Use of 6‐Methylisoxanthopterin, a Fluorescent Guanine Analog, to Probe Fob1‐Mediated Dynamics at the Stalling Fork Barrier DNA Sequences

• Published in: Chemistry, an Asian journal an Asian journal, 2019-12, Vol.14 (24), p.4760-4766
• Main Authors: Mariam, Jessy, Krishnamoorthy, G., Anand, Ruchi
• Format: Article
• Language: English
• Subjects: 6-methylisoxanthopetrin; Anisotropy; Chemistry; DNA - genetics; DNA - metabolism; DNA Replication; DNA, Cruciform; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Dynamics; Escherichia coli - genetics; Fluorescence; Fluorescent Dyes - chemistry; fluorescent probes; Gene sequencing; Protein Binding; protein–nucleic acid interactions; Saccharomyces cerevisiae - chemistry; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Stalling; time-resolved fluorescence; Xanthopterin - analogs & derivatives; Xanthopterin - chemistry
• ISSN: 1861-4728; 1861-471X
• DOI: 10.1002/asia.201901061

Fluorescent nucleic acid base mimics serve as excellent site‐specific and real‐time reporters of the local and global dynamics. In this work, using the fluorescent guanine mimic 6‐methylisoxanthopterin (6‐MI), we unravel the differential dynamics of replication fork barrier/terminator sequences (RFB1 and RFB3) mediated by fork blocking protein (Fob1). By strategic and site‐specific incorporation of this probe, we show that 6‐MI is able to capture the changes in global dynamics exhibited by Fob1 and aids in distinguishing between varied architectural forms like double‐stranded DNA versus Holliday junctions (HJs). This is important as these barriers are hotspots for recombination. Fluorescence lifetime and anisotropy decay studies further revealed that Fob1 strongly dampens the dynamics in double‐stranded RFB1, and the sequence inherently possesses lesser flexibility in comparison to RFB3. We show that 6‐MI can probe the differential oligomeric status of Fob1 in response to various architectures, that is, double‐stranded versus HJs. This work highlights the unique advantages of 6‐MI as a probe when incorporated in nucleic acid frameworks. 6‐Methylisoxanthopterin (6‐MI), a fluorescent guanine mimic was employed to capture the dynamics in duplex replication fork barrier sequences and Holliday junctions. In the presence of fork‐blocking protein, the dynamics of duplex barriers were dampened dramatically. This paper highlights the powerful features of 6‐MI in comparison to the fluorescent adenine analog, 2‐aminopurine.