Multiple factors insulate Msh2-Msh6 mismatch repair activity from defects in Msh2 domain I

Abstract

DNA mismatch repair (MMR) is a highly conserved mutation avoidance mechanism that corrects DNA polymerase misincorporation errors. In initial steps in MMR, Msh2-Msh6 binds mispairs and small insertion/deletion loops, and Msh2-Msh3 binds larger insertion/deletion loops. The msh2Δ1 mutation, which deletes the conserved DNA-binding domain I of Msh2, does not dramatically affect Msh2-Msh6-dependent repair. In contrast, msh2Δ1 mutants show strong defects in Msh2-Msh3 functions. Interestingly, several mutations identified in patients with hereditary non-polyposis colorectal cancer map to domain I of Msh2; none have been found in MSH3. To understand the role of Msh2 domain I in MMR, we examined the consequences of combining the msh2Δ1 mutation with mutations in two distinct regions of MSH6 and those that increase cellular mutational load (pol3-01 and rad27). These experiments reveal msh2Δ1-specific phenotypes in Msh2-Msh6 repair, with significant effects on mutation rates. In vitro assays demonstrate that msh2Δ1-Msh6 DNA binding is less specific for DNA mismatches and produces an altered footprint on a mismatch DNA substrate. Together, these results provide evidence that, in vivo, multiple factors insulate MMR from defects in domain I of Msh2 and provide insights into how mutations in Msh2 domain I may cause hereditary non-polyposis colorectal cancer.

Figure 1

a. Schematic of the domain structure of Msh2 and Msh6, based on homology with the human proteins. Domains I-V are defined by the MutS and MutSα crystal structures.^, The N-terminal Region is defined based on Clark et al. b. Structure of Domain I of hMSH2 (green) and hMH6 (red) with locations of HNPCC mutations in hMSH2 Domain I are indicated by the purple spheres. Lysine 6 of hMSH2 and the mispair-binding Phenylalanine 432 of hMSH6 are shown in black. The DNA mispair is colored pink. Figure was generated in WebLab Viewer using data from Warren et al.

Figure 2

Quantitative and comparative Western blots for Msh2 and msh2Δ1, using polyclonal anti-Msh2 antibody. Representative western blots of Msh2 are shown. Lanes 1–3: three independent MSH2 lysates from mid-log phase cultures; Lanes 4–9: msh2Δ lysates + 0, 0.26 ng, 0.52 ng, 1.04 ng, 1.56 ng, and 2.08 ng of purified Msh2-Msh6, respectively. Lanes 10–11, MSH2 lysate from independent mid-log phase cultures (FY23); Lanes 12–14: msh2Δ1 lysates from independent mid-log phase cultures (EAY2039: lanes 12–13; EAY2040, lane 14).

Figure 3

Gel mobility shift assays of Msh2-Msh6 and msh2Δ1-Msh6. A. Titration of Msh2-Msh6 (top) and msh2Δ1-Msh6 (bottom) incubated with homoduplex and +1 substrates, as described in Materials and Methods. B. Quantification of gel mobility shift experiments, showing average binding of Msh2-Msh6 (top) and msh2Δ1-Msh6 (bottom) to a homoduplex or +1 substrate. The error bars indicate the standard error of the mean for four (Msh2-Msh6) or five (msh2Δ1-Msh6) independent experiments.

Figure 4

In situfootprinting of DNA-protein complexes with 1, 10-phenanthroline-copper (OP-Cu). Msh2-Msh6-DNA complexes or msh2Δ1-Msh6-DNA complexes were separated by gel electrophoresis and treated with OP-Cu as described in Materials and Methods. a. Histograms of representative protection patterns of the +1 loop bottom strand substrate. Only the central portion of each substrate is shown. The signal of each band of bound DNA was normalized to the equivalent band produced in the absence of protein. Values greater than 1.0 represent enhanced cleavage; values lower than 1.0 represent protection from cleavage. The asterisk in the top panel indicates the position of the +1 nucleotide (+A). The arrow on the bottom panel indicates the position of the +A nucleotide, which is on the opposite strand. b. Summary of OP-Cu cleavage of top and bottom strands of the +1 loop substrate in the presence of Msh2-Msh6 or msh2Δ1-Msh6, based on at least three independent experiments. Bands that were protected from cleavage are indicated by the arrowhead symbols. Enhanced cleavage is indicated by the circle symbols. The extent of enhanced cleavage or protection is indicated by the shading.

Figure 4

In situfootprinting of DNA-protein complexes with 1, 10-phenanthroline-copper (OP-Cu). Msh2-Msh6-DNA complexes or msh2Δ1-Msh6-DNA complexes were separated by gel electrophoresis and treated with OP-Cu as described in Materials and Methods. a. Histograms of representative protection patterns of the +1 loop bottom strand substrate. Only the central portion of each substrate is shown. The signal of each band of bound DNA was normalized to the equivalent band produced in the absence of protein. Values greater than 1.0 represent enhanced cleavage; values lower than 1.0 represent protection from cleavage. The asterisk in the top panel indicates the position of the +1 nucleotide (+A). The arrow on the bottom panel indicates the position of the +A nucleotide, which is on the opposite strand. b. Summary of OP-Cu cleavage of top and bottom strands of the +1 loop substrate in the presence of Msh2-Msh6 or msh2Δ1-Msh6, based on at least three independent experiments. Bands that were protected from cleavage are indicated by the arrowhead symbols. Enhanced cleavage is indicated by the circle symbols. The extent of enhanced cleavage or protection is indicated by the shading.

Figure 5

DNaseI protection upon binding of Msh2-Msh6 or msh2Δ1-Msh6 to a 99-mer substrate with a single +T insertion in the middle. The proteins were incubated with the DNA substrate and then treated with DNaseI, as described in the Materials and Methods. The left panel shows the protection pattern on the lower strand of the substrate (opposite the insertion). The right panel shows the pattern of protection on the top strand of the substrate. The arrows indicate regions of the footprint that are different in the msh2Δ1-Msh6 footprint compared to that of Msh2-Msh6. The asterisk indicates the position of the +1 extrahelical nucleotide.

Figure 6

DNA bending assay of msh2Δ1-Msh6. The left panel is a cartoon of the results expected if protein binding lead to DNA bending. The right panel shows the unbound substrates and msh2Δ1-Msh6 (100 nM) bound to 90-mer substrates with +1 insertions at position 15, 45 and 75, as described in the Materials and Methods.