Intramolecular Telomeric G-Quadruplexes Dramatically Inhibit DNA Synthesis by Replicative and Translesion Polymerases, Revealing their Potential to Lead to Genetic Change

Abstract

Recent research indicates that hundreds of thousands of G-rich sequences within the human genome have the potential to form secondary structures known as G-quadruplexes. Telomeric regions, consisting of long arrays of TTAGGG/AATCCC repeats, are among the most likely areas in which these structures might form. Since G-quadruplexes assemble from certain G-rich single-stranded sequences, they might arise when duplex DNA is unwound such as during replication. Coincidentally, these bulky structures when present in the DNA template might also hinder the action of DNA polymerases. In this study, single-stranded telomeric templates with the potential to form G-quadruplexes were examined for their effects on a variety of replicative and translesion DNA polymerases from humans and lower organisms. Our results demonstrate that single-stranded templates containing four telomeric GGG runs fold into intramolecular G-quadruplex structures. These intramolecular G quadruplexes are somewhat dynamic in nature and stabilized by increasing KCl concentrations and decreasing temperatures. Furthermore, the presence of these intramolecular G-quadruplexes in the template dramatically inhibits DNA synthesis by various DNA polymerases, including the human polymerase δ employed during lagging strand replication of G-rich telomeric strands and several human translesion DNA polymerases potentially recruited to sites of replication blockage. Notably, misincorporation of nucleotides is observed when certain translesion polymerases are employed on substrates containing intramolecular G-quadruplexes, as is extension of the resulting mismatched base pairs upon dynamic unfolding of this secondary structure. These findings reveal the potential for blockage of DNA replication and genetic changes related to sequences capable of forming intramolecular G-quadruplexes.

The DNA structure and sequence preferences of WRN underlie its function in telomeric recombination events

“…4b ). Notably, single-stranded DNA containing four telomeric (GGGTTA) repeats forms intramolecular G-quadruplexes in NaCl solutions 47 49 , suggesting that intramolecular G-quadruplex formation within this particular substrate might be responsible for its reduced unwinding. Therefore, unwinding of *3-way 5′G3.5X and *3-way 5′G4X substrates with 3.5 and 4 repeats, respectively, was analysed in LiCl, which disfavours G-quadruplex formation 45 50 , versus NaCl.…”

Regulation of DNA Replication through Natural Impediments in the Eukaryotic Genome

“…G-quadruplexes were shown to hinder DNA synthesis by human DNA polymerase δ and several translesion polymerases [169]. Consistently, early studies demonstrated that, in E. coli , repeat loss occurs preferentially during lagging-strand synthesis [170].…”

Replicating through telomeres: a means to an end

“…This would be consistent with the observation that WRN-deficient cells show telomere loss during replication. The lost telomere is often in the chromatid derived from lagging strand synthesis, which is more prone to collapse due to G quadruplex formation [102,103,105]. Further work is needed to elucidate whether the role of WRN in proper telomere replication is mediated either by its potential ability to resolve G quadruplexes in the G-rich strand or by its speculated role in telomeric BIR [102,103].…”