Miyoko Ikejima

hMutSβ, a heterodimer of hMSH2 and hMSH3, binds to insertion/deletion loops in DNA

In human cells, mismatch recognition is mediated by a heterodimeric complex, hMutSalpha, comprised of two members of the MutS homolog (MSH) family of proteins, hMSH2 and GTBP [1,2]. Correspondingly, tumour-derived cell lines defective in hMSH2 and GTBP have a mutator phenotype [3,4], and extracts prepared from these cells lack mismatch-binding activity [1]. However, although hMSH2 mutant cell lines showed considerable microsatellite instability in tracts of mononucleotide and dinucleotide repeats [4,5], only mononucleotide repeats were somewhat unstable in GTBP mutants [4,6]. These findings, together with data showing that extracts of cells lacking GTBP are partially proficient in the repair of two-nucleotide loops [2], suggested that loop repair can be GTBP-independent. We show here that hMSH2 can also heterodimerize with a third human MSH family member, hMSH3, and that this complex, hMutSbeta, binds loops of one to four extrahelical bases. Our data further suggest that hMSH3 and GTBP are redundant in loop repair, and help explain why only mutations in hMSH2, and not in GTBP or hMSH3, segregate with hereditary non-polyposis colorectal cancer (HNPCC) [7].

Studies of nuclear ADP-ribosylation

Abstract Recent studies on poly(ADP-ribose) are reviewed. Poly(ADP-ribose) was shown to have α (1″ → 2′) ribose-ribose bonds. High molecular weight poly(ADP-ribose) with a branched structure was demonstrated. The structure of the branch linkage was determined as 2″-[1′-ribosyl-2″ (1‴-ribosyl)]-adenosine 5′,5″,5‴-tris(phosphate). The known sites of ADP-ribosylation of proteins were summarized. The enzymatic synthesis of poly(ADP-ribose) was classified into three steps, initiation, elongation and branching. Degradation of poly(ADP-ribose) may be mainly performed by α (1″ → 2′)-poly(ADP-ribose) ribohydrolase. Specific antibodies against poly(ADP-ribose) and its monomer, 2′-ribosyl adenosine 5′,5″-bis(phosphate), were obtained and used for quantitative measurement of naturally occurring poly(ADP-ribose). High antibody activity was found in the serum of the patients with systemic lupus erythematosus. Poly(A)·poly(U) could also raise specific antibody against poly(ADP-ribose) in rabbits. Nicotinamide, an inhibitor of poly(ADP-ribose) polymerase, increased sister chromatid exchanges. Among the nicotinamide analogues tested, N ′-methylnicotinamide was the best inducer of differentiation of Friend erythroleukemia cells. Purified poly(ADP-ribose) induced differentiation of mouse myelogenous leukemia cells.

Expression of human poly(ADP-ribose) polymerase with DNA-dependent enzymatic activity inEscherichia coli

A cDNA for human poly(ADP-ribose) polymerase was inserted into a plasmid, transfected and expressed in E. coli. A lysate of the E. coli cells containing the expression plasmid reacted with antibody against human poly(ADP-ribose) polymerase and synthesized poly(ADP-ribose). The partially purified poly(ADP-ribose) polymerase expressed in E. coli had the same molecular weight and enzymological properties as human placental poly(ADP-ribose) polymerase, including affinity for NAD, turnover number and DNA-dependency for activity. This expression system should be useful for structure-function analysis of poly(ADP-ribose) polymerase.

Decreased UV sensitivity, mismatch repair activity and abnormal cell cycle checkpoints in skin cancer cell lines derived from UVB-irradiated XPA-deficient mice.

Xeroderma pigmentosum group A gene (XPA)-deficient mice are defective in nucleotide excision repair (NER) and are therefore highly sensitive to ultraviolet (UV)-induced skin carcinogenesis. We established cell lines from skin cancers of UVB-irradiated XPA-deficient mice to investigate the phenotypic changes occurring during skin carcinogenesis. As anticipated, the skin cancer cell lines were devoid of NER activity but were less sensitive to killing by UV-irradiation than the XPA(-/-) fibroblast cell line. The lines were also more resistant to 6-thioguanine (6-TG) than XPA(-/-) and XPA(+/+) fibroblasts, which was suggestive of a mismatch repair (MMR) defect. Indeed, in vitro mismatch binding and MMR activity were impaired in several of these cell lines. Moreover, these cell lines displayed cell cycle checkpoint derangements following UV-irradiation and 6-TG exposure. The above findings suggest that MMR downregulation may help cells escape killing by UVB, as was seen previously for methylating agents and cisplatin, and thus that MMR deficient clones are selected for during the tumorigenic transformation of XPA(-/-) cells.

Association between single nucleotide polymorphisms in the hMSH3 gene and sporadic colon cancer with microsatellite instability

AbstractThe association between three single nucleotide polymorphisms (SNPs) in the hMSH3 gene and sporadic colon cancer with microsatellite instability (MSI) was analyzed. Of the three SNPs observed in this population, SNPs at residues 235 and 693 were novel, while that at residue 3133 was previously described. The SNPs at residues 235 and 3133 caused amino acid substitutions, V79I and T1045A, respectively. We analyzed the allele frequencies of the three SNPs in samples from 19 patients with sporadic colon cancer with MSI and 90 healthy controls. We found that the V79 allele frequency was significantly higher in the tumor samples than in controls. In addition, the frequency of the G693 allele showed a higher trend in the tumor samples than in controls. These results indicated that some SNPs in the hMSH3 gene were associated with colon cancer with MSI.

