Mechanisms of tandem repeat instability in bacteria

Hypermutable tandem repeat sequences (TRSs) are present in the genomes of both prokaryotic and eukaryotic organisms. Numerous studies have been conducted in several laboratories over the past decade to investigate the mechanisms responsible for expansions and contractions of microsatellites (a subset of TRSs with a repeat length of 1-6 nucleotides) in the model prokaryotic organism Escherichia coli. Both the frequency of tandem repeat instability (TRI), and the types of mutational events that arise, are markedly influenced by the DNA sequence of the repeat, the number of unit repeats, and the types of cellular pathways that process the TRS. DNA strand slippage is a general mechanism invoked to explain instability in TRSs. Misaligned DNA sequences are stabilized both by favorable base pairing of complementary sequences and by the propensity of TRSs to form relatively stable secondary structures. Several cellular processes, including replication, recombination and a variety of DNA repair pathways, have been shown to interact with such structures and influence TRI in bacteria. This paper provides an overview of our current understanding of mechanisms responsible for TRI in bacteria, with an emphasis on studies that have been carried out in E. coli. In addition, new experimental data are presented, suggesting that TLS polymerases (PolII, PolIV and PolV) do not contribute significantly to TRI in E. coli.     

Two opposing effects of mismatch repair on CTG repeat instability in Escherichia coli

The expansion of normally polymorphic CTG microsatellites in certain human genes has been identified as the causative mutation of a number of hereditary neurological disorders, including Huntington's disease and myotonic dystrophy. Here, we have investigated the effect of methyl-directed mismatch repair (MMR) on the stability of a (CTG)43 repeat in Escherichia coli over 140 generations and find two opposing effects. In contrast to orientation-dependent repeat instability in wild-type E. coli and yeast, we observed no orientation dependence in MMR- E. coli cells and suggest that, for the repeat that we have studied, orientation dependence in wild-type cells is mainly caused by functional mismatch repair genes. Our results imply that slipped structures are generated during replication, causing single triplet expansions and contractions in MMR- cells, because they are left unrepaired. On the other hand, we find that the repair of such slipped structures by the MMR system can go awry, resulting in large contractions. We show that these mutS-dependent contractions arise preferentially when the CTG sequence is encoded by the lagging strand. The nature of this orientation dependence argues that the small slipped structures that are recognized by the MMR system are formed primarily on the lagging strand of the replication fork. It also suggests that, in the presence of functional MMR, removal of 3 bp slipped structures causes the formation of larger contractions that are probably the result of secondary structure formation by the CTG sequence. We rationalize the opposing effects of MMR on repeat tract stability with a model that accounts for CTG repeat instability and loss of orientation dependence in MMR- cells. Our work resolves a contradiction between opposing claims in the literature of both stabilizing and destabilizing effects of MMR on CTG repeat instability in E. coli.     

Mechanisms of trinucleotide repeat instability during human development

Trinucleotide expansion underlies several human diseases. Expansion occurs during multiple stages of human development in different cell types, and is sensitive to the gender of the parent who transmits the repeats. Repair and replication models for expansions have been described, but we do not know whether the pathway involved is the same under all conditions and for all repeat tract lengths, which differ among diseases. Currently, researchers rely on bacteria, yeast and mice to study expansion, but these models differ substantially from humans. We need now to connect the dots among human genetics, pathway biochemistry and the appropriate model systems to understand the mechanism of expansion as it occurs in human disease.     

Two modes of microsatellite instability in human cancer: differential connection of defective DNA mismatch repair to dinucleotide repeat instability

Microsatellite instability (MSI) is associated with defective DNA mismatch repair in various human malignancies. Using a unique fluorescent technique, we have observed two distinct modes of dinucleotide microsatellite alterations in human colorectal cancer. Type A alterations are defined as length changes of ≤6 bp. Type B changes are more drastic and involve modifications of ≥8 bp. We show here that defective mismatch repair is necessary and sufficient for Type A changes. These changes were observed in cell lines and in tumours from mismatch repair gene-knockout mice. No Type B instability was seen in these cells or tumours. In a panel of human colorectal tumours, both Type A MSI and Type B instability were observed. Both types of MSI were associated with hMSH2 or hMLH1 mismatch repair gene alterations. Intriguingly, p53 mutations, which are generally regarded as uncommon in human tumours of the MSI⁺ phenotype, were frequently associated with Type A instability, whereas none was found in tumours with Type B instability, reflecting the prevailing viewpoint. Inspection of published data reveals that the microsatellite instability that has been observed in various malignancies, including those associated with Hereditary Non-Polyposis Colorectal Cancer (HNPCC), is predominantly Type B. Our findings indicate that Type B instability is not a simple reflection of a repair defect. We suggest that there are at least two qualitatively distinct modes of dinucleotide MSI in human colorectal cancer, and that different molecular mechanisms may underlie these modes of MSI. The relationship between MSI and defective mismatch repair may be more complex than hitherto suspected.     

