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

<div><p>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.</p></div

G-quadruplex Structure.

<p>A) Structure of a G-quartet in the presence of a monovalent cation (M⁺). Hydrogen-Hoogsteen bonds are indicated by red dashes. B) An intramolecular G-quadruplex structure, consisting of three planar G-quartets. C) G-quadruplex classifications, based on the number of strands involved.</p

Intramolecular G-quadruplex-induced Misincorporation and Extension by Human Pol η.

<p>A) Structure and sequence of ext-4×GGG/*P34 substrate. B) Protocol for determining ability of human pol η to promote mutagenesis when encountering an intramolecular G-quadruplex. C) A primer extension assay was performed in two steps. Initially, 4×GGG/*P34 (0.4 nM) was incubated with human pol η (1.0 nM) with all dNTPs (N), dATP (A), dTTP (T), or dGTP (G) (100 µM each) at 18°C for 5 min. Upon completion of step 1, the reaction was supplemented with 100 µM dCTP (C) and incubated at 37°C for 25 min. Aliquots (4 µL) of the reaction were removed immediately after completion of each step for analysis by denaturing PAGE. The asterisk (lane 4) highlights products generated due to misincorporation putatively across from the first guanine that could be involved in an intramolecular G-quadruplex. The arrowhead highlights products generated by incorporation by human pol η upon encountering the intramolecular G-quadruplex in the presence of all dNTPs.</p

Primer/Template Substrates.

<p>To generate primer/template substrates, a template strand (4×GGG, ext-4×GGG, or 3×GGG) is annealed to a 5′ radiolabeled primer strand, *P31 or *P34 (not shown). The 4×GGG²² oligomer (top), which is capable of forming an intramolecular G-quadruplex, was used as a model for the single-stranded template regions of 4×GGG/*P31 and ext-4×GGG/*P31. Conditions favoring (or disfavoring) G-quadruplex formation are indicated; notably, these conditions do not affect the structure of the template regions of control 3×GGG/*P31 and 3×CCC/*P31 substrates (also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080664#pone-0080664-g003" target="_blank">Fig. 3C</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080664#pone-0080664-g004" target="_blank">Fig. 4</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080664#pone.0080664.s006" target="_blank">Fig. S1</a>).</p

Inhibition of Replicative and Translesion Polymerases by Intramolecular G-quadruplexes.

<p>In primer extension assays containing 75(3×GGG/*P31) and G-quadruplex-forming (4×GGG/*P31 or ext-4×GGG/*P31) substrates (0.2–0.3 nM) were incubated with increasing concentrations of the following polymerases: Kexo⁻ (10 and 20 U/L), T4 DNA polymerase (50 and 100 U/L), pol δ (3.4, 8.6, and 17.1 nM), human pol η (0.10 and 0.26 nM), pol κ (1.3 and 2.5 nM), pol μ (5.5, 10.9, and 27.3 nM), or pol β (11.6, 23.2, and 46.3 nM). Incubation temperatures and times for individual polymerases are specified above the gel. Markers (M) generated by 3×GGG/*P31 extension using dATP and dTTP only and Kexo⁻ (lanes 4, 15, 39, and 49) or T4 DNA polymerase (lanes 5, 16, 40, and 50) are denoted. Positions of partial extension products indicating polymerase stalling are denoted with brackets. White dashed lines indicate where separated groups of lanes from a single gel have been spliced together.</p

Template Sequence of 4×GGG/*P31 Forms an Intramolecular G-quadruplex.

