Breathing fluctuations in position-specific DNA base pairs are involved in regulating helicase movement into the replication fork

Abstract

We previously used changes in the near-UV circular dichroism and fluorescence spectra of DNA base analogue probes placed site specifically to show that the first three base pairs at the fork junction in model replication fork constructs are significantly opened by "breathing" fluctuations under physiological conditions. Here, we use these probes to provide mechanistic snapshots of the initial interactions of the DNA fork with a tight-binding replication helicase in solution. The primosome helicase of bacteriophage T4 was assembled from six (gp41) helicase subunits, one (gp61) primase subunit, and nonhydrolyzable GTPγS. When bound to a DNA replication fork construct this complex advances one base pair into the duplex portion of the fork and forms a stably bound helicase "initiation complex." Replacement of GTPγS with GTP permits the completion of the helicase-driven unwinding process. Our spectroscopic probes show that the primosome in this stable helicase initiation complex binds the DNA of the fork primarily via backbone contacts and holds the first complementary base pair of the fork in an open conformation, whereas the second, third, and fourth base pairs of the duplex show essentially the breathing behavior that previously characterized the first three base pairs of the free fork. These spectral changes, together with dynamic fluorescence quenching results, are consistent with a primosome-binding model in which the lagging DNA strand passes through the central hole of the hexagonal helicase, the leading strand binds to the "outside" surfaces of subunits of the helicase hexamer, and the single primase subunit interacts with both strands.T4 DNA replication complex | helicase mechanisms | 2-aminopurine | DNA structure N ucleic acid bases at single-strand/double-strand (ss-ds) DNA junctions of replication forks and polymerase elongation sites are prime binding targets for proteins and enzymes that manipulate and modify the DNA genome. Determining the conformational changes that occur at these junctions during DNA replication, repair, and transcription can illuminate the mechanisms of these central processes of gene expression. A unique feature of base pairs at and near these ss-dsDNA junctions is that they undergo substantial position-dependent spontaneous opening and closing ("breathing" or "fraying") processes that are driven by thermal fluctuations (1). Proteins that bind more strongly to ss-than to dsDNA sequences can, in principle, use this differential binding free energy to bind these transiently open sequences as a first step in nucleic acid helicase activity, without the immediate expenditure of the chemical free energy of hydrolysis of nucleoside triphosphates (NTPs) (2). Thus, the interactions of genome regulatory proteins with thermally driven DNA breathing events are likely to be important in initiating reactions that involve the exposure and manipulation of single-stranded template sequences at replication forks or transcription bubbles within the normal dsDNA genome. Here, we examine the relations...

Single-molecule FRET and linear dichroism studies of DNA breathing and helicase binding at replication fork junctions

“…The faster decay time for the duplex sample suggests that the closing rate of a spontaneously formed bubble in the duplex region of dsDNA is faster than that of a similarly formed unstable conformation near the ss-dsDNA fork junction. DNA breathing fluctuations have been shown to occur much more frequently at replication fork junctions than at sequences positioned well within duplex DNA regions (5,6). The relative magnitudes of the lifetimes we have observed for the fork-and duplex-labeled DNA constructs are consistent with previous observations (7).…”

“…Additional examples of data sets are presented in SI Text, Control Experiments, and show those of the same DNA construct in the presence of gp41 helicase hexamers that had been assembled using nonhydrolyzable GTPγS as the NTP ligand. These helicase hexamers, assembled with nonhydrolyzable NTPs in the absence of primase, bind weakly to the ss-dsDNA junction of the replication fork constructs, but cannot catalyze duplex unwinding (5,13,14).…”

“…More-complex breathing fluctuations could be described as combinations of the above (or other) breathing modes. Recently, equilibrium and steady-state spectroscopic approaches have been used to monitor position-dependent DNA breathing that occurs in the proximity of ss-dsDNA forks and primer-template (p/t) junctions of DNA constructs (5,6). Such motions are believed to occur in the microsecond range (7), a difficult regime to access experimentally.…”

Watching DNA breath one molecule at a time

“…Compared with the well-documented chemical modifications of DNA and their functions in shaping gene expression patterns (1-3), the impact of physical properties of DNA is much less characterized, partially because they are relatively difficult to describe and measure directly. Nevertheless, it is now more and more appreciated that physical properties, such as DNA bending (4,5) and DNA breathing (6)(7)(8), play important roles in gene expression by affecting binding and assembly of molecular machineries on DNA. In PNAS, Phelps et al introduce to us a unique single-molecule approach to study DNA breathing dynamics and investigate the role of DNA breathing in protein assembly on DNA (9).…”

DNA Replication and Telomere Shortening: Key Factors Related to the Production of C3-Aldehydes and the Interaction of One of them with DNA Guanine Residues

“…Today, there is no doubt that electron hole (radical cation) migration in DNA is possible and can occur over long molecular distances [18]. More precisely, breathing fluctuations in position-specific DNA base pairs are involved in regulating helicase movement into the replication fork [19]. Owing to this specific electronic structure, a replisome, which is a complex of enzymes involved in replicating DNA, attaches to the DNA, and the first of these enzymes is a helicase, which unwinds and unzips the DNA by breaking the H bonds between the nitrogenous bases.…”