Inhibition of mutation and combating the evolution of antibiotic resistance

The emergence of drug-resistant bacteria poses a serious threat to human health. In the case of several antibiotics, including those of the quinolone and rifamycin classes, bacteria rapidly acquire resistance through mutation of chromosomal genes during therapy. In this work, we show that preventing induction of the SOS response by interfering with the activity of the protease LexA renders pathogenic Escherichia coli unable to evolve resistance in vivo to ciprofloxacin or rifampicin, important quinolone and rifamycin antibiotics. We show in vitro that LexA cleavage is induced during RecBC-mediated repair of ciprofloxacin-mediated DNA damage and that this results in the derepression of the SOS-regulated polymerases Pol II, Pol IV and Pol V, which collaborate to induce resistance-conferring mutations. Our findings indicate that the inhibition of mutation could serve as a novel therapeutic strategy to combat the evolution of antibiotic resistance.     

Coordinated functions of WSS1, PSY2 and TOF1 in the DNA damage response

The stabilization and processing of stalled replication forks is required to maintain genome integrity in all organisms. In an effort to identify novel proteins that might be involved in stabilizing stalled replication forks, Saccharomyces cerevisiae mutant wss1Δ was isolated from a high-throughput screening of ~5000 deletion strains for genes involved in the response to continuous, low-intensity UV irradiation. Disruption of WSS1 resulted in synergistic increases in UV sensitivity with null mutants of genes involved in recombination (RAD52) and cell cycle control (RAD9 and RAD24). WSS1 was also found to interact genetically with SGS1, TOP3, SRS2 and CTF4, which are involved in recombination, repair of replication forks and the establishment of sister chromatid cohesion. A yeast two-hybrid screen identified a potential physical interaction between Wss1 and both Psy2 and Tof1. Genetic interactions were also detected between PSY2 and TOF1, as well as between each gene and RAD52 and SRS2, and between WSS1 and TOF1. Tof1 is known to be involved in stabilizing stalled replication forks and our data suggest that Wss1 and Psy2 similarly function to stabilize or process stalled or collapsed replication forks.     

The evolution of DNA polymerases with novel activities

DNA and RNA polymerases have evolved in nature to function in specific environments with specific substrates. Thus, although the commercial availability of these enzymes has revolutionized the biotechnology industry, their applications are limited. The availability of polymerases that have unnatural properties would be of even greater utility. Towards this goal, several activity-based screening and selection approaches have been developed. Using these techniques, polymerases that synthesize a variety of different polymers, including those containing 2'-O-methyl-modified nucleotides or unnatural base pairs, have been evolved. These results suggest that polymerases tailored for any specific application could soon be available.     

Myosin light-chain expression during avian muscle development

Monoclonal antibodies to adult chicken myosin light chains were generated and used to quantitate the types of myosin light-chain (MLC) isoforms expressed during development of the pectoralis major (PM), anterior latissimus dorsi (ALD), and medial adductor (MA) muscles of the chicken. These are muscles which, in the adult, are composed predominantly of fast, slow, and a mixture of fiber types, respectively. Three distinct phases of MLC expression characterized the development of the PM and MA muscles. The first identifiable pase occurred during the period of 5-7 d of incubation in ovo. Extracts of muscles from the pectoral region (which included the presumptive PM muscle) contained only fast MLC isoforms. This period of exclusive fast light-chain synthesis was followed by a phase (8- 12 d of incubation in ovo) in which coexpression of both fast and slow MLC isoforms was apparent in both PM and MA muscles. During the period, the composition of both fast and slow MLC isoforms in the PM and MA muscles was identical. Beginning at day 12 in ovo, the ALD was also subjected to immunochemical analyses. The proportion of fast and slow MLCs in this muscle at day 12 was similar to that present in the other muscles studied. The third development phase of MLC expression began at approximately 12 d of incubation in ovo and encompassed the transition in MLC composition to the isoform patterns incubation in ovo and encompassed the transition in MLC composition to the isoform patterns typical of adult muscle. During this period, the relative proportion of slow MLC rose in both the MA and ALD and fell in the PM. By day 16, the third fast light chain, LC(3f), was apparent in extracts of both the PM and MA. These results show that there is a developmental progression in the expression of MLC in the two avian muscles studied from day 5 in ovo; first, only fast MLCs are accumulated, then both fast and slow MLC isoforms are expressed. Only during the latter third of development in ovo is the final MLC isoform pattern characteristic of a particular muscle type expressed.     

SOS regulatory elements are essential for UPEC pathogenesis

Epithelial cells are highly regarded as the first line of defense against microorganisms, but the mechanisms used to control bacterial diseases are poorly understood. A component of the DNA damage repair regulon, SulA, is essential for UPEC virulence in a mouse model for human urinary tract infection, suggesting that DNA damage is a key mediator in the primary control of pathogens within the epithelium. In this study, we examine the role of DNA damage repair regulators in the intracellular lifestyle of UPEC within superficial bladder epithelial cells. LexA and RecA coordinate various operons for repair of DNA damage due to exogenous and endogenous agents and are known regulators of sulA. UPEC strains defective in regulation of the SOS response mediated by RecA and LexA display attenuated virulence in immunocompetent mice within the first 6 h post infection. RecA and LexA regulation of the SOS regulon is dispensable in immunocompromised mice. These data suggest that epithelial cells produce sufficient levels of DNA damaging agents, such that the bacterial DNA damage repair response is essential, as a means to control invading bacteria. Since many pathogens interact with the epithelium before exposure to professional phagocytes, it is likely that adaptation to oxidative radicals during intracellular growth provides additional protection from killing by innate immune phagocytes.     

Esc4/Rtt107 and the control of recombination during replication

When replication forks stall during DNA synthesis, cells respond by assembling multi-protein complexes to control the various pathways that stabilize the replication machinery, repair the replication fork, and facilitate the reinitiation of processive DNA synthesis. Increasing evidence suggests that cells have evolved scaffolding proteins to orchestrate and control the assembly of these repair complexes, typified in mammalian cells by several BRCT-motif containing proteins, such as Brca1, Xrcc1, and 53BP1. In Saccharomyces cerevisiae, Esc4 contains six such BRCT domains and is required for the most efficient response to a variety of agents that damage DNA. We show that Esc4 interacts with several proteins involved in the repair and processing of stalled or collapsed replication forks, including the recombination protein Rad55. However, the function of Esc4 does not appear to be restricted to a Rad55-dependent process, as we observed an increase in sensitivity to the DNA alkylating agent methane methylsulfonate (MMS) in a esc4Deltarad55Delta mutant, as well as in double mutants of esc4Delta and other recombination genes, compared to the corresponding single mutants. In addition, we show that Esc4 forms multiple nuclear foci in response to treatment with MMS. Similar behavior is also observed in the absence of damage when either of the S-phase checkpoint proteins, Tof1 or Mrc1, is deleted. Thus, we propose that Esc4 associates with ssDNA of stalled forks and acts as a scaffolding protein to recruit and/or modulate the function of other proteins required to reinitiate DNA synthesis.     

Beyond A, C, G and T: augmenting nature's alphabet

Efforts to expand the genetic alphabet are predicated upon a stable and replicable third base pair. Recent progress has resulted in the development of several candidates that are both stable in duplex DNA and replicated by DNA polymerases with various degrees of efficiency and fidelity. The candidate base pairs draw upon unnatural hydrogen-bonding topologies as well as upon shape complementarity and hydrophobic forces. This review provides a critical comparison of the third base pair candidates and discusses the further work required to expand the genetic alphabet.