DNA Break? Call RNF8!Cellular damage that is severe enough to cause a doublestrand break in DNA requires an immediate and efficientrepair response. Cells exposed to ionizing radiation suffersuch damage and respond by building a complex ofproteins, the ionizing radiation-induced focus (IRIF), at thedamage site. IRIF formation is initiated by the ATM kinase,which phosphorylates histone H2AX, forming γ-H2AX. γ-H2AXtriggers the accumulation of additional proteins, includingMDC1, NBS1, 53BP1, and BRCA1. Now, in nearly simulta-neous reports from four research groups encompassing sixindependent laboratories, we learn of the role of a keyprotein, RNF8, in the assembly and function of the IRIF [Kolaset al. (2007) Science 318, 1637; Mailand et al. (2007) Cell131, 887; Huen et al. (2007) Cell 131, 901; and Wang andElledge (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 20759.

The four groups took different approaches to their dis-covery of RNF8. Kolas et al. screened an siRNA library insearch of proteins that are required for the incorporationof 53BP1 into the IRIF. Wang and Elledge used the fact thatthe E2 ubiquitin-conjugating enzyme, UBC13, is involved inIRIF formation, so they searched for E3 ubiquitin ligases thatinteract with UBC13. Both Huen et al. and Mailand et al.capitalized on the knowledge that FHA- and RING domain-containing proteins are frequently involved in DNA repair,so they focused on proteins bearing these characteristics.In every case, the single protein identified was RNF8.

Extensive studies on the role of RNF8 in IRIF formation ledto the following conclusions. Upon initiation of a doublestrand break, formation of γ-H2AX leads to the rapidaccumulation of MDC1, NBS1, and RNF8 to the damagesite. MDC1 is required for the binding of the other two

proteins, but NBS1 and RNF8 bind independently of oneanother. An intact FHA domain in RNF8 is required for itsinteraction with MDC1, which contains four TQXF motifs towhich the FHA domain binds tightly following phosphory-lation of the threonine residue by ATM. Once bound, RNF8works together with UBC13 to promote K63 ubiquitylationof histones. Both H2 and H2AX histones are substrates forubiquitylation, which requires an intact RNF8 RING domain.

Rap80 is an adaptor molecule bearing two UIM domainsthat mediate binding to K63 polyubiquitylated proteins.Rap80 also bears an AIR (abraxas interacting region)domain, through which it binds abraxas, which then servesas an adaptor to bind BRCA1. 53BP1 binding to the IRIFdoes not depend on Rap80, but it is dependent on RNF8,UBC13, and ubiquitylation at the damage site. Thus, fol-lowing the rapid aggregation of MDC1, NBS1 and RNF8 atthe IRIF, 53BP1, and BRCA1 subsequently appear afterhistone ubiquitylation catalyzed by RNF8. However, it is notclear if their arrival depends upon histone ubiquitinylationor some other ubiquitinylation substrate.

These conclusions were reached through the applicationof multiple methods in the six laboratories, and each

Probing for Cysteine SulfenicAcidEndogenous and exogenous reactive oxygen species(ROS) are known causes of cellular damage. Whenexcessive ROS production overwhelms cellular defensemechanisms, oxidative stress results. The study ofoxidative stress requires accurate measurement of thechemical consequences of ROS exposure, such as theone proposed by Takanishi et al. [(2007) Biochemistry46, 14275]. Yap1 is a transcription factor responsiblefor regulating oxidant-dependent transcription in Sac-charomyces cerevisiae. Cys598 of the Yap1 c-terminalcysteine-rich domain (cCRD) detects oxidant damagethrough disulfide bond formation with cysteine sulfen-ic acids, such as those generated in peroxiredoxinsupon oxidant exposure. Takanishi et al. expressed

His6-tagged cCRD in Escherichia coli and demon-strated that H2O2 exposure led to complex forma-tion between the cCRD and other cellular proteins.Dimedone, which reacts with cysteine sulfenic acid,prevented complex formation. When cell lysateswere passed over a nickel affinity column, thecomplexes bound to the column. Following elutionof nonadherent proteins, the researchers used dithio-threitol to break the disulfide bonds and then elutethe complexed proteins. This method allowed theidentification of six E. coli proteins known toexhibit cysteine sulfenic acid formation upon oxi-dation plus nine other proteins known to havereactive cysteine residues. This interesting methodshould be readily adaptable to the study of oxi-dative stress in mammalian cells as well. • CarolA. Rouzer

Reprinted from Mailand et al. (2007) Cell 131, 887, with permission from Elsevier.

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laboratory contributed additional unique insights into IRIFformation and function. For full appreciation, it is well worthchecking out all four of these articles. • Carol A. Rouzer

Do Not Cross Your DNAWhile the cancer-causing ability of many chemicals is firmlylinked to their mutagenic activity, the DNA lesions causingthe mutations are not yet fully understood. Research onchemically induced DNA damage has largely been fo-cused on binary carcinogen-DNA adducts producedwhen DNA and the chemical of interest are reacted in atest tube. However, DNA lesions in cells are not created insuch an isolated environment. One large class of DNAadducts that has been almost completely ignored bymutagenesis research includes DNA–protein and DNA-peptide cross-links.

