C refer to iM-1. Identical set of interactions are found in iM-2. Bases marked with an apostrophe belong to chain B and those marked with an asterisk are from a symmetry mate.

Figure 4.

Water networks in A) the major groove and B) loop region of iHRAS. Water molecules are colored purple. Hydrogen bonds are indicated by dashed lines. The water molecule with strong electron density is marked with an asterisk in panel B. Nucleotides are colored by atoms with P in orange, O in red, N in blue, and C in beige. The stacking of the G6-G19’ base pair and C20’ onto the iM-1 C5-C18’ base pair is clearly visible in panel B.

Figure 5.

The pathway to the iHRAS monomer. A) A proposed transition between iHRAS iM dimer and monomer. The intramolecular model of iHRAS, depicted in blue (right), is a regularized structural model and not a real structure. B) Schematics of the monomeric iHRAS