The generation of genetic variation (somatic hypermutation) is an essential process for the adaptive immune system in vertebrates. We demonstrate the targeted single-nucleotide substitution of DNA using hybrid vertebrate and bacterial immune systems components. Nuclease-deficient type II CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated) and the activation-induced cytidine deaminase (AID) ortholog PmCDA1 were engineered to form a synthetic complex (Target-AID) that performs highly efficient target-specific mutagenesis. Specific point mutation was induced primarily at cytidines within the target range of five bases. The toxicity associated with the nuclease-based CRISPR/Cas9 system was greatly reduced. Although combination of nickase Cas9(D10A) and the deaminase was highly effective in yeasts, it also induced insertion and deletion (indel) in mammalian cells. Use of uracil DNA glycosylase inhibitor suppressed the indel formation and improved the efficiency.

Mitochondrial cytochrome c oxidase plays an essential role in aerobic cellular respiration, reducing dioxygen to water in a process coupled with the pumping of protons across the mitochondrial inner membrane. An aspartate residue, Asp-51, located near the enzyme surface, undergoes a redox-coupled x-ray structural change, which is suggestive of a role for this residue in redoxdriven proton pumping. However, functional or mechanistic evidence for the involvement of this residue in proton pumping has not yet been obtained. We report that the Asp-51 3 Asn mutation of the bovine enzyme abolishes its proton-pumping function without impairment of the dioxygen reduction activity. Improved x-ray structures (at 1.8͞1.9-Å resolution in the fully oxidized͞ reduced states) show that the net positive charge created upon oxidation of the low-spin heme of the enzyme drives the active proton transport from the interior of the mitochondria to Asp-51 across the enzyme via a water channel and a hydrogen-bond network, located in tandem, and that the enzyme reduction induces proton ejection from the aspartate to the mitochondrial exterior. A peptide bond in the hydrogen-bond network critically inhibits reverse proton transfer through the network. A redoxcoupled change in the capacity of the water channel, induced by the hydroxyfarnesylethyl group of the low-spin heme, suggests that the channel functions as an effective proton-collecting region. Infrared results indicate that the conformation of Asp-51 is controlled only by the oxidation state of the low-spin heme. These results indicate that the low-spin heme drives the proton-pumping process.

Bovine heart cytochrome c oxidase (CcO) pumps four proton equivalents per catalytic cycle through the H-pathway, a protonconducting pathway, which includes a hydrogen bond network and a water channel operating in tandem. Protons are transferred by H 3 O ؉ through the water channel from the N-side into the hydrogen bond network, where they are pumped to the P-side by electrostatic repulsion between protons and net positive charges created at heme a as a result of electron donation to O 2 bound to heme a 3 . To block backward proton movement, the water channel remains closed after O 2 binding until the sequential four-proton pumping process is complete. Thus, the hydrogen bond network must collect four proton equivalents before O 2 binding. However, a region with the capacity to accept four proton equivalents was not discernable in the x-ray structures of the hydrogen bond network. The present x-ray structures of oxidized/reduced bovine CcO are improved from 1.8/1.9 to 1.5/1.6 Å resolution, increasing the structural information by 1.7/1.6 times and revealing that a large water cluster, which includes a Mg 2؉ ion, is linked to the H-pathway. The cluster contains enough proton acceptor groups to retain four proton equivalents. The redox-coupled x-ray structural changes in Glu 198 , which bridges the Mg 2؉ and Cu A (the initial electron acceptor from cytochrome c) sites, suggest that the Cu A -Glu 198 -Mg 2؉ system drives redox-coupled transfer of protons pooled in the water cluster to the H-pathway. Thus, these x-ray structures indicate that the Mg 2؉ -containing water cluster is the crucial structural element providing the effective proton pumping in bovine CcO.Cytochrome c oxidase (CcO) 3 reduces molecular oxygen (O 2 ) in a reaction coupled with a proton pumping process. After binding of O 2 to the O 2 reduction site (which includes two redox-active metal sites, heme a 3 and Cu B ), four electron equivalents are sequentially donated from cytochrome c via two additional redox active metal sites, Cu A and heme a. Each of the four electron transfers is coupled to the pumping of a single proton equivalent (1, 2).High resolution x-ray structural studies together with mutational analyses for bovine CcO show a possible proton pumping pathway, known as the H-pathway, which includes a hydrogen bond network and a water channel functioning in tandem (1, 2). The water channel provides access of water molecules (or H 3 O ϩ ions) inside the mitochondrial inner membrane (the N-side) to one end of the hydrogen bond network that extends to the outside of the mitochondrial inner membrane (the P-side). The hydrogen bond network interacts tightly with heme a by forming two hydrogen bonds between the formyl group of heme a and Arg 38 in the hydrogen bond network and between the A-ring propionate of heme a and a fixed water molecule in the hydrogen bond network (1, 2). The net positive charge increase in heme a, which occurs upon electron donation from heme a to the O 2 reduction site to be delocalized to the formyl and propionate gro...

A new human breast cancer cell line, KPL-4, was recently isolated from the malignant pleural effusion of a breast cancer patient with an inflammatory skin metastasis. This cell line can be cultured under serum-free conditions and is tumorigenic in female athymic nude mice. Flow cytometric analysis revealed the expression of Erb B-1, -2 and -3. Dot blot hybridization showed a 15-fold amplification of the erbB-2. Reverse transcription-polymerase chain reaction analysis showed a detectable level of mRNA expression of all the Erb B family receptors. In addition, all the receptors were autophosphorylated under a serum-supplemented condition. Unexpectedly, transplanted KPL-4 tumours induced cachexia of recipient mice. A high concentration of interleukin-6 (IL-6) was detected in both the culture medium and the serum of mice. The weight of tumours significantly correlated with the serum IL-6 level. The antiproliferative effect of a humanized anti-Erb B-2 monoclonal antibody, rhuMAbHER2, was investigated. This antibody significantly inhibited the growth of KPL-4 cells in vitro but modestly in vivo. Loss of mouse body weight was partly reversed by rhuMAbHER2. These findings suggest that KPL-4 cells may be useful in the development of new strategies against breast cancer overexpressing the Erb B family receptors and against IL-6-induced cachexia. © 1999 Cancer Research Campaign

Effects of changes in oxidation state at the other metal centers on oxidized heme a3 cyanide of bovine heart cytochrome c oxidase have been investigated. Only one CN- binds, giving Fe3+a3CN, in fully-oxidized cytochrome c oxidase and its 1-, 2-, and 3-electron reduction products. Soret/visible spectra for the heme a3 cyanide are independent of overall redox level, whereas distinct shifts in C-N infrared stretch band frequency occur upon reduction, reflecting changes in the polarity of the ligand (CN-) environment. Catalysis of O2 reduction can be critically dependent upon such changes in polarity at the reduction site. These findings indicate that CuB, when reduced, exists in two forms whose relative stabilities are independent of Fea and CuA oxidation states and, when oxidized, is in only one stable form. These results are consistent with the oxidation of Cu+B triggering proton pumping and with the involvement of a CuB ligand in respiratory control. Electron equivalents introduced into the enzyme are distributed equally among Fea, CuA, and CuB, which raises the possibility that all four electrons used in O2 reduction are donated via Cu+B, which is favorably positioned with respect to Fea3 (the O2 binding site) in order to carry out this role in electron transfers.