A Comparative Study of G-Quadruplex Unfolding and DNA Reeling Activities of Human RECQ5 Helicase

Abstract

RECQ5 is one of five members of the RecQ family of helicases in humans, which include RECQ1, Bloom (BLM), Werner (WRN), RECQ4, and RECQ5. Both WRN and BLM have been shown to resolve G-quadruplex (GQ) structures. Deficiencies in unfolding GQ are known to result in DNA breaks and genomic instability, which are prominent in Werner and Bloom syndromes. RECQ5 is significant in suppressing sister chromatid exchanges during homologous recombination but its GQ unfolding activity are not known. We performed single-molecule studies under different salt (50-150 mM KCl or NaCl) and ATP concentrations on different GQ constructs including human telomeric GQ (with different overhangs and polarities) and GQ formed by thrombin-binding aptamer to investigate this activity. These studies demonstrated a RECQ5-mediated GQ unfolding activity that was an order of magnitude weaker than BLM and WRN. On the other hand, BLM and RECQ5 demonstrated similar single-stranded DNA (ssDNA) reeling activities that were not coupled to GQ unfolding. These results demonstrate overlap in function between these RecQ helicases; however, the relatively weak GQ destabilization activity of RECQ5 compared to BLM and WRN suggests that RECQ5 is not primarily associated with GQ destabilization, but could substitute for the more efficient helicases under conditions where their activity is compromised. In addition, these results implicate a more general role for helicase-promoted ssDNA reeling activity such as removal of proteins at the replication fork, whereas the association of ssDNA reeling with GQ destabilization is more helicase-specific.

Figure 1

smFRET measurements on pd-hGQ12T-3′ construct in KCl. (A) Measurement in 150 mM KCl. Red-open histogram in top panel shows folding of hGQ and gray-solid histogram shows a small shift in the distribution upon binding of 1 μM RECQ5 to the 12T overhang in the absence of ATP. Adding 200 nM (middle panel) and 1 μM (bottom panel) RECQ5 in 2 mM ATP results in 17% and 23% of hGQ molecules to unfold, respectively. The letters A and D on the DNA construct represent the donor and acceptor fluorophores, respectively. (B) Measurement in 50 mM KCl. Red-hollow histogram in top panel shows folding of hGQ and gray-solid histogram shows a small shift in the distribution upon binding of 20 nM RECQ5 to the 12T overhang in the absence of ATP. Adding 50 nM (middle panel) and 200 nM (bottom panel) RECQ5 in 2 mM ATP results in 19% and 32% of hGQ molecules to unfold, respectively. To see this figure in color, go online.

Figure 2

smFRET measurements on pd-hGQ12T-3′ construct in NaCl. (A) Measurement in 150 mM NaCl. Red-open histogram in top panel shows folding of hGQ and gray-solid histogram shows a small shift in the distribution upon binding of 50 nM RECQ5 to the 12T overhang in the absence of ATP. Adding 200 nM, 400 nM (middle panels) and 1 μM (bottom panel) RECQ5 in 2 mM ATP results in 19%, 37% and 37% of hGQ molecules to unfold, respectively. (B) Measurement in 50 mM NaCl. Red-open histogram in top panel shows folding of hGQ and gray-solid histogram shows a small shift in the distribution upon binding of 50 nM RECQ5 to the 12T overhang in the absence of ATP. Adding 50 nM (middle panel) and 200 nM (bottom panel) RECQ5 in 2 mM ATP results in 68% and 70% of hGQ molecules to unfold, respectively. To see this figure in color, go online.

Figure 3

smFRET measurements on pd-TBAGQ12T-3′ construct in KCl. (A) Measurement in 150 mM KCl. Red-open histogram in top panel shows folding of hGQ. Adding 80 nM (second panel), 400 nM (third panel), and 1 μM (bottom panel) RECQ5 in 2 mM ATP results in 20%, 26%, and 35% of hGQ molecules to unfold, respectively. (B) Measurement in 50 mM KCl. Red-open histogram in top panel shows folding of hGQ. Adding 20 and 80 nM (mid panels), and 400 nM (bottom panel) RECQ5 in 2 mM ATP results in 23%, 37%, and 55% of hGQ molecules to unfold, respectively. To see this figure in color, go online.

Figure 4

Probing the reeling activity of RECQ5 in pd-35T-5′ construct. (A) smFRET histogram of pd-35T-5′ in 150 mM KCl shows a peak at E_FRET = 0.37. (B) Adding 20 nM RECQ5 and 5 μM ATP results in reeling of the ssDNA overhang, as manifested by the FRET rise up to E_FRET = 0.80–0.90 range. Similar measurements were also performed at other ATP concentrations and the reeling rate was determined by counting the FRET rise events per second. (C) A schematic of the various steps during ssDNA reeling. RECQ5 binds in the vicinity of ssDNA/dsDNA junction and pulls the ssDNA overhang toward the junction, resulting in the observed FRET increases. Upon completion of a reeling event, RECQ5 could either release the ssDNA and reel it again or dissociate from the DNA. To see this figure in color, go online.

Figure 5

Probing GQ unfolding after reeling in of ssDNA overhang in pd-20ThGQ construct. (A) smFRET histogram on pd-20ThGQ-5′ at 150 mM KCl. This construct has a long enough spacer ssDNA (20T) between the duplex and GQ that enables RECQ5 to reel in the DNA before interacting with GQ. The two GQ conformations are manifested as two peaks in the histogram, the lower peak F1 representing the antiparallel (or hybrid) conformation while the higher peak F2 represents the parallel conformation. (B) Two sample smFRET traces showing an initial FRET level at F1 (top) and F2 (bottom).The FRET rise (in dashed rectangle) in both traces demonstrates a reeling event. However, we did not observe a dip in the FRET level, which is a signature of GQ unfolding, after this rise, suggesting that RECQ5 does not unfold hGQ after reeling in an intervening ssDNA overhang. To see this figure in color, go online.

Figure 6

RecQ5-mediated GQ unfolding on pd-12ThGQ construct, which was also used for our earlier BLM measurements. (A) smFRET histogram on pd-12ThGQ-5′ at 150 mM KCl. The two GQ conformations are manifested as two peaks in the histogram, the lower peak F1 representing the antiparallel (or hybrid) conformation while the higher peak F2 represents the parallel conformation. (B) A sample smFRET trace showing RECQ5-mediated GQ unfolding events in 20 nM RECQ5 and 2 mM ATP. The folded GQ FRET level is at E_FRET ≈ 0.70 in this case and the transitions to E_FRET ≈ 0.30 represent unfolding of the GQ. RECQ5 is capable of maintaining the unfolded state for extended periods of time as demonstrated in the bottom panel. The spacer ssDNA between the duplex and GQ in this case is only 12 nt long, and does not result in any significant reeling activity. To see this figure in color, go online.