Homologous recombination (HR) plays a critical role in facilitating replication fork progression when the polymerase complex encounters a blocking DNA lesion, and it also serves as the primary mechanism for error-free repair of DNA double strand breaks. Rad51 is the central catalyst of HR in all eukaryotes, and to this point studies of human Rad51 have focused exclusively on events occurring within the nucleus. However, substantial amounts of HR proteins exist in the cytoplasm, yet the function of these protein pools has not been addressed. Here, we provide the first demonstration that Rad51 and the related HR proteins Rad51C and Xrcc3 exist in human mitochondria. We show stress-induced increases in both the mitochondrial levels of each protein and, importantly, the physical interaction between Rad51 and mitochondrial DNA (mtDNA). Depletion of Rad51, Rad51C, or Xrcc3 results in a dramatic decrease in mtDNA copy number as well as the complete suppression of a characteristic oxidative stress-induced copy number increase. Our results identify human mtDNA as a novel Rad51 substrate and reveal an important role for HR proteins in the maintenance of the human mitochondrial genome.

Exposure of cells to DNA-damaging agents results in a rapid increase in the formation of subnuclear complexes containing Rad51. To date, it has not been determined to what extent DNA damage-induced cytoplasmic to nuclear transport of Rad51 may contribute to this process. We have analyzed subcellular fractions of HeLa and HCT116 cells and found a significant increase in nuclear Rad51 levels following exposure to a modest dose of ionizing radiation (2 grays). We also observed a DNA damage-induced increase in nuclear Rad51 in the Brca2-defective cell line Capan-1. To address a possible Brca2-independent mechanism for Rad51 nuclear transport, we analyzed subcellular fractions for two other Rad51-interacting proteins, Rad51C and Xrcc3. Rad51C has a functional nuclear localization signal, and although we found that the subcellular distribution of Xrcc3 was not significantly affected by DNA damage, there was a damage-induced increase in nuclear Rad51C. Furthermore, RNA interference-mediated depletion of Rad51C in HeLa and Capan-1 cells resulted in lower steady-state levels of nuclear Rad51 as well as a diminished DNA damage-induced increase. Our results provide important insight into the cellular regulation of Rad51 nuclear entry and a role for Rad51C in this process.

The information required for the development and growth of an organism is encoded in its deoxyribonucleic acid (DNA), the genetic material. Mutations, or changes to the information content of a cell’s DNA, can result in developmental defects or genetic diseases that may occur at any point in one’s life. Cancers are genetic diseases resulting from mutations that disrupt the ability of cells and tissues to control their own growth and proliferation. Given the critical importance of maintaining the integrity of the information encoded by DNA, evolution has resulted in various means by which a cell can either avoid mutations resulting from DNA damage, or repair damage once it has occurred. Despite the fact that all cells suffer continuous DNA damage caused either by normal metabolic processes, e.g., DNA replication and cell division, or by exposure to various environmental carcinogens, e.g. sunlight, tobacco and car exhaust, cells have a remarkable ability to both minimize the rate of mutation and to repair DNA damage. Human cells have five distinct enzyme systems that repair DNA damage, and the importance of each of these “DNA repair pathways” is highlighted by the fact that defects in any of the five result in higher incidences of specific types of cancers. In this Chapter we describe how each of these pathways operates to protect cells from acquiring mutations. Additionally, we briefly discuss recent ideas supporting an important role for stem cells in limiting the cancer potential of cells and tissues that have accumulated DNA damage. Gildemeister, O.S., Sage, J.M. and Knight, K.L. (2009) Mutations and Cell Defenses, in Pardee, A.D. and Stein, G.S. (eds.), The Biology and Treatment of Cancer: Understanding Cancer, Hoboken, New Jersey: John Wiley & Sons, Inc., 123-142. ISBN 0470009586, 9780470009581.

