Responses of DNA Mismatch Repair Proteins to a Stable G-Quadruplex Embedded into a DNA Duplex Structure

Abstract

DNA mismatch repair (MMR) plays a crucial role in the maintenance of genomic stability. The main MMR protein, MutS, was recently shown to recognize the G-quadruplex (G4) DNA structures, which, along with regulatory functions, have a negative impact on genome integrity. Here, we studied the effect of G4 on the DNA-binding activity of MutS from Rhodobacter sphaeroides (methyl-independent MMR) in comparison with MutS from Escherichia coli (methyl-directed MMR) and evaluated the influence of a G4 on the functioning of other proteins involved in the initial steps of MMR. For this purpose, a new DNA construct was designed containing a biologically relevant intramolecular stable G4 structure flanked by double-stranded regions with the set of DNA sites required for MMR initiation. The secondary structure of this model was examined using NMR spectroscopy, chemical probing, fluorescent indicators, circular dichroism, and UV spectroscopy. The results unambiguously showed that the d(GGGT)4 motif, when embedded in a double-stranded context, adopts a G4 structure of a parallel topology. Despite strong binding affinities of MutS and MutL for a G4, the latter is not recognized by E. coli MMR as a signal for repair, but does not prevent MMR processing when a G4 and G/T mismatch are in close proximity.

Impact of G-Quadruplexes on the Regulation of Genome Integrity, DNA Damage and Repair

“…Steric factors, determined by quadruplex topology, strongly influence the ability of G4 to coexist with a neighboring duplex in the same DNA structure. Parallel intramolecular G4s, usually formed in promotor regions, have 5′- and 3′-ends on opposite sides of the G4 core, thereby facilitating its placement inside the double helix without any structured linker [ 35 ]. In contrast, antiparallel G4s enchain ends on the same side, which requires a duplex linker to accommodate the G4 structure in B-DNA [ 66 ].…”

“…The binding of E. coli MutS (ecMutS) protein and the human homolog MutSα to tetrameric polymorphic DNA G4s has been reported [ 35 , 139 , 140 ], and the affinity of these proteins for G4 turned out to be 2–4-times higher than for DNA with a G/T mismatch, a specific substrate of the MutS. The binding of MutSα to G4 motifs of immunoglobulin class-switching regions was directly visualized by electron microscopy [ 140 ].…”

“…To answer the questions of whether G4 formation interferes with mismatch-induced DNA cleavage caused by the coordinated actions of MutS, MutL and MutH proteins, and whether G4 itself activates MMR responses, our lab has designed new DNA constructs containing a biologically relevant intramolecular parallel G4 stabilized in the context of double-stranded DNA with a set of DNA sites required to initiate the MMR pathway (G/T mismatch and MutH recognition site). These DNA models were created by the hybridization of partly complementary strands, one of which contained a G4 motif d(GGGT) 4 flanked by oligonucleotides, while the opposite strand lacked the site complementary to the G4-forming insert [ 35 ]. Using NMR spectroscopy, chemical probing, fluorescent indicators, circular dichroism and UV spectroscopy, the coexistence of parallel-stranded intramolecular G4 and duplex domains in the developed DNA models has been unequivocally proved.…”

“…For the most studied MutS homologs, it was found that the mode of MutS binding to intermolecular and intramolecular G4s is apparently common for prokaryotic and eukaryotic organisms, regardless of the strand discrimination mechanism [ 35 , 139 ]. Their involvement in DNA repair and recombination is also well known [ 142 , 143 , 144 ].…”

“…In recent years, the deleterious effects of G4s on genome integrity and their potential role in regulating the DNA repair machinery have become the subject of intense research. It has been shown that, among identified G4-binding proteins, there are many G4-resolving and repair proteins, such as special helicases and proteins involved in homologous recombination and other canonical repair pathways [ 34 , 35 ]. Recent findings have revealed novel functions of G4 structures in various methods of epigenetic regulation.…”