G-Quadruplex Formed by the Promoter Region of the hTERT Gene: Structure-Driven Effects on DNA Mismatch Repair Functions

Abstract

G-quadruplexes (G4s) are a unique class of noncanonical DNAs that play a key role in cellular processes and neoplastic transformation. Herein, we focused on the promoter region of human TERT oncogene, whose product is responsible for the immortality of cancer cells. It has been shown by chemical probing and spectroscopic methods that synthetic 96-nt DNAs modeling the wild-type G-rich strand of the hTERT promoter and its variants with G>A point substitutions corresponding to somatic driver mutations fold into three stacked parallel G4s with sites of local G4 destabilization caused by G>A substitutions in the G4 motif. These models were used to elucidate how the hTERT multiG4 affects the binding affinity and functional responses of two key proteins, MutS and MutL, involved in the initial stage of DNA mismatch repair (MMR) in Escherichiacoli and Neisseriagonorrhoeae with different MMR mechanisms. We have shown for the first time that (i) point substitutions do not affect the effective binding of these proteins to the hTERT G4 structure, and (ii) the endonuclease activity of MutL from N. gonorrhoeae is significantly suppressed by the stable G4 scaffold. It is likely that some of the genomic instability associated with G4 may be related to the blockage of human intrinsic methyl-independent MMR attempting to operate near G4 structures.

New DNA Plasmid Model for Studying DNA Mismatch Repair Response to the G4 Structure

“…Strong specific interactions of G4s with MMR proteins MutS and MutL from various organisms including E. coli , N. gonorrhoeae , R. sphaeroides , and humans have been described previously [ 14 , 15 , 31 , 32 ]. However, the biological significance of G4 recognition by cellular proteins is not always clear.…”

“…In addition, we aimed to answer the question of whether a single G4 inserted into a DNA plasmid can affect the activity of ngMutL with an endonuclease function in the methyl-independent MMR inherent in eukaryotic organisms. It has previously been shown that ngMutL recognizes and preferentially binds isolated DNA G4 with high affinity, but, nevertheless, inefficiently cleaves the phosphodiester bonds inside of the G4-motif in TERT promoter fragment [ 32 ]. To clarify this issue, we analyzed ngMutL-mediated cleavage of the G4-pUC plasmid, which contained an extrahelical stable G4, as well as reference plasmids, G/C-pUC and G/T-pUC.…”

pilE G-Quadruplex Is Recognized and Preferentially Bound but Not Processed by the MutL Endonuclease from Neisseria gonorrhoeae Mismatch Repair Pathway

“…Recently, we have tested the ngMutL ability to cleave the single-stranded DNA containing a tandem three-quadruplex structure of the hTERT promoter and revealed that it is significantly suppressed by the stable G4 scaffold [ 29 ]. Herein, we evaluated the efficiency of ngMutL-mediated cleavage of the single parallel G4 formed by the d(GGGT)4 sequence, which is stabilized in a DNA duplex context due to the lack of the site complementary to the G4-forming motif in the opposite strand, (95G4/76M).…”

“…In contrast, the ngMutL endonuclease binds nonspecifically to DNA, showing no preferential affinity for mismatch sites [ 25 , 26 , 27 , 28 ]. Nevertheless, we recently discovered the ability of this enzyme to recognize and effectively bind the single parallel G4 formed by the d(GGGT) 4 sequence, as well as the three-quadruplex structure formed by the promoter region of the human telomerase reverse transcriptase ( hTERT ) gene; at the same time, ngMutL has been shown to not hydrolyze DNA within the tandem G4s [ 29 ].…”

“Dynamical Docking” of Cyclic Dinuclear Au(I) Bis-N-heterocyclic Complexes Facilitates Their Binding to G-Quadruplexes

“…The G4-stabilizing properties of the isolated bis-NHC Au­(I) complexes, AuB2 , AuB3 , and AuC1–3 , were assessed via the FRET DNA melting assay using a panel of five G4s in a 5:1 stoichiometry [Au­(I) NHC complex: G4, see the Experimental Section for details]. The G4s were chosen to represent both the telomeric (hTelo) and promoter ( cKIT1 , cKIT2 , BCL2 , and hTERT ) regions of the polynucleotide, as well as different G4 strand orientations such as hybrid-mixed (3 + 1) (hTelo and BCL2 ) and parallel ( cKIT1 , cKIT2 , and hTERT ). − The bis-caffeine complex AuTMX 2 was tested as a benchmark, also because its cKIT2 and BCL2 G4-stabilizing properties have never been reported so far. , …”