Abstract
DNA sequences that can form intramolecular quadruplex structures are found in promoters of proto-oncogenes. Many of these sequences readily fold into parallel quadruplexes. Here we characterize the ability of yeast Pif1 to bind and unfold a parallel quadruplex DNA substrate. We found that Pif1 binds more tightly to the parallel quadruplex DNA than single-stranded DNA or tailed duplexes. However, Pif1 unwinding of duplexes occurs at a much faster rate than unfolding of a parallel intramolecular quadruplex. Pif1 readily unfolds a parallel quadruplex DNA substrate in a multiturnover reaction and also generates some product under single cycle conditions. The rate of ATP hydrolysis by Pif1 is reduced when bound to a parallel quadruplex compared with single-stranded DNA. ATP hydrolysis occurs at a faster rate than quadruplex unfolding, indicating that some ATP hydrolysis events are non-productive during unfolding of intramolecular parallel quadruplex DNA. However, product eventually accumulates at a slow rate.
Highlights
Some G-rich sequences fold into intramolecular quadruplex structures
Pif1 Unfolds Very Little Parallel Quadruplex in a Single Binding Event—The DNA backbone in duplex DNA and tetramolecular G4DNA is in a helical conformation that presents a similar track for sequential helicase movement (Fig. 2A)
The trapped product migrates more slowly than the corresponding G4DNA allowing for separation by PAGE (Fig. 2B)
Summary
Raney From the Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205
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