Figure 4

Integrity of DNA replication intermediates (RIs) in fission yeast cryo-ground cells. (a) Schematic depiction (based on the pioneering study by Brewer and Fangman [25]) of Southern blot signals from two-dimensional (2D) gel-electrophoretic resolution of a restriction-endonuclease-digested, asynchronously-replicating DNA population. Axis labels indicate the primary resolution criteria in each electrophoretic direction, with font size conveying relative influence of given factors. Color-matched cartoon renderings depict the comparative structures and sizes of probe-targeted fragments responsible for specific features of the signal pattern. Green: linear fragments ("arc of linears"), 1N: migration position of single-length simple linear version of probe-targeted fragment; 2N: migration position of double-length, simple linear version of probe-targeted fragment; Yellow: fragments bearing single, externally-initiated replication forks ("Y arc"); Orange: fragments bearing two converging, externally initiated replication forks ("X line"); Red: fragments bearing diverging replication forks, initiated from a common internally located origin ("Bubble Arc"). (b) Autoradiogram from 2D analysis of the 3-kb fission yeast rDNA Kpn I/Hin dIII digestion fragment using HMW DNA prepared from our 972 h-wild-type cryo-ground cells. (c) Left: Autoradiogram from 2D analysis of DNA from panel B after enrichment for RIs by Benzoylated Naphthoylated DEAE-cellulose chromatography. Center: Schematic representation of selective retention of bubble-bearing DNA fragments in Low-Gelling-Temperature (LGT) agarose (blue filaments) by topological capture during gel polymerization. Right: Autoradiograph from 2D analysis of topologically purified bubble-bearing fragments.