NINE-bp REPEAT POLYMORPHISM IN EXON 1 OF THE hMSH3 GENE

SummaryWe have identified a polymorphic 9-bp repeat sequence in exon 1 of the hMSH3 gene using polymerase chain reaction (PCR). Five alleles were observed in unrelated Japanese individuals with heterozygosity of 0.57.

A novel missense mutation and frameshift mutations in the type II receptor of transforming growth factor-β gene in sporadic colon cancer with microsatellite instability

Microsatellite instability of DNA samples of 79 sporadic colon cancer patients were analyzed. These samples were also screened to search mutations in the repeat sequences in the gene for the type II receptor of transforming growth factor-beta (TGF-beta RII) using polymerase chain reaction (PCR), electrophoresis with urea gel, and PCR-single strand conformation polymorphism (PCR-SSCP) method. The incidence of microsatellite instability, defined as severe replication error phenotype (RER) with microsatellite alterations in more than three loci, was 6%. Deletion and insertion of an A residue in the (A)10 region, which cause frameshift mutation, were found in four samples and their incidence in the samples with microsatellite instability was 80%. A novel nucleotide substitution of T for G at 1918, which causes missense mutation of arginine to leucine at codon 528, was found in a sample with microsatellite instability. The mutation at 1918 was in highly conservative amino acid residue.

Frameshift mutations and a length polymorphism in the hMSH3 gene and the spectrum of microsatellite instability in sporadic colon cancer

Mutations in the hMSH3 gene in sporadic colon cancer with microsatellite instability (MSI) were investigated, since several mismatch repair genes were known to be mutated in cancers with MSI, but only deletions in the (A)8 region in the hMSH3 gene have been reported. We also analyzed the relationships between hMSH3 mutations and the spectrum of MSI. We screened MSI in 79 sporadic colon cancer samples using mono‐ and dinucleotide repeat markers and the samples with MSI were further analyzed for tri‐ and tetranucleotide repeat instability and mutations in the hMSH3 gene by polymerase chain reaction‐single strand conformation polymorphism (PCR‐SSCP) analysis. Five (6%) out of 79 tumors were MSI‐H and 15 (19%) were MSI‐L. Two MSI‐H tumors showed insertion in the (C)8 region in the hMSH6 gene and one tumor showed insertion and deletion in the (A)8 region in the hMSH3 gene, and two of the three above tumors showed MSI in tri‐and tetranucleotide repeats. One MSI‐L tumor showed somatic alteration in a 9‐bp repeat sequence in hMSH3. No frameshift mutations were found in the (A)7 and (A)6 regions in hMSH3. Thus, we confirmed that the (A)8 region in hMSH3 is a hot spot and mutations in the (A)7 and (A)6 regions in hMSH3 are not common. The hMSH3 mutation may enhance genomic instability in some colorectal cancers.

Identification of the protein components of mismatch binding complexes in human cells using a gel-shift assay

In eukaryotes, mismatch recognition is thought to be mediated by two heterodimers, hMutSα (hMSH2+hMSH6), which preferentially binds to base‐base mismatches and hMutSβ (hMSH2+hMSH3), which binds to insertion/deletion loops. We studied these mismatch binding activities in several human cell lines with a gel‐shift assay using various mismatch oligonucleotides as substrates. Both hMutSα and hMutSβ activities could be detected in various human cell lines. In cells with amplified copies of the hMSH3 gene, a large increase in hMutSβ and a reduction in hMutSα were observed. To identify the composition of each mismatch binding complex, the protein‐DNA complexes were transferred from gel‐shift polyacrylamide gel to a polyvinylidene difluoride membrane and were subjected to immunoblot analysis with an enhanced chemiluminescence protein detection system. The results clearly demonstrated that hMutSα detected by the gel‐shift assay was composed of hMSH2 and hMSH6, while hMutSβ was composed of hMSH2 and hMSH3. Our data, therefore, support a model whereby formation of hMutSα and hMutSβ is mutually regulated. Combination of a gel‐shift assay with immunoblotting (shift‐Western assay) proved to be a highly sensitive technique and should be useful for studying the interactions between DNA and binding proteins, including DNA mismatch recognition.

Regulation of DNA polymerase ß by poly(ADP-ribose) polymerase

The involvement of poly(ADP-ribose) polymerase in the DNA repair process has been suggested by various in vitro and in vivo studies (Althaus & Richter, 1987). However, the mechanism of the involvement of poly(ADP-ribose) polymerase in DNA repair is still unclear. Recently we isolated the 5’-flanking region of human poly(ADP-ribose) polymerase gene and found that it has a structural similarity to the 5’-flanking region of the genes of human and mouse DNA polymerase s which is one of the key enzymes of DNA repair (Ogura et al., 1990a). We also found a striking similarity in the distribution patterns of the transcript and enzymatic activity of both enzymes in various mouse organs (Ogura et al., 1990b). Recently we found that both poly(ADP-ribose) polymerase and DNA polymerase (s activities dramatically increased during rat liver regeneration and after stimulation of human peripheral blood lymphocytes by phytohemagglutinin (to be published elsewhere). It is interesting to note that both enzymes utilize DNA nick for their enzymatic activities (Benjamin & Gill, 1980, Wang & Korn, 1980). All these characteristics of both enzymes suggest that there might be a similar regulatory mechanism of the expression of both genes and that both enzymes might functionally interact with each other. In this work, we report the interaction of poly(ADP-ribose) polymerase and DNA polymerase s in vitro and a possible interaction of in vivo.