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. 2023 Feb 25;51(6):2725–2739. doi: 10.1093/nar/gkad096

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© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 2. — An excess of MutLα is required to promote DNA–DNA associations. (A) Schematic depicting DNA pull-down experiment. Biotinylated 4.3 kb covalently closed (cc) plasmid is immobilized on streptavidin beads. MutLα is bound to immobilized DNA and a 2.7 kb unbiotinylated plasmid is added to determine if the MutLα that is already bound to DNA can pull-down additional DNA molecules. Fractions that are analyzed by agarose gel are shown in green and fractions that are denatured and analyzed by SDS-PAGE are shown in grey. (B) Agarose gel assaying supernatant and bead-bound fractions. Lane 1 contains 1 kb plus DNA ladder (NEB), lane 2 contains a 4.3 kb DNA marker and lane contains DNA inputs for 2.7 kb (135 ng, 77 fmol) plasmids. In lanes 6 and 10, 8.0 pmol MutLα was added. In lanes 7–9 and 11–13, MutLα titration amounts are 2.6 pmol, 8 pmol, and 13.3 pmol respectively. Amount of recovered 2.7 kb DNA (fmol) relative to the input is shown in bar graph below the gel. Number of replicates is 2. (C) SDS-PAGE analysis of supernatant and bead-bound fractions. Lane 1 contains Precision Plus protein ladder (BioRad). Lane 2 contains the MutLα input for lane 9 (8.0 pmol). The Mlh1 subunit weighs 87 kDa and the Pms1 subunit weighs 99 kDa.