Evidence that hMLH3 functions primarily in meiosis and in hMSH2-hMSH3 mismatch repair

Abstract

The MutS (MSH) and MutL (MLH) homologs are conserved proteins that function in mismatch repair (MMR) and meiosis. We examined mRNA and protein expression of hMLH3 compared to other human MSH and MLH in a panel of human tissues and the HeLa cell line. Quantitative PCR suggests that MSH and MLH transcripts are expressed ubiquitously. hMLH3 mRNA is present at low levels in numerous tissues. Protein expression appears to correlate with a threshold of mRNA expression with hMLH3 present at high levels in testis. In addition, we have found and mapped interactions between hMLH1 and hMLH3 with hMSH3. These data are consistent with yeast studies and suggest a role for hMLH3 in meiosis as well as hMSH2-hMSH3 repair processes and little if any role in Hereditary Non-Polyposis Colorectal Cancer (HNPCC).

Fig. 1. mRNA expression of MSH and hRAD51 DNA repair genes in multiple tissues

Quantitative representation of (A) hMSH2, (B) hMSH3, (C) hMSH4, (D) hMSH5, (E) hMSH6, and (F) hRAD51 mRNA in human tissues and HeLa cells. Copy number was determined by qPCR with gene specific primer sets and total RNA reversed transcribed to cDNA. The charts represent an average of three separate experiments and quantities are displayed in 10⁷ copies/µg of total RNA.

Fig. 2. mRNA expression of MLH DNA repair genes in multiple tissues

Quantitative representation of (A) hMLH1, (B) hPMS1, (C) hMLH3, and (D) hPMS2 mRNA in human tissues and HeLa cells. Copy number was determined by qPCR with gene specific primer sets and total RNA reversed transcribed to cDNA. The charts represent an average of three separate experiments and quantities are displayed in 10⁷ copies/µg of total RNA.

Fig. 3. Western analysis of DNA repair protein expression in multiple tissues

Blots of 15µg total protein from multiple human tissue types were probed with antibodies specific for (Top) hMLH3, hMSH6, hMSH2, hPMS2, and hMLH1 with GAPDH as a loading control; and (Bottom) hRAD51 with actin as a loading control.

Fig. 4. hMLH1 and hMLH3 interact with hMSH3

A GST interaction assay was used to identify interactions between MSH and MLH proteins. The MSH proteins were tagged with an N-terminal GST and hMLH1 and hMLH3 were in vitro transcribed/translated in the presence of ³⁵S-methionine. (A) hMLH3 interaction with hMLH1 is a positive control. Interactions of other MSH proteins with hMLH3 are relative to the hMLH3 interaction with hMLH1 (Int_rel-hMLH1). Phosphorimager analysis was used for quantitation. Interaction with GST-alone is subtracted as background. hMLH1 specifically interacts with hMSH3, but not other MSH proteins. (B) hMLH1 interaction with hPMS1 is a positive control and the reference interaction with other MSH proteins (Int_rel-hPMS2). hMLH1 interacts with hMSH3 but not hMSH2, hMSH6, hMSH4, or hMSH5.

Fig. 5. Region of hMLH1 required for interaction with hMSH3

(A) The region of hMLH1 required for its interaction with hMSH3 was determined via the GST interaction assay. Deletion constructs of hMLH1 were in vitro transcribed/translated and tested for interaction to GST-hMSH3. The interaction between hPMS1 and hMLH1 is a positive control and the reference for all other interactions(Int_rel-hMLH1). Phosphorimager analysis was used for quantitation. The minimal region of hMLH1 required for interaction with hMSH3 consisted of amino acids 382–756 (lane 7). (B) The GST interaction assay was used to define the region of hMSH3 required for interaction with hMLH1. hMLH1 was tested against in vitro transcribed/translated deletion constructs of hMSH3. The interaction between hMLH1 and in vitro transcribed/translated hMLH3 served as a positive control and reference for all other interactions (Int_rel-hMSH3). Phosphorimager analysis was used for quantitation. hMSH3 amino acids 1–250 is the minimal region required for interaction with hMLH1 (lane 6).

Fig. 6. Diagram of the interaction regions of MSH and MLH proteins

Based on Schmutte et. al. . The C-terminus of hMLH1 is required for interaction with the N-terminus of hMSH3 (yellow rectangle and lines). This region of hMLH1 is also required for its interaction with hEXOI and heterodimer formation with hMLH3, hPMS1, and hPMS2 , , . In addition, the N-terminal region of hMSH3 required for interaction with hMLH1 includes the interaction regions of hMSH3 with hMSH2 and hEXOI , .