An activated Mlh1-Mlh3-DNA complex can nick DNA in trans.

<p>0.7 nM 7.2 kb closed circular M13mp18 phagemid and 1.8 nM 2.7 kb linear pUC18 substrate were incubated with 300 nM Mlh1-Mlh3 under standard endonuclease assay conditions either in isolation or within the same reaction as indicated. (A) Reaction products were run on an alkaline agarose gel. The 7.2 kb substrate was linearized with <i>Hin</i>dIII prior to loading in the alkaline agarose gel. The material in lane 4 and 5 was mixed and run as a control in lane 11 to demonstrate the readout for a negative result. The fraction of DNA nicked was measured by determining the band density in either the 7.2 kb linear band or the 2.7 kb linear band by subtracting the density in a region immediately above the band as background and comparing it to the band densities in the negative controls. (B) Prior to linearization with <i>Hin</i>dIII, 10 μL of each reaction was removed and run on a native agarose gel.