DNA Repair Enzyme

Cellular DNA is constantly challenged by various endogenous and exogenous genotoxic factors that inevitably lead to DNA damage: structural and chemical modifications of primary DNA sequence. These DNA lesions are either cytotoxic, because they block DNA replication and transcription, or mutagenic due to the miscoding nature of the DNA modifications, or both, and are believed to contribute to cell lethality and mutagenesis. Studies on DNA repair in Escherichia coli spearheaded formulation of principal strategies to counteract DNA damage and mutagenesis, such as: direct lesion reversal, DNA excision repair, mismatch and recombinational repair and genotoxic stress signalling pathways. These DNA repair pathways are universal among cellular organisms. Mechanistic principles used for each repair strategies are fundamentally different. Direct lesion reversal removes DNA damage without need for excision and de novo DNA synthesis, whereas DNA excision repair that includes pathways such as base excision, nucleotide excision, alternative excision and mismatch repair, proceeds through phosphodiester bond breakage, de novo DNA synthesis and ligation. Cell signalling systems, such as adaptive and oxidative stress responses, although not DNA repair pathways per se, are nevertheless essential to counteract DNA damage and mutagenesis. The present review focuses on the nature of DNA damage, direct lesion reversal, DNA excision repair pathways and adaptive and oxidative stress responses in E. coli.

DNA exonucleases, enzymes that hydrolyze phosphodiester bonds in DNA from a free end, play important cellular roles in DNA repair, genetic recombination and mutation avoidance in all organisms. This article reviews the structure, biochemistry, and biological functions of the 17 exonucleases currently identified in the bacterium Escherichia coli. These include the exonucleases associated with DNA polymerases I (polA), II (polB), and III (dnaQ/mutD); Exonucleases I (xonA/sbcB), III (xthA), IV, VII (xseAB), IX (xni/xgdG), and X (exoX); the RecBCD, RecJ, and RecE exonucleases; SbcCD endo/exonucleases; the DNA exonuclease activities of RNase T (rnt) and Endonuclease IV (nfo); and TatD. These enzymes are diverse in terms of substrate specificity and biochemical properties and have specialized biological roles. Most of these enzymes fall into structural families with characteristic sequence motifs, and members of many of these families can be found in all domains of life.

This chapter provides an overview of what is known about the regulation and defenses against reactive oxygen species and nitrogen species. In Escherichia coli and a subset of enteric bacteria, SoxR only controls the expression of SoxS, an AraC-type transcription regulator. Structural and spectral studies of oxidized SoxR bound to DNA show that the activated protein introduces a significant distortion into the DNA in order to activate transcription, as has been observed for other MerR family members. In Salmonella enterica and E. coli, OxyR regulates numerous genes whose expression is induced by exposure to H2O2 and that make sense in terms of a defense against peroxides, including genes encoding catalase and the AhpC peroxiredoxin together with the AhpF reductase, the iron-sequestering protein Dps, and the disulfide bond reducing enzymes thioredoxin and glutaredoxin. In the denitrifying organism Ralstonia eutropha, the single subunit quinol-oxidizing respiratory free radical nitric oxide (NO) reductase is encoded by the norB gene, which is co-transcribed with norA under the control of the transcription activator encoded by the divergently transcribed norR gene. In higher eukaryotes, the major receptor for NO is the soluble guanylate cyclase (sGC). DosS (DevS) and DosT are paralogous histidine kinases from Mycobacterium tuberculosis that each contain a heme cofactor bound to an N-terminal GAF domain. In all cases, the redox-active centers contain reactive metals or cysteine or histidine residues, although the types of modifications that occur are surprisingly varied.

The chapter describes the abilities of Cryptococcus neoformans to sense and adapt to a subset of environmental conditions it encounters. The mechanisms for sensing extracellular signals such as stress, light, nitrogen, carbon dioxide, and oxygen are emerging. There are several future paths toward understanding the light responses of C. neoformans. First, the functions of the opsin and phytochrome in light sensing, if any, are currently unknown. Second, the conformational effects of light on the white collar complex are also unknown in C. neoformans or in detail from any fungal system since the proteins are notoriously difficult to purify in abundance for structural and other studies. Third, the genes that are regulated by light remain to be fully elucidated, although this is being addressed through transcript profiling microarray and genetic screens, particularly since these genes should control C. neoformans ability to proliferate in the wild and cause disease in humans. The current model of the CO2-sensing system in C. neoformans suggests that under limiting concentrations this molecule diffuses into the cell and is subsequently hydrated to HCO3 - and fixed inside the cell by the CA Can2p. Analysis of deletion mutants identified two genes in addition to SRE1 and SCP1 that were required for full growth under hypoxic conditions. It is of interest to note that mutation of SRE1 and SCP1 leads to an increased sensitivity to ROS, which led Ingavale et al. to propose that there is a link in oxygen sensing between hypoxia, mitochondrial function, and ROS generation.

Nitrate reduction to ammonia via nitrite occurs widely as an anabolic process through which bacteria, archaea, and plants can assimilate nitrate into cellular biomass. Escherichia coli and related enteric bacteria can couple the eight-electron reduction of nitrate to ammonium to growth by coupling the nitrate and nitrite reductases involved to energy-conserving respiratory electron transport systems. In global terms, the respiratory reduction of nitrate to ammonium dominates nitrate and nitrite reduction in many electron-rich environments such as anoxic marine sediments and sulfide-rich thermal vents, the human gastrointestinal tract, and the bodies of warm-blooded animals. This review reviews the regulation and enzymology of this process in E. coli and, where relevant detail is available, also in Salmonella and draws comparisons with and implications for the process in other bacteria where it is pertinent to do so. Fatty acids may be present in high levels in many of the natural environments of E. coli and Salmonella in which oxygen is limited but nitrate is available to support respiration. In E. coli, nitrate reduction in the periplasm involves the products of two seven-gene operons, napFDAGHBC, encoding the periplasmic nitrate reductase, and nrfABCDEFG, encoding the periplasmic nitrite reductase. No bacterium has yet been shown to couple a periplasmic nitrate reductase solely to the cytoplasmic nitrite reductase NirB. The cytoplasmic pathway for nitrate reduction to ammonia is restricted almost exclusively to a few groups of facultative anaerobic bacteria that encounter high concentrations of environmental nitrate.

