A comparison of experimental and theoretical melting maps for replicative form ofφX174 DNA

Abstract

A previously elaborated technique for fixing a chosen partially melted state of DNA with glyoxal was used in a study of the melting process of the replicative form (RF III) of phi X174 DNA. Electron-microscopic maps corresponding to five points of the melting curve of RF III were obtained and compared with the theoretical melting maps obtained in (4) and (6). This comparison clearly shows that only rigorous calculations (4) and not the ones proposed by Azbel (6,7) correctly predict the course of RF III melting.

DNA Overstretching in the Presence of Glyoxal: Structural Evidence of Force-Induced DNA Melting

“…In the case of thermal DNA melting, this selectivity was attributable to the heterogeneity of DNA basepair stability. Thus, at some temperatures below the average T m , certain AT-rich segments are predominantly opened and fixed by glyoxal, whereas the rest remain closed (42,47,48). It is the extremely slow rate of the glyoxal/ guanine covalent bonding that makes this reaction practically unobservable for hydrogen-bonded basepairs (44).…”

“…Increasing the temperature exponentially facilitates the rate of glyoxal-promoted unwinding of the DNA duplex (46). Therefore, selective fixation of the low temperature-melting segments requires very specific glyoxal exposure conditions (41,47,48). In the case of FIM, the force becomes an equivalent of the temperature, whereas the fractional extension into the plateau region, f, is an equivalent of fractional DNA melting (7).…”

“…More specifically, when DNA was incubated with glyoxal at some fixed temperature close to but lower than the average melting temperature, T m , only certain AT-rich fragments of DNA were permanently fixed in the single-stranded state with glyoxal (40,41). Determining the location of these sites, by either single-strand digestion (40,46) or electron microscopy (42,47,48), led to the identification of these low-melting sites, with the sites predicted by direct calculations for the particular DNA sequence (48).…”

The duplex DNA is very underwound in the three‐stranded RecA protein‐mediated synaptic complex

“…Potassium permanganate is generally considered a probe for ssDNA; the mechanism of its selectivity, however, is different from other ssDNAspecific reagents. Some react with positions involved in hydrogen bond formation [halogenoacetaldehydes (Kohwi & Kohwi 1992), glyoxal (Lyubchenko et al 1982), carbodiimides (Smooker & Cotton 1993), dimethyl sulphate (Kirkegaard et al 1983), hydroxylamine and methoxylamine ( Johnston 1992)], while others are not capable of approaching reactive sites in the major groove of the double helix because of steric hindrance [diethyl pyrocarbonate (Kohwi & Kohwi 1992), bulky alkylating reagents (Mazin et al 1989)]. Potassium permanganate, as well as osmium tetroxide ( Jelen et al 1991) and sodium bisulphite (Chen & Shaw 1993), however, have a specific stereochemistry for pyrimidine bases.…”

Strand Dissociation and Cooperative Melting of Double-Stranded DNAs Detected by Denaturant Gradient Gel Electrophoresis1

“…' They also inferred that the separation of DNA fragments by their sequences came from the retardation caused by their partial melting. Lyamichev et al devel oped the technique so that the partially melted regions in DNAs at a certain temperature which were fixed with glyoxal before gel electrophoresis could be identified because the melted regions caused a remarkable retardation in gel electropho resis (10,11). Their method can, however, only detect the partial melting of DNAs in one dimen sion and does not offer further information.…”