Mechanisms of replication fork restart in Escherichia coli

Replication of the genome is crucial for the accurate transmission of genetic information. It has become clear over the last decade that the orderly progression of replication forks in both prokaryotes and eukaryotes is disrupted with high frequency by encounters with various obstacles either on or in the template strands. Survival of the organism then becomes dependent on both removal of the obstruction and resumption of replication. This latter point is particularly important in bacteria, where the number of replication forks per genome is nominally only two. Replication restart in Escherichia coli is accomplished by the action of the restart primosomal proteins, which use both recombination intermediates and stalled replication forks as substrates for loading new replication forks. These reactions have been reconstituted with purified recombination and replication proteins.     

Involvement of the nucleotide excision repair protein UvrA in instability of CAG*CTG repeat sequences in Escherichia coli.

Several human genetic diseases have been associated with the genetic instability, specifically expansion, of trinucleotide repeat sequences such as (CTG)(n).(CAG)(n). Molecular models of repeat instability imply replication slippage and the formation of loops and imperfect hairpins in single strands. Subsequently, these loops or hairpins may be recognized and processed by DNA repair systems. To evaluate the potential role of nucleotide excision repair in repeat instability, we measured the rates of repeat deletion in wild type and excision repair-deficient Escherichia coli strains (using a genetic assay for deletions). The rate of triplet repeat deletion decreased in an E. coli strain deficient in the damage recognition protein UvrA. Moreover, loops containing 23 CTG repeats were less efficiently excised from heteroduplex plasmids after their transformation into the uvrA(-) strain. As a result, an increased proportion of plasmids containing the full-length repeat were recovered after the replication of heteroduplex plasmids containing unrepaired loops. In biochemical experiments, UvrA bound to heteroduplex substrates containing repeat loops of 1, 2, or 17 CAG repeats with a K(d) of about 10-20 nm, which is an affinity about 2 orders of magnitude higher than that of UvrA bound to the control substrates containing (CTG)(n).(CAG)(n) in the linear form. These results suggest that UvrA is involved in triplet repeat instability in cells. Specifically, UvrA may bind to loops formed during replication slippage or in slipped strand DNA and initiate DNA repair events that result in repeat deletion. These results imply a more comprehensive role for UvrA, in addition to the recognition of DNA damage, in maintaining the integrity of the genome.     

Localized production of human E-cadherin-derived first repeat in Escherichia coli

E-cadherin is a cell surface adhesion molecule that is expressed in both epithelial and endothelial tissues. In this study, an improved method for the simple production of the human E-cadherin-derived first repeat E-CAD1 was developed by exporting it into the periplasmic space of Escherichia coli. Localization of the recombinant protein into the periplasm allowed the isolation of E-CAD1 without cell lysis. The N-terminus of E-CAD1 is fused to a streptavidin-derived peptide to allow single-step purification using a Streptag affinity column. Optimal expression in LB medium produced 3.2 mg/L while expression in minimal medium containing 15NH(4)Cl as the sole source of nitrogen produced 4.2 mg/L purified (15)N-labeled E-CAD1. Heteronuclear NMR spectroscopy confirmed that the purified E-CAD1 produced in this manner was correctly folded. The expression and purification protocol for unlabeled and isotopically labeled E-CAD1 permits rapid preparative production of this protein for mechanistic and structural studies.     

Nitrite reductase of Escherichia coli specific for reduced nicotinamide adenine dinucleotide

Kemp, John D. (University of California, Los Angeles), and Daniel E. Atkinson. Nitrite reductase of Escherichia coli specific for reduced nicotinamide adenine dinucleotide. J. Bacteriol. 92:628-634. 1966.-A nitrite reductase specific for reduced nicotinamide adenine dinucleotide (NADH(2)) appears to be responsible for in vivo nitrite reduction by Escherichia coli strain Bn. In extracts, the reduction product is ammonium, and the ratio of NADH(2) oxidized to nitrite reduced or to ammonium produced is 3. The Michaelis constant for nitrite is 10 mum. The enzyme is induced by nitrite, and the ability of intact cells to reduce nitrite parallels the level of NADH(2)-specific nitrite reductase activity demonstrable in cell-free preparations. Crude extracts of strain Bn will also reduce hydroxylamine, but not nitrate or sulfite, at the expense of NADH(2). Kinetic observations indicate that hydroxylamine and nitrite may both be reduced at the same active site. The high apparent Michaelis constant for hydroxylamine (1.5 mm), however, seems to exclude hydroxylamine as an intermediate in nitrite reduction. In vitro activity is enhanced by preincubation with nitrite, and decreased by preincubation with NADH(2).     

