Identification of Mismatch Repair Protein Complexes in HeLa Nuclear Extracts and Their Interaction with Heteroduplex DNA

Abstract

Deficiencies in DNA mismatch repair (MMR) have been found in hereditary colon cancers (hereditary nonpolyposis colon cancer, HNPCC) as well as in sporadic cancers, illustrating the importance of MMR in maintaining genomic integrity. We have examined the interactions of specific mismatch repair proteins in human nuclear extracts. Western blot and co-immunoprecipitation studies indicate two complexes as follows: one consisting of hMSH2, hMSH6, hMLH1, and hPMS2 and the other consisting of hMSH2, hMSH6, hMLH1, and hPMS1. These interactions occur without the addition of ATP. Furthermore, the protein complexes specifically bind to mismatched DNA and not to a similar homoduplex oligonucleotide. The protein complex-DNA interactions occur primarily through hMSH6, although hMSH2 can also become cross-linked to the mismatched substrate when not participating in the MMR protein complex. In the presence of ATP the binding of hMSH6 to mismatched DNA is decreased. In addition, hMLH1, hPMS2, and hPMS1 no longer interact with each other or with the hMutS␣ complex (hMSH2 and hMSH6). However, the ability of hMLH1 to co-immunoprecipitate mismatched DNA increases in the presence of ATP. This interaction is dependent on the presence of the mismatch and does not appear to involve a direct binding of hMLH1 to the DNA.The DNA mismatch repair system acts to recognize and repair mispaired nucleotides and plays an important role in maintaining genomic integrity (for reviews see Refs. 1-3). The importance of this mutation avoidance system can be seen in HNPCC 1 kindreds, where germ-line mutations in the genes associated with mismatch repair lead to microsatellite instability, elevated mutation rates, and predisposition to cancer (4, 5). Similar defects in MMR have also been found in spontaneous colon cancers and other solid tumors (6). Although the precise functions of the human MMR proteins are just beginning to be understood, the human MMR genes are homologous to those in the relatively well characterized Escherichia coli MutHLS strand-specific mismatch repair pathway (for review see Ref. 7). Indeed, the degree of conservation between such divergent species reflects the essential role of MMR. In bacteria, the MutS protein recognizes mismatches and initiates the repair process. Next, MutS interacts with MutL and translocates along the DNA strand, forming an ␣-loop structure (8). This process requires ATP hydrolysis and MutL interaction, although the exact role MutL plays is unclear. Following MutH incision of the unmethylated strand, a segment of DNA is excised by Rec I, exonuclease I, or exonuclease VII, and the DNA is re-synthesized by the polymerase III holoenzyme.In humans, the MMR pathway is more complex than that of E. coli. At least six genes have now been demonstrated to be involved in eukaryotic MMR. These include the MutS homologs hMSH2, hMSH6, and hMSH3 and the MutL homologs hMLH1, hPMS2, and hPMS1 (for reviews see Refs. 9 and 10). hMSH2 and hMSH6 have been shown to interact with each other in the hMutS␣ heterodimer that binds t...

Interactions of the DNA mismatch repair proteins MLH1 and MSH2 with c-MYC and MAX

“…Since MAX and MSH2 are coprecipitated from the MLH1/PMS2-deficient A2780/cp70 line, this argues that neither MLH1 nor PMS2 are required for the MAX: MSH2 interaction detected. Coimmunoprecipitation of MSH2 and MLH1 has been observed from human cell extracts under certain experimental conditions (Gu et al, 1998;Matton et al, 2000). However, as shown in Fig.…”

Recognition and binding of mismatch repair proteins at an oncogenic hot spot

“…In confirmation of hMutSα binding to a G:T mismatch at this oncogenic site, the hMutSα :DNA complex was efficiently interrupted by goat anti-hMSH6 and with rabbit anti-hMSH2 (Figure 1, lanes 3 and 5), but not by BSA, nonspecific goat IgG, or nonspecific rabbit IgG (lanes 1, 2, and 4, respectively). As further evidence of specific hMutsα:DNA mismatch binding, addition of 1.5 mM ATP completely disrupted the gel shifted band (results not shown) [24,30]. Thus, MMR protein binding to a G:T mismatch located at H- ras codon 12 within the nuclear environment appears to be primarily, if not exclusively, hMutSα.…”

“…The primary human homologs for MutS that play instrumental roles in MMR include hMSH2, hMSH6, and hMSH3 [20,21]. The hMutSα heterodimer (hMSH2 and hMSH6) has been demonstrated to recognize and bind DNA mispairs and short IDLs [14,21-24]. The hMutSβ heterodimer (hMSH2 and hMSH3) preferentially recognizes and binds IDLs of up to 12 nucleotides.…”

“…We have previously demonstrated that the efficiency of correct mismatch repair within the cell can differ significantly, depending on exact type of mismatch, site-specific location, phase of cell cycle, or cell type [23,24,37,38]. Within this report, we have focused on a more precise understanding of the differences between MMR protein interactions with a G:A (least repaired) or G:T (best repaired) mismatch located at H- ras codon 12 to better understand molecular events leading to activating mutations at this site.…”

DNA Mismatch Repair in Advanced Metastatic Prostate Carcinoma: Clinical Perspective

“…However, MutS homolog 2 (MSH2) complexes with MSH6 or MSH3 to generate heterodimers of the MSH2/ MSH6 (MutSα) or MSH2/MSH3 (MutSβ) [11]. These complexes then help in recognition and digestion of mismatched DNA segments and create error-free DNA helix.…”