<p>A) Intramolecular G-quadruplex formation from single-stranded DNA containing four GGG runs. Conditions that impact formation and dissociation of G-quadruplexes are indicated. B) To detect G-quadruplex formation, a DMS protection assay was performed on *4×GGG²² (lanes 1 and 2) or 3×GGG (lanes 3 and 4) in extension buffer conditions plus or minus 75 mM KCl as indicated. Products resulting from cleavage at unmethylated guanine bases are analyzed by denaturing PAGE. Representative lengths and positions of key guanines (G) for DNA fragments are indicated. C) Primer/template substrates (4×GGG/*P31, 3×CCC/*P31 and 3×GGG/*P31, pictured at top) were incubated in 75 mM KCl or LiCl for 1 h at 25°C. The structures of these substrates were analyzed by their relative migration on native PAGE (15%) containing 75 mM KCl or LiCl in the gel matrix and running buffer. For lanes 1 and 5, 4×GGG/*P31 substrate was heat-denatured before electrophoresis. The dotted line indicates migration of primer/template substrates with unstructured templates.</p

Identification of WRN acetylation sites.

<p><b>A</b>, acetylation assay in cells with different acetyltransferases. Western blot analysis was performed with anti-acetylated lysine antibody (Upper panel) or with anti-WRN antibody. The experiment was repeated at least two times. <b>B</b>, mass-spectrometry analyses identify the WRN acetylation sites. Acetylated WRN protein was purified by co-transfection of FLAG-tagged WRN with both CBP and p300. The protein was digested with trypsin and analyzed by both MALDI-TOF and LC-ESI MS/MS. Scores of ESI or MALDI QIT were shown. Mascot scores greater than 40 or found in both MALDI and ESI were most confident for the true detection of acetylation.</p

WRN acetylation inhibits its ubiquitination.

<p><b>A</b>, Flag-WRN alone or with HA-ub was transfected into HEK293 cells. After immunoprecipitation with FLAG M2 beads, the IP products were subjected to SDS-PAGE and Western blot with anti-HA and anti-WRN antibodies. <b>B</b>, Flag-WRN alone or with HA-ub was transfected into HEK293 cells. Cells were treated with or without MG132 (5 µM, 8 hr). The same assay described in A was performed. <b>C</b>, FLAG-WRN-WT or 6KR was transfected with vector, HA-ub into HEK293 cells. The same assay described in A was performed. <b>D</b>, FLAG-WRN was transfected with vector, HA-ub, CBP and HA-ub, or SIRT1 and HA-ub into HEK293 cells. The same assay described in A was performed. All the experiments were repeated at least three times.</p

Confirmation of WRN acetylation sites in cells using acetylation assay.

<p><b>A</b>, WRN-WT or single mutant was transfected with CBP into HEK293 cells. After immunoprecipitation with FLAG M2 beads, the IP products were subjected to SDS-PAGE and Western blotting with anti-acetylated lysine and anti-WRN antibodies. <b>B</b>, WRN-WT or double mutant or triple mutant or quadruple mutant or 6KR mutant was transfected with CBP into cells. The same assay described in A was performed. All the experiments were repeated at least three times. The bands of WRN proteins were quantified by ImageJ software.</p

CBP stabilizes WRN protein.

<p><b>A</b>, Flag-WRN alone or with CBP was transfected into HEK293 cells. After immunoprecipitation with FLAG M2 beads, the IP products were subjected to SDS-PAGE and Western blotting with anti-WRN and anti-acetylated lysine antibodies. <b>B</b>, endogenous WRN protein level was increased by CBP. Vector or CBP were transfected into HEK293 cells. Cell lysates were analyzed by Western blot with anti-WRN and anti-β-actin antibodies. <b>C</b>, endogenous WRN protein level was decreased by CBP knockdown. HEK293 cells were transfected with siRNA for CBP or GFP. Cell lysates were analyzed by Western blot with anti-WRN, anti-CBP, and anti-β-actin antibodies. <b>D</b>, WRN protein levels from <b>C</b> were quantified. <b>E</b>, Vector or CBP was transfected into HEK293 cells. Total RNA was isolated and cDNA were synthesized using the Superscript III enzyme. Real-time PCR was performed to detect the WRN mRNA level. All the experiments were repeated at least three times. The bands of WRN proteins were quantified by ImageJ software.</p