Abstract
Various helicases and single stranded DNA (ssDNA) binding proteins unfold G-quadruplex (GQ) structures. However, the underlying mechanisms of this activity have only recently come to focus. We report kinetic studies on Bloom (BLM) helicase and human telomeric GQ interactions using single-molecule Förster resonance energy transfer (smFRET). Using partial duplex DNA (pdDNA) constructs with different 5′ ssDNA overhangs, we show that BLM localizes in the vicinity of ssDNA/double-stranded DNA (dsDNA) junction and reels in the ssDNA overhang in an ATP-dependent manner. A comparison of DNA constructs with or without GQ in the overhang shows that GQ unfolding is achieved in 50–70% of reeling attempts under physiological salt and pH conditions. The unsuccessful attempts often result in dissociation of BLM from DNA which slows down the overall BLM activity. BLM-mediated GQ unfolding is typically followed by refolding of the GQ, a pattern that is repeated several times before BLM dissociates from DNA. BLM is significantly less processive compared to the highly efficient GQ destabilizer Pif1 that can repeat GQ unfolding activity hundreds of times before dissociating from DNA. Despite the variations in processivity, our studies point to possible common patterns used by different helicases in minimizing the duration of stable GQ formation.
Highlights
Human Bloom helicase (BLM) is classified as a superfamily 2 helicase [1,2]
We have shown that the efficiency of BLM-mediated GQ unfolding activity was enhanced in the presence of nonhydrolyzable ATP analogs, ATP␥ S and AMP-PNP, and reduced in the presence of the hydrolysis product, ADP, which correlates with the effect these nucleotides have on BLM-single stranded DNA (ssDNA) binding stability
Quantification of the timedependence of loss of Cy3 spots for different BLM and ATP concentrations (Figure 1D) confirms that Cy3 spots are removed faster at higher BLM concentrations. These results suggest that BLM is capable of unfolding the GQ as evidenced by unwinding of the 12 bp duplex DNA in this construct
Summary
Human Bloom helicase (BLM) is classified as a superfamily 2 helicase [1,2]. BLM is a member of RecQ family of helicases and contains two RecA-like domains that are involved in binding and hydrolysis of ATP and enable BLM to translocate on ssDNA in the 3 to 5 direction [2,3,4,5]. We have shown that the efficiency of BLM-mediated GQ unfolding activity was enhanced in the presence of nonhydrolyzable ATP analogs, ATP␥ S and AMP-PNP, and reduced in the presence of the hydrolysis product, ADP, which correlates with the effect these nucleotides have on BLM-ssDNA binding stability. A similar correlation between DNA-binding stability and protein-mediated GQ unfolding was observed in ssDNA-binding proteins such as Replication Protein A (RPA) and Protection of Telomere 1 (POT1) [22,23]. This similarity suggested a common initiating mechanism for GQ destabilization in the absence of ATP for ssDNA-binding proteins and enzymes that need to bind to the vicinity of GQ before unfolding it. For both groups of proteins an ssDNA overhang in the vicinity of GQ is required for GQ unfolding, and whether this overhang is placed 3 or 5 to the GQ made a significant difference depending on the polarity of the protein orientation [20]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