Many human carcinogens are known to cause DNA-peptide and DNA–protein cross-links (which may be pro-teolytically processed into DNA-peptide cross-links) in cellsor in vitro. Despite this, there is a dearth of research intothe effects of these lesions. In a new study, Minko et al.[(2008) Mutat. Res. 637, 161] provide important insight intothe mutagenic properties of DNA-peptide cross-links. Theauthors modeled cross-links formed after acrolein exposureand found that mutagenic effects of these modificationsin mammalian cells were dependent on the site of attach-ment to DNA. They discovered that peptides attached atthe N6 position of deoxyadenosine in the major DNA groovewere not significantly mutagenic. In contrast, cross-links atthe N2 position of deoxyguanosine in the minor DNA groovecaused almost 10 times more mutations than binary DNAadducts. The authors’ findings have opened an avenue ofinvestigation into a previously overlooked form of DNAdamage that is expected to contribute considerably to themutagenicity of other bifunctional DNA-damaging car-cinogens. • Elizabeth Bartley and Anatoly Zhitkovich

Downfall of LumiracoxibSelective inhibitors of cyclooxygenase-2 (COX-2) wereproposed as the ideal substitutes for nonsteroidal anti-inflammatory drugs (NSAIDS) for the treatment of pain andinflammation. The selective inhibition of the COX-2 isoform,which is predominantly associated with inflammation, whileleaving the constitutively expressed COX-1 unaffected,leads to fewer gastrointestinal side effects than are ob-served with the nonselective NSAIDS. However, clinical useof COX-2 inhibitors has revealed unexpected toxicitiesleading to the removal of rofecoxib (Vioxx), eterocoxib(Bextra), and, most recently, lumiracoxib (Prexige) fromglobal markets.

Rofecoxib and eterocoxib cause cardiovascular andepidermal toxicities, respectively, effects that are believedto be mechanism-based. In contrast, lumiracoxib is hepa-totoxic, and now, Li et al. [(2007) Drug Metab. Dispos.published online Nov. 12, DOI:10.1124/dmd.107.019018]propose a metabolism-based mechanism for lumiracoxib’stoxicity, based on its similarity in structure to diclofenac.Incubation of lumiracoxib with human liver microsomes orhuman hepatocytes in the presence of N-acetylcysteine(NAC) yields two products, 3′-NAC-4′-hydroxy lumiracoxiband 4′-hydroxy-6′-NAC-desfluoro lumiracoxib. In both cases,the reactions are attributed to cytochrome P450 2C9, andboth involve the generation of a reactive quinone imineintermediate. Thus, as in the case of diclofenac, a likelymechanism for lumiracoxib hepatotoxicity is P450-catalyzedbioactivation to an electrophilic intermediate that dam-ages surrounding tissue. • Carol A. Rouzer

Searching for 5-MethylcytosineEvidence is growing for the role of DNA base methylationin the epigenetic control of chromatin structure and func-tion. Aberrant methylation patterns have been associatedwith various disease states, such as cancer and inflamma-tion, and 5-methycytosine (5MedC) residues, present at CpGsequences, are particularly prone to mutagenesis. Conse-quently, the ability to identify 5MedC residues in DNA is keyto fully understanding the role of DNA methylation in themechanism of toxic species that act in the nucleus.

Bareyt and Carell [(2008) Angew. Chem. Int. Ed. 120, 187]address this issue in work aimed at finding conditions thatwill selectively react with 5MedC but no other DNA bases.Because of reactivity at the C5-C6 double bond, the redoxpotential of 5MedC is slightly lower than that of dC or dT,suggesting that selective oxidation should be possible.Reaction of a test methylated DNA strand with V2O5 at pH3-5 showed that, indeed, 5MedC reacted whereas dT anddC did not. However, reaction with dG also occurred,attributed to direct electron abstraction, rather than elec-trophilic attack on a double bond. The latter reaction couldbe suppressed by the addition of LiBr and/or by theremoval of oxygen. However, a search for more “userfriendly” reaction conditions led to the identification ofNaIO4/LiBr at pH 5 and 40 °C as ideal for the highly efficientand selective oxidation of 5MedC residues. Coupled withhot piperidine-induced strand breaks at the modifiedbases, this reaction was used to identify the 5MedC residuesin a 40 base pair fragment from the promoter of p16, animportant gene in the regulation of basal cell carcinomaproliferation. These results form the framework for thedevelopment of a new, robust assay for the detection of5MedC in complex DNA samples. • Carol A. Rouzer

TX8000048

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Published online 02/18/2008 • DOI: 10.1021/tx8000048 $40.75 Vol. 21, No. 2, • CHEMICAL RESEARCH IN TOXICOLOGY 275© 2008 American Chemical Society