The unstructured linker arms of Mlh1-Pms1 are important for interactions with DNA during mismatch repair - PubMed (original) (raw)

The unstructured linker arms of Mlh1-Pms1 are important for interactions with DNA during mismatch repair

Aaron J Plys et al. J Mol Biol. 2012.

Abstract

DNA mismatch repair (MMR) models have proposed that MSH (MutS homolog) proteins identify DNA polymerase errors while interacting with the DNA replication fork. MLH (MutL homolog) proteins (primarily Mlh1-Pms1 in baker's yeast) then survey the genome for lesion-bound MSH proteins. The resulting MSH-MLH complex formed at a DNA lesion initiates downstream steps in repair. MLH proteins act as dimers and contain long (20-30 nm) unstructured arms that connect two terminal globular domains. These arms can vary between 100 and 300 amino acids in length, are highly divergent between organisms, and are resistant to amino acid substitutions. To test the roles of the linker arms in MMR, we engineered a protease cleavage site into the Mlh1 linker arm domain of baker's yeast Mlh1-Pms1. Cleavage of the Mlh1 linker arm in vitro resulted in a defect in Mlh1-Pms1 DNA binding activity, and in vivo proteolytic cleavage resulted in a complete defect in MMR. We then generated a series of truncation mutants bearing Mlh1 and Pms1 linker arms of varying lengths. This work revealed that MMR is greatly compromised when portions of the Mlh1 linker are removed, whereas repair is less sensitive to truncation of the Pms1 linker arm. Purified complexes containing truncations in Mlh1 and Pms1 linker arms were analyzed and found to have differential defects in DNA binding that also correlated with the ability to form a ternary complex with Msh2-Msh6 and mismatch DNA. These observations are consistent with the unstructured linker domains of MLH proteins providing distinct interactions with DNA during MMR.

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Figures

Fig. 1

Fig. 1

Location of TEV cleavage sites in Mlh1-Pms1 linker arms. Cartoon of predicted structures of TEV-containing Mlh1-Pms1 constructs, based on structural and biochemical data., Mlh1 is in magenta and Pms1 is in blue. Linker arms are illustrated by a series of unconnected dots. Approximate positions of the TEV cleavage site (black dashed line), FLAG-tag (star) and HA-tag (filled circle) are shown. The exact position of each tag is described in the Materials and Methods.

Fig. 2

Fig. 2. Mlh1(TEV)-Pms1 complexes remain intact after TEV cleavage

(a). Wild-type and the indicated Mlh1-Pms1 complexes wereexpressed and purified from S. cerevisiae (see Materials and Methods). 0.5 μg of each complex were loaded into each lane. Proteins were electrophoresed in 8% SDS-PAGE and the gel was stained with Coomassie blue. The sizes of the relevant molecular weight standards (M) are indicated. (b). Immunoprecipitation (IP) of the Mlh1(TEV448, FLAG499)-Pms1(HA565) complex using an anti-HA antibody. Mlh1-Pms1 was untreated or treated with TEV protease prior to IP (Materials and Methods). Input lanes show TEV untreated and treated complexes prior to IP. Control reactions were performed in parallel with Mlh1(TEV448, FLAG499)-Pms1 lacking an HA tag on Pms1. Bands arising from BSA (**) and IgG (*) present in the IP reactions are indicated.

Fig. 3

Fig. 3

Schematic diagram of Mlh1 and Pms1 linker arm deletion series. (a). Outline of amino acid deletions (ΔX-Y) created within the Mlh1 linker arm domain. The location of the FLAG epitope tag, after Y499 in Mlh1, is indicated by the red bar. Equal amounts of crude cellular extracts (20 μg) from strains bearing the indicated MLH1 allele were loaded onto and separated in 8% SDS-PAGE and then probed with an anti-FLAG antibody. (b). Outline of amino acid deletions (ΔX-Y) created within the Pms1 linker arm domain. The location of the HA-epitope tag, after D565 in Pms1, is indicated by the blue bar. Equal amounts of crude cellular extracts (20 μg) from strains expressing the indicated PMS1 allele were loaded onto and separated in 8% SDS-PAGE and then probed with anti-HA antibody. For Panels (a) and (b), MMR function, as assayed in lys2A14 mutator assays, is described as similar to wild-type (+++), a weak mutator (++), or a null phenotype (−). The domains of Mlh1 and Pms1 are not drawn to scale.

Fig. 4

Fig. 4

Mlh1 and Pms1 linker arm deletion mutants display synergistic defects in MMR. Mutation rates of mlh1Δ348-378, pms1Δ584-634 and pms1Δ600 -625 single and double mutant strainswere determined in the lys2A14 assay as described in the Materials and Methods. Rates are shown as a percentage of the corresponding null (Table 4).

Fig. 5

Fig. 5

mlh1 and pms1 linker arm deletions display altered DNA binding affinities. EMSA was performed as described in Materials and Methods. All reactions contained 60 nM 40-bp homoduplex substrate. Titration reactions contained the indicated amounts of Mlh1-Pms1, Mlh1-pms1Δ390-610, mlh1Δ336-480-Pms1, and mlh1Δ348-373-pms1Δ584-634 complexes. Free and bound substrates are indicated. % bound was calculated using ImageJ software as the amount bound divided by the total (bound + free) and is indicated below each lane.

Fig. 6

Fig. 6

mlh1-pms1 complexes that bind to DNA also form ternary complexes with Msh2-Msh6 at a DNA mismatch. EMSA was performed as described in the Materials and Methods. Binding reactions contained 60 nM 40-bp (+1) mismatch substrate and 1 mM ATP. 150 nM Msh2-Msh6, and 100 nM Mlh1-Pms1 or mutant derivatives were included as indicated.

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