(CAG)n-hairpin DNA binds to Msh2–Msh3 and changes properties of mismatch recognition

Abstract

Cells have evolved sophisticated DNA repair systems to correct damaged DNA. However, the human DNA mismatch repair protein Msh2-Msh3 is involved in the process of trinucleotide (CNG) DNA expansion rather than repair. Using purified protein and synthetic DNA substrates, we show that Msh2-Msh3 binds to CAG-hairpin DNA, a prime candidate for an expansion intermediate. CAG-hairpin binding inhibits the ATPase activity of Msh2-Msh3 and alters both nucleotide (ADP and ATP) affinity and binding interfaces between protein and DNA. These changes in Msh2-Msh3 function depend on the presence of A.A mispaired bases in the stem of the hairpin and on the hairpin DNA structure per se. These studies identify critical functional defects in the Msh2-Msh3-CAG hairpin complex that could misdirect the DNA repair process.

Conformational trapping of Mismatch Recognition Complex MSH2/MSH3 on repair-resistant DNA loops

“…To test whether nucleotide binding to MSH2/MSH3 altered its association with DNA, we labeled each DNA template with fluorescein at the 5′-end of the bottom strand, and measured the DNA-binding affinity by fluorescence anisotropy (FA). In the absence of bound nucleotide, the apparent affinity of MSH2/MSH3 for both the ðCAÞ 4 loop and hairpin templates was in the low nanomolar range (Table 2), and was in good agreement with previous measurements (24,26,31 13 DNA in that the latter forms a hairpin comprising G-C hydrogen bonded base pairs and A/A mispaired bases every third nucleotide in the stem (24,26). To test for conformation differences between the two templates, we measured the proteininduced DNA conformational dynamics using smFRET.…”

“…Neither the ðCAÞ 4 loop nor the CAG stem had complementary sequences within the duplex portion of the template. Thus, the junction templates folded into stable extrahelical loops, which have been previously characterized in solution (26,31). Folding of the CAG loops creates A/A mismatches every third base pair in the stem, for a total of three mismatches in the ðCAGÞ 7 template or a total of six mismatches in the ðCAGÞ 13 template.…”

“…A small ðCAÞ 4 loop of DNA can be faithfully repaired by MSH2/MSH3 both in vitro (24,26) and in vivo (25,27,28). In contrast, hydrogen bonded CAG hairpin loops are not excised, and confer genomic instability through insertion and amplification of CAG repetitive tracts (29)(30)(31). Although ðCAÞ 4 loops and CAG hairpins both harbor three-way junctions, MSH2/ MSH3 interacts with them distinctly (24,26).…”

“…Although ðCAÞ 4 loops and CAG hairpins both harbor three-way junctions, MSH2/ MSH3 interacts with them distinctly (24,26). Why one template is repaired better than the other is not known, but the consequence is remarkable: Inefficient repair of CAG loops results in mutations that underlie more than 20 hereditary neurodegenerative or neuromuscular diseases (30)(31)(32)(33).…”

DNA mismatch repair: Molecular mechanism, cancer, and ageing

“…Repeat instability is reduced in MSH3-null mice but elevated in MSH6-null mice pointing to a role for MSH3 in promoting repeat instability (van den Broek et al, 2002). MSH2-MSH3 has been shown in vitro to target CAG-hairpin DNA and to be modulated by an A-A mismatch in the hairpin stem (Owen et al, 2005).…”