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. 2014 May 14;42(11):7104–7112. doi: 10.1093/nar/gku420

Figure 4.

Figure 4.

Mismatch-provoked excision in reconstituted systems requires the Exo1 hydrolytic function. (A) G-T heteroduplexes used in this study contain a NheI site that is located 5 bp distal to the mismatch in the 5′-heteroduplex and 5 bp proximal to the mispair in the 3′-heteroduplex. Mismatch provoked excision, which renders this region single-stranded and NheI-resistant (9,10), was scored by cleavage with NheI and ClaI. Arrows designate excision products in panels B–D. (B) 5′-directed excision on the G-T heteroduplex was determined in the 4-protein system. Reactions contained RPA with MutSα and MutLα present as indicated. Excision was scored in the presence of Exo1-D173A, Exo1-E109K or wild type enzyme (Materials and Methods). (C) 5′-directed excision in the 6-protein system was determined in reactions containing MutSα, MutLα, RPA, RFC, PCNA (indicated as 5P in lane labels), in the absence (lane 2) or presence of Exo1-D173A, Exo1-E109K or wild type Exo1 (lanes 3–5). Lanes 6–9 correspond to reactions containing wild type Exo1 with omissions as indicated. (D) Reactions were as in panel C except the heteroduplex strand break was located 3′ to the mismatch.