Regulation of origin recognition complex conformation and ATPase activity:differential effects of single-stranded and double-stranded DNA binding

The Saccharomyces cerevisiae origin recognition complex (ORC) is bound to o rigins of DNA replication throughout the cell cycle and directs the assembl y of higher-order protein-DNA complexes during G(1.) To examine the fate of ORC when origin DNA is unwound during replication initiation, we determine d the effect of single-stranded DNA (ssDNA) on ORC. We show that ORC can bi nd ssDNA and that ORC bound to ssDNA is distinct from that bound to double- stranded origin DNA, ssDNA stimulated ORC ATPase activity, whereas double-s tranded origin DNA inhibited the same activity. Electron microscopy studies revealed two alternative conformations of ORC: an extended conformation st abilized by origin DNA and a bent conformation stabilized by ssDNA, Therefo re, ORC appears to exist in two distinct states with respect to its conform ation and ATPase activity. Interestingly, the effect of ssDNA on these prop erties of ORC is correlated with ssDNA length. Since double-stranded origin DNA and ssDNA differentially stabilize these two forms of ORC, we propose that origin unwinding triggers a transition between these alternative state s.