■ PROTECTING AGAINST OXIDATIVE DAMAGE IN AMDAge-related macular degeneration (AMD) is the leading cause of blindness in the elderly, affecting tens of millions worldwide.The hallmark of “wet” AMD, the most aggressive form, is choroidal neovascularization (CNV), which leads to bleeding andexudation in the retina. Among a number of well-characterized heritable risk factors for AMD is a single nucleotide polymorphismwhich codes for histidine as opposed to the more common tyrosine at position 420 in complement factor H (CFH). CFHrecognizes pathogen-associated and damage-associated molecular patterns that are generated during infection or tissue damage,respectively. CFH plays a regulatory role in the inflammatory response by suppressing activation of the pro-inflammatory plasmacomplement protein system. Since exposure to sunlight and high retinal O2 content are associated with oxidative stress, a likelycontributor to the pathogenesis of AMD, Shaw et al. [(2012) Proc. Natl. Acad. Sci. U.S.A., 109, 13757] hypothesized that CFH helpsto prevent AMD by suppressing the inflammatory response to oxidative stress in the retina.To test their hypothesis, Shaw et al. focused on oxidative damage to phospholipids. They showed that CFH binds to oxidized

low-density lipoproteins (oxLDL) much more strongly than to native LDL, and that CFH420Y binds to oxLDL twice as stronglyas CFH420H. They measured the levels of oxidized phospholipids (oxPL) in the plasma of people homozygous for both CFHalleles, finding higher levels in those bearing CFH420H.Retinal pigment epithelial cells (RPE) and macrophages exposed to oxLDL responded by increased expression of multiple pro-

inflammatory cytokines and receptors. Binding of oxLDL to these cells was inhibited more strongly by preincubation with plasmafrom individuals homozygous for CFH420Y than for CFH420H.Injection of oxLDL into the subretinal space of mice led to exudation and leakage characteristic of CNV as detected by fundus

fluorescence angiography and histologic examination. Both oxPL and CFH were present in the extracellular plaques (drusen)found in the retinas of AMD patients. Together, the results indicate that CFH binds oxPL, thereby preventing its binding to RPEand macrophages and inducing an inflammatory response. CFH may play a role in protecting against other toxic effects mediatedby oxPL under conditions of oxidative stress. Carol A. Rouzer

■ BISPHENOL−RECEPTOR INTERACTIONS

Reprinted with permission from Delfosse et al. (2012) Proc. Natl.Acad. Sci. U.S.A., 109, 14930. Copyright 2012 Delfosse et al.

As a result of their wide use in manufacturing, bisphenols (BPs)are present in many consumer products and can be releasedfrom these materials. The most prevalent BP, BPA, is present infood, water, wastewater, air, and dust, and has also beendetected in human body fluids and tissues. Many BPs bind toand modulate the activity of a range of hormone receptors,classifying them as endocrine disrupters, and raising concernregarding their reproductive and developmental toxicity. Thisled Delfosse et al. [(2012) Proc. Natl. Acad. Sci. U.S.A, 109,14930] to more closely examine the interaction of three BPs,BPA, BPAF, and BPC, with the estrogen receptors, ERα andERβ, which are the prime targets of BPA.

Studies using ERα- and ERβ-expressing reporter cells anddirect ER receptor binding assays indicated binding affinities ofthe three BPs that decreased in the order BPC > BPAF > BPA.Using induction of luciferase expression to measure ER activation,all of the BPs acted as partial agonists at both ERs, with BPAdemonstrating the highest and BPC the lowest efficacy.The ligand binding domain (LBD) of the ER comprises 12

α-helices that form a hydrophobic binding pocket. Uponbinding of an agonist, helix H12 covers the pocket, exposing abinding site for coactivator proteins. Delfosse et al. used afluorescently labeled fragment of the coactivator SRC-1 thatbinds to activated ERs to show that E2 and, to a lesser extent,BPA enhanced, while the ER antagonist 4-hydroxytamoxifen(OHT) and BPC inhibited the binding of SRC-1 to ERα.Fluorescent anisotropy measurements of a fluorescein moietyattached to helix H12 of the ERα LBD revealed a graded effectof BPs on the stability of the active site conformation thataccounts for their differential impact on SRC-1 recruitment.Crystal structures of BPA, BPAF, and BPC in complex with theERα LBD and the SRC-1 fragment revealed that BPA binds inthe canonical active conformation, with H12 capping the ligandbinding pocket, and the SRC-1 fragment bound as expected. Incontrast, BPC bound in an antagonist conformation similar tothat previously observed for OHT. BPAF adopted bothconformations, with one in each subunit of the homodimericcomplex (see the Figure).Delfosse et al. used these results to construct a model of BPA

binding to nuclear receptors that correctly predicted relativepotencies and efficacies of seven BPs at four receptors. Thiswork provides new insights into the mechanism of action ofBPs at nuclear receptors and lays the foundation for designing

Published: November 19, 2012

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new BPs that have reduced endocrine disrupting activity.Carol A. Rouzer

■ DEFINING THE FATE OF 8-OXOG

Reprinted from Fleming et al. (2012) J. Am. Chem. Soc., 134,15091. Copyright 2012 American Chemical Society.