Patients with any of the acute porphyrias may suffer from acute attacks. If these patients are treated with certain drugs, such as barbiturates, the likelihood of developing an attack is increased. Patients treated with antidepressants or benzodiazepine-type anxiolytics also could be placed at increased risk of developing porphyric attacks because little is known about the potential for some of these drugs to induce attacks. Primary cultures of chick embryo liver cells were used to study the effects of selected antidepressants and anxiolytics on porphyrin accumulation. Cells were treated with desferrioxamine (to partially block heme synthesis, simulating conditions encountered in porphyric patients) and increasing concentrations (3.16-1000 microM) of the evaluated drugs. Twenty hours later, porphyrin accumulation was measured. The drugs included four antidepressants and five benzodiazepine-type anxiolytics. The antidepressants bupropion and nefazodone significantly increased porphyrin accumulation when given with desferrioxamine, whereas neither fluoxetine nor paroxetine increased porphyrin accumulation. The benzodiazepine-type anxiolytic agents oxazepam, lorazepam, diazepam, triazolam, and midazolam all significantly increased porphyrin accumulation when given with desferrioxamine. Dose-response studies showed that diazepam, midazolam, and triazolam produced significant increases even at the lowest concentration tested (3.16 microM), whereas lorazepam and oxazepam required higher concentrations (>/=10 microM). These studies suggest that patients with acute porphyrias may be at greater risk for developing porphyric attacks when treated with bupropion or nefazodone compared with fluoxetine or paroxetine, and that the evaluated benzodiazepine derivatives should be administered with caution. Among the latter, low doses of lorazepam and oxazepam may be safer than those of diazepam, midazolam, and triazolam.

Heme oxygenase catalyzes the first and rate-controlling step in heme catabolism. One of the two forms of heme oxygenase (heme oxygenase-1) has been shown to be increased by heme, metals, and in some systems, by certain environmental stresses. However, it remains uncertain whether heme induces hepatic heme oxygenase-1 by a general stress response, or a specific heme-dependent cellular response. The work communicated here explores this issue by examining possible mechanisms whereby heme and other metalloporphyrins induce heme oxygenase-1 in normal liver cells. Primary cultures of chick embryo liver cells were tested for their ability to increase heme oxygenase mRNA after exposure to selected metalloporphyrins (heme, chromium mesoporphyrin, cobalt protoporphyrin and manganese protoporphyrin). The ability of antioxidants to decrease metalloporphyrin-mediated induction of heme oxygenase-1 mRNA was also tested. Our results indicate that: 1) the increase in heme oxygenase-1 mRNA mediated by heme or other metalloporphyrins may involve a short-lived protein(s) since the increase was prevented by several inhibitors of protein synthesis; and 2) in normal liver cells, heme-dependent oxidative stress does not play a key role in the heme-mediated induction of heme oxygenase-1. We conclude that heme and other non-heme metalloporphyrins induce heme oxygenase-1 through a mechanism requiring protein synthesis, not because metalloporphyrins increase cellular oxidative or other stress.

Hepatic 5-aminolevulinic acid synthase, the first and normally rate-controlling enzyme of heme biosynthesis, is regulated by heme. One of the known mechanisms whereby increased cellular heme regulates 5-aminolevulinic acid synthase is by decreasing the stability of its mRNA. In primary cultures of chick embryo liver cells, we tested whether a decrease in cellular heme might increase 5-aminolevulinic acid synthase mRNA stability and whether heme or other metalloporphyrins could reverse this stabilization. We found that: (a) The stability of 5-aminolevulinic acid synthase mRNA was markedly increased by inhibitors of heme biosynthesis, namely, 4,6-dioxoheptanoic acid or deferoxamine; (b) This increased stability of 5-aminolevulinic acid synthase mRNA was reversed by the addition of heme (10 microM) or by the combination of zinc mesoporphyrin (50 nM), an inhibitor of heme oxygenase, and heme (200 nM); (c) Repression of 5-aminolevulinic acid synthase mRNA levels by zinc mesoporphyrin (10 microM) was due to inhibition of heme oxygenase, rather than a direct, heme-like, effect of zinc mesoporphyrin on 5-aminolevulinic acid synthase mRNA; (d) Among the several non-heme metalloporphyrins tested, only zinc mesoporphyrin and chromium mesoporphyrin significantly decreased 5-aminolevulinic acid synthase mRNA without increasing heme oxygenase mRNA.