The cells of higher eukaryotes typically contain several thousand copies of mitochondrial DNA (mtDNA). This chapter first talks about mitochondrial mutagenesis and DNA damage in the mitochondrial genome. Several factors can facilitate preferential mtDNA damage. While a number of early studies provided suggestive hints about mtDNA repair, the first definitive study demonstrating active base excision repair (BER) in mitochondria in mammalian cells documented the formation and repair of N-methylpurines in an insulinoma cell line exposed to the naturally occurring nitrosamine streptozotocin. The chapter discusses loss of specific types of base damage from mtDNA and repair of oxidative damage. Other covered topics are enzymes for BER repair in mitochondrial extracts, short-patch BER of mitochondrial DNA, age-related studies of mitochondrial DNA repair, alternative excision repair pathway, and recombinational repair in mtDNA. The determination of the number and type of distinct DNA repair pathways that operate in mtDNA in mammalian cells remains an important challenge and that results obtained with lower eukaryotes cannot be extrapolated to higher organisms.

This chapter focuses on the sources, the regulation, the spectrum of activities, and the viral and microbial targets of reactive oxygen intermediates (ROIs) and reactive nitrogen intermediates (RNIs) generated by mammalian host cells. With respect to the control of infectious agents, the two most important oxygen-dependent pathways for the generation of antiviral or antimicrobial effector molecules are the phagocyte NADPH oxidase (Phox) and the inducible nitric oxide synthase (iNOS) pathways. One of the most intriguing discoveries in the field of ROIs in recent years was the observation by Wentworth and colleagues that antibodies, independent of their source or antigen specificity, can catalyze the generation of ROIs. Members of all groups of infectious pathogens (viruses, bacteria, protozoa, helminths, and fungi) were found to be controlled by RNIs. The significant improvement of certain infectious diseases after inhibition or genetic deletion of iNOS, which was without negative effects on the pathogen clearance, was unexpected. It can be explained by the inhibition of T-cell proliferation or induction of T-cell apoptosis via iNOS-positive suppressor cells (macrophages and dendritic cells) or by the tissue-damaging properties of RNIs. Transgenic mouse models have been extremely helpful to elucidate the relative contributions of ROIs and RNIs for the control of infectious pathogens. Viruses, bacteria, parasites, and fungi have developed multiple strategies to evade killing by oxygen-dependent effector mechanisms. Current research projects aim at the development of ROI or RNI precursors that enter only certain types of host cells and are activated by the infectious pathogens themselves.

This chapter provides an overview of the current understanding of the molecular mechanism of retroviral DNA integration and points to some of the issues that are not yet well understood. The mechanism of retroviral DNA integration is closely related to the mechanism by which many transposons and retrotransposons move from one location to another in the genome of the host cell. Retroviral integrase is encoded by the 3' part of the pol gene and is assembled into virus particles as the Gag-Pol polyprotein precursor. Retroviral integrases share a common domain structure, and the biochemical activities of integrase proteins from different retroviruses are fundamentally similar. Integrase has a nonspecific nuclease activity that is most easily monitored by observing nicking of closed circular DNA. Indeed, this nonspecific nuclease activity was the first biochemical activity detected for a retroviral integrase. This activity may reflect an inefficient 3' processing reaction acting on an aberrant DNA substrate. Complementation experiments demonstrate that HIV-1 integrase functions as a multimer. Individual proteins lacking either the N-terminal or the C-terminal domain are inactive both for 3' end processing and DNA strand transfer. Mu-mediated PCR footprinting has been used to probe the nucleoprotein organization within preintegration complexes (PICs). Several cellular proteins have been implicated to play a role in retroviral DNA integration. Further studies of retroviral DNA integration will be needed to address these and other questions for which there are only partial answers at this time.

Escherichia coli has served as the principal model for investigations into DNA repair mechanisms. The DNA repair systems identified in this paradigm have also been discovered in most other organisms studied. This chapter attempts to look at these repair systems with respect to differentiation processes and developmental biology as studied in the gram-positive bacterium Bacillus subtilis. Originally, DNA repair systems were considered integral parts of an organism's ability to survive the effects of environmental insults and metabolic processes. However, as the molecular characterization of these repair systems proceeded it became obvious that in addition to determining mutation frequency and cell survival, DNA repair systems also play important roles in viral activation, DNA replication, genetic recombination, metabolism, and cancer. In order to investigate systematically the interrelationship(s) between DNA repair systems and these other phenomena, an appropriate model system must be identified. These systems are generally linked in their expression and activity with one or more of the developmental states that have been identified for the bacterium. It is in the elucidation of this linkage that B. subtilis becomes a critical model for the understanding of how organisms respond at the molecular level to stressful situations. Similar characterizations of these repair systems in other gram-positive bacteria, especially among the extremely resistant bacteria, will determine whether or not B. subtilis represents a paradigm for gram-positive bacteria or for bacteria that have distinct developmental cycles.