Mechanisms of acid resistance in enterohemorrhagic Escherichia coli.

Enterohemorrhagic strains of Escherichia coli must pass through the acidic gastric barrier to cause gastrointestinal disease. Taking into account the apparent low infectious dose of enterohemorrhagic E. coli, 11 O157:H7 strains and 4 commensal strains of E. coli were tested for their abilities to survive extreme acid exposures (pH 3). Three previously characterized acid resistance systems were tested. These included an acid-induced oxidative system, an acid-induced arginine-dependent system, and a glutamate-dependent system. When challenged at pH 2.0, the arginine-dependent system provided more protection in the EHEC strains than in commensal strains. However, the glutamate-dependent system provided better protection than the arginine system and appeared equally effective in all strains. Because E. coli must also endure acid stress imposed by the presence of weak acids in intestinal contents at a pH less acidic than that of the stomach, the ability of specific acid resistance systems to protect against weak acids was examined. The arginine- and glutamate-dependent systems were both effective in protecting E. coli against the bactericidal effects of a variety of weak acids. The acids tested include benzoic acid (20 mM; pH 4.0) and a volatile fatty acid cocktail composed of acetic, propionic, and butyric acids at levels approximating those present in the intestine. The oxidative system was much less effective. Several genetic aspects of E. coli acid resistance were also characterized. The alternate sigma factor RpoS was shown to be required for oxidative acid resistance but was only partially involved with the arginine- and glutamate-dependent acid resistance systems. The arginine decarboxylase system (including adi and its regulators cysB and adiY) was responsible for arginine-dependent acid resistance. The results suggest that several acid resistance systems potentially contribute to the survival of pathogenic E. coli in the different acid stress environments of the stomach (pH 1 to 3) and the intestine (pH 4.5 to 7 with high concentrations of volatile fatty acids). Of particular importance to the food industry was the finding that once induced, the acid resistance systems will remain active for prolonged periods of cold storage at 4 degrees C.     

Transport of nicotinamide adenine dinucleotide in an unc mutant of Escherichia coli.

The presence and some properties of an NAD+ transport system were examined in PA5, a Mg, Ca-ATPase [EC 3.6.1.3]-defective mutant strain of Escherichia coli W2252. NAD+ uptake was stimulated by exogenous energy sources and dependent on external substrate concentrations with an apparent Km of about 25 micrometer. Most of the radioactivity from [14C]-NAD+ accumulated in the cells was identified as NAD+. [14C]NAD+ uptake was competively inhibited by unlabeled NAD+, NADP+, NMN+ or nicotinamide. Similar uptake activity was also observed in W2252.     

Mechanisms of the radioprotective effect of cysteamine in Escherichia coli

The values of the oxygen effect (m) and the maximal protective effect of cysteamine (DMF*) were estimated for four Escherichia coli strains: AB1157 (wild type), AB1886 (uvrA), AB2463 (recA), and p3478 (polA). A correlation made between DMF* and m as well as the kinetics of the increase of DMF with oxygen depletion showed that the protective effect of cysteamine is realized by three mechanisms: (i) anoxia achieved by oxygen reduction, with the DMF varying from 2.2 to 4.2 for different E. coli strains (this protection is the major contribution to the entire mechanism); (ii) lowering of the indirect radiation effect; i.e., for 50 mM cysteamine DMF does not exceed 1.1; and (iii) increase of the efficiency of enzymatic repair. The latter effect of cysteamine is registered only with the wild-type E. coli, the DMF being not less than 1.4.     

Mechanisms of action of ostrich beta-defensins against Escherichia coli

To understand their mechanism of antimicrobial activity against Gram-negative bacteria, ostrich beta-defensins, ostricacins-1 and 2 (Osp-1 and Osp-2), were compared with those of sheep myeloid antimicrobial peptide (SMAP)-29 and human neutrophil peptide (HNP)-1, well-characterized sheep alpha-helical and human alpha-defensin peptides, respectively. Fluorescence-based biochemical assays demonstrated that the ostricacins bound lipopolysaccharides and disrupted both outer and cytoplasmic membrane integrity. The ostricacins' permeabilizing ability was weaker than that of SMAP-29, but stronger than HNP-1. As ostricacins have previously shown the ability to inhibit bacterial growth, these peptides were suggested to be bacteriostatic to Gram-negative bacteria, which are caused by the interaction between the peptides and cytoplasmic targets causing the inhibition of DNA, RNA, and protein synthesis as well as enzymatic activities. These findings indicated promising possibilities for the peptides to be used in the development of therapeutic and topical products.