Active site mutations in mammalian DNA polymerase delta alter accuracy and replication fork progression.

DNA polymerase δ (pol δ) is one of the two main replicative polymerases in eukaryotes; it synthesizes the lagging DNA strand and also functions in DNA repair. In previous work, we demonstrated that heterozygous expression of the pol δ L604G variant in mice results in normal life span and no apparent phenotype, whereas a different substitution at the same position, L604K, is associated with shortened life span and accelerated carcinogenesis. Here, we report in vitro analysis of the homologous mutations at position Leu-606 in human pol δ. Four-subunit human pol δ variants that harbor or lack 3' → 5'-exonucleolytic proofreading activity were purified from Escherichia coli. The pol δ L606G and L606K holoenzymes retain catalytic activity and processivity similar to that of wild type pol δ. pol δ L606G is highly error prone, incorporating single noncomplementary nucleotides at a high frequency during DNA synthesis, whereas pol δ L606K is extremely accurate, with a higher fidelity of single nucleotide incorporation by the active site than that of wild type pol δ. However, pol δ L606K is impaired in the bypass of DNA adducts, and the homologous variant in mouse embryonic fibroblasts results in a decreased rate of replication fork progression in vivo. These results indicate that different substitutions at a single active site residue in a eukaryotic polymerase can either increase or decrease the accuracy of synthesis relative to wild type and suggest that enhanced fidelity of base selection by a polymerase active site can result in impaired lesion bypass and delayed replication fork progression.

LOH-proficient embryonic stem cells: a model of cancer progenitor cells?

Cancers are thought to originate in stem cells through the accumulation of multiple mutations. Some of these mutations result in a loss of heterozygosity (LOH). A recent report demonstrates that exposure of mouse embryonic stem cells to nontoxic amounts of mutagens triggers a marked increase in the frequency of LOH. Thus, mutagen induction of LOH in embryonic stem cells suggests a new pathway to account for the multiple homozygous mutations in human tumors. This induction could mimic early mutagenic events that generate cancers in human tissue stem cells.

Mutation at the polymerase active site of mouse DNA polymerase delta increases genomic instability and accelerates tumorigenesis.

Mammalian DNA polymerase delta (Pol delta) is believed to replicate a large portion of the genome and to synthesize DNA in DNA repair and genetic recombination pathways. The effects of mutation in the polymerase domain of this essential enzyme are unknown. Here, we generated mice harboring an L604G or L604K substitution in highly conserved motif A in the polymerase active site of Pol delta. Homozygous Pold1(L604G/L604G) and Pold1(L604K/L604K) mice died in utero. However, heterozygous animals were viable and displayed no overall increase in disease incidence, indicative of efficient compensation for the defective mutant polymerase. The life spans of wild-type and heterozygous Pold1(+/L604G) mice did not differ, while that of Pold1(+/L604K) mice was reduced by 18%. Cultured embryonic fibroblasts from the heterozygous strains exhibited comparable increases in both spontaneous mutation rate and chromosome aberrations. We observed no significant increase in cancer incidence; however, Pold1(+/L604K) mice bearing histologically diagnosed tumors died at a younger median age than wild-type mice. Our results indicate that heterozygous mutation at L604 in the polymerase active site of DNA polymerase delta reduces life span, increases genomic instability, and accelerates tumorigenesis in an allele-specific manner, novel findings that have implications for human cancer.

Generation of mutator mutants during carcinogenesis.

Mutations are rare in normal cells. In contrast, multiple mutations are characteristic in most tumors. Previously we proposed a "mutator phenotype" hypothesis to explain how pre-cancer cells may acquire large number of mutations during carcinogenesis. Here we extend the "mutator phenotype" hypothesis considering recently discovered biochemical activities whose aberrant expression may result in genome-wide random mutations. The scope of this article is to emphasize that simple random point mutations can drive carcinogenesis and highlight new emerging pathways that generate these mutations. We focus specifically on random point mutations generated by replication errors, oxidative base damage, covalent base modifications by enzymes, and spontaneously generated abasic sites as a source of mutator mutants.

Mutator phenotypes caused by substitution at a conserved motif A residue in eukaryotic DNA polymerase delta.

