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. 2021 Jun 4;118(23):e2022704118. doi: 10.1073/pnas.2022704118

Fig. 3.

Fig. 3.

DNA-binding properties of MutLγ and identification of a mlh3-KERE separation of function mutant. (A) EMSA of MutLγ(CTD) with a HJ with four arms of 25 bp each, 50 bp–long dsDNA or 100 bp–long dsDNA. The quantification of the gels is from the following number of experiments: HJ (n = 3), 50mer (n = 3), and 100mer (n = 2). Values are the mean ± SEM when n = 3, the mean ± range when n = 2. (B) EMSA of Mlh1-Pms1(CTD) with the same DNA substrates as in A. Values are the mean ± SEM from four independent experiments for each condition. (C and D) Molecular modeling of full-length Mlh1-Mlh3. The surface are colored according (C) to the electrostatic potential and (D) to the conservation rate of the amino acids deduced from multiple sequence alignments of Mlh1 or Mlh3 eukaryotes sequences. The circles represent the five main DNA-binding sites proposed from the literature and the experiments presented in this study. The N1, L1, and C1 sites are respectively in the NTD, linker, and CTD of Mlh1. The C3 is in the CTD of Mlh3. (E) The C3 site contains two basic residues (K668 and R671) that are exposed at the surface of Mlh3(CTD). These residues are close to the C670 position that is involved in the endonuclease site. (F) Mutation rate as measured with the Lys+ reporter assay of the Mlh3-KERE allele compared to wild type and mlh3Δ. Values are the mean of nine independent colonies ± SEM. (G) Spore viability of diploid strains bearing the indicated MLH3 genotype at its endogenous locus. ***P < 0.001, Fisher’s exact test. Refer also to SI Appendix, Table S2. (H) Crossing over frequency at the HIS4LEU2 hotspot monitored by Southern blot. Graph shows quantification from two independent biological replicates ± range and are expressed relative to levels in MLH3. (I) EMSA of wild-type Mlh1-Mlh3 and Mlh1-Mlh3(KERE) mutant with dsDNA and HJ. The quantification of the EMSA is from two independent experiments. Values are the mean ± range. (J) Nuclease activity of wild-type Mlh1-Mlh3 and Mlh1-Mlh3(KERE) mutant on supercoiled pUC19 plasmid DNA.