RPA-Mediated Unfolding of Systematically Varying G-Quadruplex Structures

Ray, Sujay; Qureshi, Mohammad Haroon; Malcolm, Dominic W.; Budhathoki, Jagat B.; Çelik, Uğur; Balci, Hamza

doi:10.1016/j.bpj.2013.04.004

Cited by 70 publications

(74 citation statements)

References 50 publications

(57 reference statements)

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“…Another aspect of RECQ5-mediated GQ unfolding is whether this weak activity might be adequate due to a cooperation of RECQ5 with RPA, which is known to significantly enhance RECQ5's helicase activity by inhibiting RECQ5's strand annealing activity (22). RPA has been demonstrated to be a potent GQ destabilizer (38) and cooperation between RPA and RECQ5 might eliminate the need for RECQ5 to be efficient at destabilizing these structures. Second, we note that the BLM construct used for this comparison was a functional core of full-length protein and contains helicase, RQC, and Helicase and RNase D-like C-terminal (HRDC) domains.…”

Section: Discussionmentioning

confidence: 99%

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

Budhathoki

Maleki

Roy

et al. 2016

Biophysical Journal

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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.

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Section: Discussionmentioning

confidence: 99%

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

Budhathoki

Maleki

Roy

et al. 2016

Biophysical Journal

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“…The gamma parameter, obtained from a ratio of the intensity change in acceptor and donor intensities upon acceptor photobleaching, revealed the underlying mechanism of the shifting of the F1 and F2 peaks as POT1 is titrated in the 0-200 nM range ( Fig. S5) (29). We interpret the changes in the gamma parameter to be due to POT1 loading to the TTAG overhang, which spaces the donor away from the GQ.…”

Section: Pot1mentioning

confidence: 99%

G-quadruplex formation in telomeres enhances POT1/TPP1 protection against RPA binding

Ray

Bandaria

Qureshi

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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138

144

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Human telomeres terminate with a single-stranded 3′ G overhang, which can be recognized as a DNA damage site by replication protein A (RPA). The protection of telomeres (POT1)/POT1-interacting protein 1 (TPP1) heterodimer binds specifically to single-stranded telomeric DNA (ssTEL) and protects G overhangs against RPA binding. The G overhang spontaneously folds into various G-quadruplex (GQ) conformations. It remains unclear whether GQ formation affects the ability of POT1/TPP1 to compete against RPA to access ssTEL. Using single-molecule Förster resonance energy transfer, we showed that POT1 stably loads to a minimal DNA sequence adjacent to a folded GQ. At 150 mM K + , POT1 loading unfolds the antiparallel GQ, as the parallel conformation remains folded. POT1/TPP1 loading blocks RPA's access to both folded and unfolded telomeres by two orders of magnitude. This protection is not observed at 150 mM Na + , in which ssTEL forms only a lessstable antiparallel GQ. These results suggest that GQ formation of telomeric overhangs may contribute to suppression of DNA damage signals.telomere protection | DNA damage response | single molecule imaging H uman telomeres consist of 2,000-30,000 bp of doublestranded TTAGGG repeats and terminate with a 50-to 200-nt-long, single-stranded 3′ G overhang (1). Telomeric termini need to be protected against DNA damage signals to ensure genome integrity. Replication protein A (RPA) nonspecifically binds ssDNA, is highly abundant in eukaryotes, and plays a role in DNA replication and repair (2). RPA binding to ssDNA, including telomeric overhangs, activates the ataxia telangiectasia and Rad3-related checkpoint (2, 3). The protection of telomeres (POT1)/telomere protection protein (TPP1) subunit of the shelterin complex contributes to telomere protection by specifically binding to the G overhang (4, 5). RPA is 1,000-fold more abundant than POT1/TPP1 and has a similar affinity for singlestranded telomeric DNA (ssTEL) (6, 7). Therefore, POT1/TPP1 alone may not be able to effectively compete against RPA binding (8, 9). Efficient protection of ssTEL against RPA binding may require association of POT1/TPP1 to the rest of the shelterin complex along double-stranded telomeric tracts or other telomere-associated proteins (6, 9).Another potentially significant factor that could influence the competition between RPA and POT1/TPP1 is the ability of ssTEL to form G-quadruplex (GQ) structures (10, 11). Recent studies have shown that human telomeres form GQ structures in vivo (12, 13) and in cell extracts (14). At physiologically relevant ionic conditions (∼150 mM K + ), GQs are thermodynamically more stable than competing Watson-Crick pairing (15). These structures were often regarded as obstacles for recruitment of telomerase (16) and translocation of the DNA replication machinery (17), and their unfolding requires helicase activity (17-19) or ssDNA binding proteins (20,21). It remains unclear whether GQ formation of ssTEL plays any role in protection of telomeres. ssTEL sequences fold into parallel...

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“…Force spectroscopy measurements have shown that the end to end distance change upon unfolding of a GQ is in the ~ 1 nm range (You et al 2015). Therefore, it is possible to monitor GQ dynamics by smFRET in isolation (Ying et al 2003; Lee et al 2005; Shirude and Balasubramanian 2008; Okumus and Ha 2010; Kruger and Birkedal 2013; Long and Stone 2013; Tippana et al 2014) or while it interacts with single stranded DNA binding proteins (Kruger et al 2010; Hwang et al 2012; Qureshi et al 2012; Ray et al 2013; Hwang et al 2014; Ray et al 2014; Zhou et al 2014), helicases (Budhathoki et al 2014; Chatterjee et al 2014; Zhou et al 2014; Budhathoki et al 2015), or GQ-stabilizing small molecules (Jena et al 2009; Kreig et al 2015). Furthermore, monitoring more subtle features, such as different conformations of GQ, is also possible with carefully designed DNA constructs (Long and Stone 2013; Budhathoki et al 2015).…”

Section: Introductionmentioning

confidence: 99%

A practical guide to studying G-quadruplex structures using single-molecule FRET

et al. 2017

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In this article, we summarize the knowledge and best practices learned from bulk and single molecule measurements to address some of the frequently experienced difficulties in single molecule Förster Resonance Energy Transfer (smFRET) measurements on G-quadruplex (GQ) structures. The number of studies that use smFRET to investigate the structure, function, dynamics, and interactions of GQ structures has grown significantly in the last few years, with new applications already in sight. However, a number of challenges need to be overcome before reliable and reproducible smFRET data can be obtained in measurements that include GQ. The annealing and storage conditions, the location of fluorophores on the DNA construct, and the ionic conditions of the experiment are some of the factors that are of critical importance for the outcome of measurements, and many of these manifest themselves in unique ways in smFRET assays. By reviewing these aspects and providing a summary of best practices, we aim to provide a practical guide that will help in successfully designing and performing smFRET studies on GQ structures.

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RPA-Mediated Unfolding of Systematically Varying G-Quadruplex Structures

Cited by 70 publications

References 50 publications

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

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

G-quadruplex formation in telomeres enhances POT1/TPP1 protection against RPA binding

A practical guide to studying G-quadruplex structures using single-molecule FRET

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