Eukaryotic DNA polymerase (Pol) delta replicates chromosomal DNA and is also involved in DNA repair and genetic recombination. Motif A in Pol delta, containing the sequence DXXXLYPSI, includes a catalytically essential aspartic acid as well as other conserved residues of unknown function. Here, we used site-directed mutagenesis to create all 19 amino acid substitutions for the conserved Leu(612) in Motif A of Saccharomyces cerevisiae Pol delta. We show that substitutions at Leu(612) differentially affect viability, sensitivity to genotoxic agents, cell cycle progression, and replication fidelity. The eight viable mutants contained Ile, Val, Thr, Met, Phe, Lys, Asn, or Gly substitutions. Individual substitutions varied greatly in the nature and extent of attendant phenotypic deficiencies, exhibiting mutation rates that ranged from near wild type to a 37-fold increase. The L612M mutant exhibited a 7-fold elevation of mutation rate but essentially no detectable effects on other phenotypes monitored; the L612T mutant showed a nearly wild type mutation rate together with marked hypersensitivity to genotoxic agents; and the L612G and L612N strains exhibited relatively high mutation rates and severe deficits overall. We compare our results with those for homologous substitutions in prokaryotic and eukaryotic DNA polymerases and discuss the implications of our findings for the role of Leu(612) in replication fidelity.

Vertebrate tankyrase domain structure and sterile alpha motif (SAM)-mediated multimerization.

Tankyrases 1 and 2 are two highly related poly(ADP-ribose) polymerases that interact with a variety of cytoplasmic and nuclear proteins. Both proteins have been implicated in telomere length regulation, insulin signalling and centrosome function. To learn more about their mode of action, we have isolated the chicken tankyrase homologues and examined their interaction partners and subcellular location. Cross-species sequence comparison indicated that tankyrase domain structure is highly conserved and supports division of the ankyrin domain into five subdomains, which are each separated by a highly conserved LLEAAR/K motif. Glutathione S-transferase pull-down experiments demonstrated that the ankyrin domains of both proteins interact with chicken telomere repeat factor 1 (TRF1). Analysis of total cellular and nuclear proteins revealed that cells contain approximately twice as much tankyrase 1 as tankyrase 2. Although > or = 90% of each protein is present in the cytoplasm, both tankyrase 1 and 2 were detected in the nucleus. The nuclear location together with its ability to interact with TRF1, point to tankyrase 2 having a telomeric function. Yeast two-hybrid and cross-linking experiments show that both tankyrases can multimerize through their sterile-alpha motif domains. These results indicate that tankyrases may be master scaffolding proteins, capable of regulating assembly of large protein complexes.

Benzoate X receptors alpha and beta are pharmacologically distinct and do not function as xenobiotic receptors.

The Xenopus benzoate nuclear hormone receptors, BXRalpha and BXRbeta, share 82% identity within their ligand-binding domains and are classified as members of the NR1I2 subfamily that includes the mammalian steroid and xenobiotic receptor, SXR/PXR. Although alkyl benzoates have been identified as endogenous ligands, the exact role of the benzoate receptors in amphibian physiology has not been established. In this report, we show that BXRalpha and BXRbeta are pharmacologically distinct from each other: BXRalpha is more promiscuous than BXRbeta with respect to both ligand specificity and co-activator recruitment. BXRalpha can be transactivated by a number of benzoate derivatives including 4-amino-butylbenzoate (4-ABB), 4-hydroxy-butylbenzoate (4-HBB), 3-hydroxy ethyl benzoate (3-HEB), and benzyl benzoate, but only 4-HBB acts as an agonist for both receptors. Furthermore, BXRalpha-specific agonists such as 4-ABB, chlorpyrifos, and trifluralin act as antagonists on BXRbeta. BXRs are widely distributed in adult tissues but do not show any enrichment in liver and intestine, major sites of SXR/PXR expression that are critical in xenobiotic metabolism. Neither BXR shows the broad specificity toward steroids or xenobiotics exhibited by SXR/PXR. Therefore, we conclude that the BXRs are pharmacologically distinct from each other and unlikely to serve as xenobiotic sensors.