No abstract

A mismatch-binding heterodimer of hMSH2 and a 160-kilodalton polypeptide has been isolated from HeLa cells by virtue of its ability to restore mismatch repair to nuclear extracts of hMSH2-deficient LoVo colorectal tumor cells. This heterodimer, designated hMutS alpha, also restores mismatch repair to extracts of alkylation-tolerant MT1 lymphoblastoid cells and HCT-15 colorectal tumor cells, which are selectively defective in the repair of base-base and single-nucleotide insertion-deletion mismatches. Because HOT-15 cells appear to be free of hMSH2 mutations, this selective repair defect is likely a result of a deficiency of the hMutS alpha 160-kilodalton subunit, and mutations in the corresponding gene may confer hypermutability and cancer predisposition.

The human lymphoblastoid MT1 B-cell line was previously isolated as one of a series of mutant cells able to survive the cytotoxic effects ofN-methyl-N'-nitro-N-nitrosoguanidine (MNNG single-nucleotide insertions, transversions, and A-T -k G-C transitions.In vitro assay has demonstrated that the MT1 line is in fact deficient in strand-specific correction of all eight basebase mispairs. This defect, which is manifest at or prior to the excision stage of the reaction, is due to simple deficiency of a requlred activity because MT1 nuclear extracts can be complemented by a partially purified HeLa fraction to restore in vitro repair. These findings substantiate the idea that strand-specific mismatch repair contributes to alkylation-induced cytotoxicity and imply that this process serves as a barrier to spontaneous transition, transversion, and insertion/deletion mutations in mamian cells.

Human DNA polymerase ␥ is composed of a 140-kDa catalytic subunit and a smaller accessory protein variously reported to be 43-54 kDa. Immunoblot analysis of the purified, heterodimeric native human polymerase ␥ complex identified the accessory subunit as 55 kDa. We isolated the full-length cDNA encoding a 55-kDa polypeptide, expressed the cDNA in Escherichia coli and purified the 55-kDa protein to homogeneity. Recombinant Hp55 forms a high affinity, salt-stable complex with Hp140 during protein affinity chromatography. Immunoprecipitation, gel filtration, and sedimentation analyses revealed a 190-kDa complex indicative of a native heterodimer. Reconstitution of Hp140⅐Hp55 raises the salt optimum of Hp140, stimulates the polymerase and exonuclease activities, and increases the processivity of the enzyme by several 100-fold. Similar to Hp140, isolated Hp55 binds DNA with moderate strength and was a specificity for double-stranded primer-template DNA. However, Hp140⅐Hp55 has a surprisingly high affinity for DNA, and kinetic analyses indicate Hp55 enhances the affinity of Hp140 for primer termini by 2 orders of magnitude. Thus the enhanced DNA binding caused by Hp55 is the basis for the salt tolerance and high processivity characteristic of DNA polymerase ␥. Observation of native DNA polymerase ␥ both as an Hp140 monomer and as a heterodimer with Hp55 supports the notion that the two forms act in mitochondrial DNA repair and replication. Additionally, association of Hp55 with Hp140 protects the polymerase from inhibition by N-ethylmaleimide.Human mitochondrial DNA is a 16,569-base pair closed circular molecule encoding 13 polypeptides required for oxidative phosphorylation and 24 specialized tRNA and rRNAs needed for translation within the organelle (for review see Refs. 1 and 2). Point mutations or deletions in mitochondrial DNA cause of a wide range of neurological and cardiopathological diseases (3). The human mitochondrial genome is replicated by the nuclear encoded DNA polymerase ␥ (4). DNA polymerase ␥ has been characterized as a processive, salt-tolerant, dideoxynucleotide-sensitive, aphidicolin-resistant, Family A type DNA polymerase that can utilize a wide variety of DNA substrates including poly(rA)⅐oligo(dT) (5). Animal cell DNA polymerase ␥ from Drosophila melanogaster was first shown unequivocally by Wernette and Kaguni (6) to consist of two subunits of 125 and 35 kDa in the highly purified fraction. Highly purified DNA polymerase ␥ from Xenopus laevis contains two subunit of 140 and 50 kDa (7). An initial report on human HeLa cell DNA polymerase ␥ identified a 140-kDa polypeptide and a 54-kDa polypeptide in the most purified fraction (8).We have previously cloned the cDNAs for the human, chicken, Drosophila, and Schizosaccharomyces pombe DNA polymerase ␥ catalytic subunit (9, 10). The overexpressed human 140-kDa catalytic subunit contains DNA polymerase, 3Ј 3 5Ј exonuclease, and 5ЈdRP lyase activities in the absence of the accessory subunit (11, 12). Native heterodimeric Drosophila polymerase ␥ is high...