Under conditions of oxidative stress, DNA is a prime target ofreactive free radicals, with G being the primary site of oxidant-induced damage. Oxidation of G can generate a number of dif-ferent species, but in genomic DNA under physiological condi-tions, the primary product is 8-oxo-7,8-dihydroguanine (OG).OG is even more reactive than G, and one-electron oxidationproduces spiroiminodihydantoin (Sp) and/or 5-guanidinohy-dantoin (Gh) (see figure). Both Gh and Sp are highly mutagenic,leading to G > C and G > T transversions.The relative amounts of Sp and Gh formed from oxidation of

OG are highly dependent on substrate structure and reactionconditions. This led Fleming et al. [(2012) J. Am. Chem. Soc.,134, 15091] to explore the determinants of product forma-tion using OG nucleoside, OG nucleotide (OGTP), an OG-containing single-stranded oligonucleotide (ssOG) and OG-containing double-stranded oligonucleotides in which OGwas placed opposite C (OG·C), A (OG·A), or an abasic siteanalogue (OG·F). Reaction of each of these substrates withNa2IrCl6 or ONOOCO2-derived CO3

·−/·NO2 generated Spand Gh, with the relative Sp/Gh ratio decreasing from ∼100%to ∼0% Sp in the order of OG nucleoside, OGTP, ssOG, OG·F,OG·A, and OG·C. Competition studies revealed decreasingreactivity in the same order.Sp and Gh share a common intermediate 5-OH-OG. When

5-OH-OG is protonated at N1 (pKa ∼ 5.8), the reaction favorsthe formation of Gh, while deprotonated 5-OH-OG forms Sp.Studies of the effects of pH on the Sp/Gh ratio in the varioussubstrates indicated that the effective pKa in each substratecontext increased in the order OG nucleoside < OGTP < ssODN∼ OG·F < OG·A < OG·C. Changes in the bases neighboring OGin the double-stranded oligonucleotides from two purines to twopyrimidines had little effect on the Sp to Gh product ratios.Gh formation was more favored in ssOG than in the OG

nucleoside and more favored in OG·F than ssOG, suggestingthat base stacking may play a role in the reaction pathway.Consistently, cooling the reaction mixture to 4 °C to stabilizebase stacking in ssOG further increased Gh formation. Thehigher Gh formation in OGTP versus OG nucleoside suggestedthat electrostatic effects from the sugar−phosphate backbonealso play a role.The increased Gh formation in OG·C versus OG·A could

result from differences in hydrogen bonds (OG·C forms 3 andOG·A forms 2), base conformation of OG (OG·C is anti andOG·A is syn), and/or helix stability (OG·C is more stable).Lowering the temperature to increase helix stability did notmarkedly increase Gh formation in OG·A reaction mixtures.Replacing C and A with analogues that form 2 and 3 hydrogen

bonds, respectively, with OG also had relatively little effect onthe Sp/Gh product ratios. Fleming et al. conclude that acombination of base stacking, electrostatics, and base pairinginfluences the Gh to Sp ratio. The high reactivity of free OGsuggests that the nucleoside pool could serve to protect DNAfrom oxidative damage. In addition, the predominant formationof Gh in genomic DNA indicates the need to learn more aboutthe biological implications of this lesion. Carol A. Rouzer

■ QUANTIFYING TRANSCRIPTIONAL MUTAGENESISThat damaged DNA bases can lead to replication block andmutagenesis is now a familiar story. However, the effects ofsuch damage on transcription are less well known. To betterassess these effects, You et al. [(2012) Nat. Chem. Biol., 8, 817]designed a competitive transcription and adduct bypass (CTAB)assay. They used a nonreplicating double-stranded plasmid con-taining a single lesion and a control plasmid containing thecorresponding undamaged base. Competitor plasmids encodedthree more nucleotides than the test plasmids so that transcriptsfrom competitor and test plasmids could be easily distinguishedby electrophoresis or MS.You et al. mixed test and competitor plasmids at given molar

ratios and used them as templates for in vitro or in vivotranscription. The assay yielded relative bypass efficiency(RBE), calculated by dividing the ratio of lesion transcripts tocompetitor transcripts by the ratio of control transcripts tocompetitor transcripts, and the relative mutation frequency(RMF), defined as the percent of detectable mutant restrictionfragments. You et al. used their assay to study the effects of theoxidative lesions (5′S)-8,5′-cyclo-2′-deoxyadenosine or -deox-yguanosine (S-cdA or S-cdG, respectively) and the R- and S-diastereomers of the methylglyoxal adduct N2-(1-carboxyeth-yl)-2′-deoxyguanosine (N2-CEdG). Using T7 RNA polymerase(RNAP) and HeLa cell nuclear extracts containing hRNAPIIfor in vitro studies, they showed that S-cdA and S-cdG weresubstantial blocks to transcription, exhibiting RBEs of 28% and14%, respectively, with T7 RNAP and <1% with hRNAPII. MSanalysis revealed a single mutation resulting from T7 RNAP-dependent transcription, the deletion of a single baseimmediately downstream of the lesion. This error resultedwith RMFs of 8% and 12% for S-cdA and S-cdG, respectively.Both diasetereomers of N2-CEdG were transcription blocks,exhibiting RBEs of 15% and 29% for T7 RNAP and hRNAPII,respectively. The resulting transcripts all bore the wild-typesequence.Studies using intact cells proficient in nucleotide excision

repair (NER) demonstrated increasing RBEs with time forall lesions. This was not observed in cells lacking the NERprotein XPA. Cells treated with an siRNA to knock down theexpression of the global genome-NER protein XPC transcribedall of the lesion-containing plasmids the same as wild-type cells,while knock down of the transcription-coupled-NER proteinCSB resulted in a significant reduction in transcription.Together, the results confirm the value of the CTAB assay

to quantify the effects of DNA damage on transcription.Furthermore they provide valuable information on the effects ofthese specific lesions on transcription and indicate that all of thelesions are repaired by TC-NER in this model system.Carol A. Rouzer

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