Model for Mlh1-Mlh3 activation.

<p>(A) Model for Mlh1-Mlh3’s (blue and purple heterodimer) endonuclease activation dependent on polymerization of the protein. (1) Mlh1-Mlh3 binds DNA and forms a polymer with a specified polarity. (2) Polymer formation proceeds and if other factors are present, they are displaced. (3) A polymer is formed of the critical length and stability for activation. (4) The activated polymer can introduce a nick into one strand of the duplex DNA. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001164#sec002" target="_blank">Results</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001164#sec007" target="_blank">Discussion</a> for details. (B) Mlh1-Mlh3 activation in the context of in vitro substrates. Red arrow indicates nicking activity by an active polymer. (1) An Mlh1-Mlh3 polymer can form on circular or linear DNA. For linear DNA, the molecule must be a length critical for polymer stability. (2) On circular substrate that is at least 12 kb, Mlh1-Mlh3 binds to adjacent regions of duplex DNA, leading to DSB formation. (3) Top: on circular substrates with preexisting nicks, polymers form and interactions between polymers direct Mlh1-Mlh3 to nick opposite the preexisting nick. Bottom: nicking in trans. If the adjacent regions of dsDNA do not have the same polarity, interactions between polymers may occur with a different orientation, promoting nicking to the duplex that does not contain a preexisting nick. (C) Hypothesis for how in vitro observations can be applied to understand HJ resolution. Bound Msh4-Msh5 recruits Mlh1-Mlh3 to form a polymer. Polymer formation and relative polarities of the duplex arms activate endonuclease activity to cleave strands with the same polarity. Branch migration could position nicks directly at the junction. (D) Hypothesis for how Mlh1-Mlh3 may resolve dHJs in vivo if the dHJ is unligated. The mode of resolution is different depending on where synthesis terminates. The grey square with a white question mark indicates unknown occupancy of one of the junctions. Mlh1-Mlh3 is recruited by Msh4-Msh5, which is then displaced by the Mlh1-Mlh3 polymer. The relative polarities of the two duplexes and the nicks resulting from DNA synthesis direct asymmetric resolution with respect to the two junctions. Cleavage could take place near the junction or some distance away. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001164#sec007" target="_blank">Discussion</a> for additional details. (E) Two possible conformations of a dHJ. Small black arrows in the top of the panel indicate where nicks would need to form to produce the CO products in the bottom of the panel. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001164#sec007" target="_blank">Discussion</a>. (F) Hypothesis for how Mlh1-Mlh3 may resolve type II dHJs in E. Mlh1-Mlh3 is recruited by Msh4-Msh5, which could be displaced by Mlh1-Mlh3. Polymer formation activates the endonuclease to cleave. Cleavage in this model can occur symmetrically with respect to both junctions, either near the junction or at a site away from the branch point.