No abstract

Alterations in the exonuclease domain of DNA polymerase ε (Polε) cause ultramutated tumors. Severe mutator effects of the most common variant, Polε-P286R, modeled in yeast suggested that its pathogenicity involves yet unknown mechanisms beyond simple proofreading deficiency. We show that, despite producing a catastrophic amount of replication errors in vivo, the yeast Polε-P286R analog retains partial exonuclease activity and is more accurate than exonuclease-dead Polε. The major consequence of the arginine substitution is a dramatically increased DNA polymerase activity. This is manifested as a superior ability to copy synthetic and natural templates, extend mismatched primer termini, and bypass secondary DNA structures. We discuss a model wherein the cancer-associated substitution limits access of the 3’-terminus to the exonuclease site and promotes binding at the polymerase site, thus stimulating polymerization. We propose that the ultramutator effect results from increased polymerase activity amplifying the contribution of Polε errors to the genomic mutation rate.

DNA polymerase ζ (Pol ζ) and Rev1 are key players in translesion DNA synthesis. The error-prone Pol ζ can also participate in replication of undamaged DNA when the normal replisome is impaired. Here we define the nature of the replication disturbances that trigger the recruitment of error-prone polymerases in the absence of DNA damage and describe the specific roles of Rev1 and Pol ζ in handling these disturbances. We show that Pol ζ/Rev1-dependent mutations occur at sites of replication stalling at short repeated sequences capable of forming hairpin structures. The Rev1 deoxycytidyl transferase can take over the stalled replicative polymerase and incorporate an additional ‘C’ at the hairpin base. Full hairpin bypass often involves template-switching DNA synthesis, subsequent realignment generating multiply mismatched primer termini and extension of these termini by Pol ζ. The postreplicative pathway dependent on polyubiquitylation of proliferating cell nuclear antigen provides a backup mechanism for accurate bypass of these sequences that is primarily used when the Pol ζ/Rev1-dependent pathway is inactive. The results emphasize the pivotal role of noncanonical DNA structures in mutagenesis and reveal the long-sought-after mechanism of complex mutations that represent a unique signature of Pol ζ.

DNA replication fidelity relies on base selectivity of the replicative DNA polymerases, exonucleolytic proofreading, and postreplicative DNA mismatch repair (MMR). Ultramutated human cancers without MMR defects carry alterations in the exonuclease domain of DNA polymerase ε (Polε). They have been hypothesized to result from defective proofreading. However, modeling of the most common variant, Polε-P286R, in yeast produced an unexpectedly strong mutator effect that exceeded the effect of proofreading deficiency by two orders of magnitude and indicated the involvement of other infidelity factors. The in vivo consequences of many additional Polε mutations reported in cancers remain poorly understood. Here, we genetically characterized 13 cancer-associated Polε variants in the yeast system. Only variants directly altering the DNA binding cleft in the exonuclease domain elevated the mutation rate. Among these, frequently recurring variants were stronger mutators than rare variants, in agreement with the idea that mutator phenotype has a causative role in tumorigenesis. In nearly all cases, the mutator effects exceeded those of an exonuclease-null allele, suggesting that mechanisms distinct from loss of proofreading may drive the genome instability in most ultramutated tumors. All mutator alleles were semidominant, supporting the view that heterozygosity for the polymerase mutations is sufficient for tumor development. In contrast to the DNA binding cleft alterations, peripherally located variants, including a highly recurrent V411L, did not significantly elevate mutagenesis. Finally, the analysis of Polε variants found in MMR-deficient tumors suggested that the majority cause no mutator phenotype alone but some can synergize with MMR deficiency to increase the mutation rate.

Aggregation substance (AS) is anEnterococcus faecalis surface protein that may contribute to virulence. Using a recently described system for controlled expression of AS in E. faecalis and the heterologous host Lactococcus lactis, experiments were designed to assess the effect of AS on bacterial internalization by HT-29 and Caco-2 enterocytes. AS expression was associated with increased internalization of E. faecalis by HT-29 enterocytes and of L. lactis by HT-29 and Caco-2 enterocytes. Compared to enterocytes cultivated under standard conditions, either cultivation in hypoxia or 1-h pretreatment of enterocytes with calcium-free medium resulted in increased internalization of both E. faecalis and L. lactis (with and without AS expression). Also, AS expression augmented these increases when E. faecalis was incubated with pretreated HT-29 enterocytes and when L. lactis was incubated with pretreated Caco-2 and HT-29 enterocytes. These data indicated that AS might facilitate E. faecalis internalization by cultured enterocytes.Although Enterococcus faecalis is a component of the normal human intestinal flora, enterococci number among the top three nosocomial microbial pathogens (8), and strains resistant to all useful antimicrobial agents are increasingly involved in fatal infections (12). Thus, it is important to clarify the mechanisms involved in extraintestinal dissemination of enterococci. Aggregation substance (AS) protein may be involved in virulence and is expressed on the surface of E. faecalis. AS molecules are encoded by different pheromone-responsive plasmids; e.g., Asc10 is encoded by pCF10, and Asa1 is encoded by pAD1 (6). Pheromones produced by potential recipients induce expression of AS on the surfaces of plasmid-containing donor cells. AS facilitates aggregation of donor and recipient bacteria and aids conjugative plasmid transfer (6).The gene for Asc10 was recently cloned in a vector containing a nisin-inducible promoter, resulting in surface expression of Asc10 on E. faecalis and the heterologous host Lactococcus lactis. E. faecalis OG1SSp and L. lactis NZ9800 were transformed with plasmid pMSP7517 that encodes Asc10 (9). We have used these transformants to clarify the effect of AS on bacterium-enterocyte interactions. Because pMSP7517 contains a gene for erythromycin resistance, bacteriological media were supplemented with 10 g of erythromycin (Sigma Chemical Co., St. Louis, Mo.) per ml. For experiments, E. faecalis was cultivated overnight in Todd-Hewitt broth (Difco Laboratories, Detroit, Mich.) in the absence of nisin or in broth supplemented with 25 ng of nisin (Sigma) per ml; nisin was present either throughout the incubation period or only during the final 2 h. Following incubation at 35°C with nisin, E. faecalis cells clumped, confirming Asc10 expression (6, 9). To obtain single-cell suspensions (verified by light microscopy), these inocula were sonicated, typically with 20 W for 10 s using a 40-W high-intensity ultrasonic processor (Sonics and Materials, Danbury, Conn.). L. lactis was cult...