Photochemical Approach to Probing Different DNA Structures

DNA StructuresDOI: 10.1002/anie.200501962

Photochemical Approach to Probing Different DNAStructuresYan Xu and Hiroshi Sugiyama*

Keywords:DNA structures · DNA · hydrogen abstraction ·photochemistry · uracil

1. Introduction

Polymorphic DNA exists in a variety of distinct confor-mations.[1] Duplex DNA can adopt a variety of sequence-dependent secondary structures that range from the canonicalright-handed B form through to the left-handed Z form.[2,3]

Triplex and tetraplex structures of DNA are also known toexist.[3–5] Figure 1 shows the characteristic deviation of DNAstructures: the A, B, and Z forms, the protein-induced DNAkink, and the G-quartet form. These conformations arethought to play important biological roles in processes such asDNA replication, gene expression and regulation, and therepair of DNA damage.[6, 7] For example, human telomericDNA, which forms G-quartet (quadruplex) structures, servesto protect the cell from recombination and degradation.[7,8]

Disruption of telomere maintenance leads to eventual celldeath, which can be exploited for therapeutic intervention incancer treatment.[8–14] Recently, Rich and co-workers shedmore light on the biological relevance of Z-DNA in a report

that double-stranded RNA adenosinedeaminase (ADAR1), the tumor-asso-ciated protein DLM-1, and the E3Lprotein of vaccinia virus bind specifi-cally to Z-form DNA.[6] Furthermore,Liu et al. presented evidence that Z-DNA-forming sequences are required

for chromatin-dependent activation of the CSF1 (colony-stimulating factor 1) promoter.[15] Besides the structure itselfof DNA, its binding of a transcription factor such as a TATA

The various conformations of DNA—the A, B, and Z forms, theprotein-induced DNA kink, and the G-quartet form—are thought toplay important biological roles in processes such as DNA replication,gene expression and regulation, and the repair of DNA damage. Theinvestigation of local DNA conformational changes associated withbiological events is therefore essential for understanding the functionof DNA. In this Minireview, we discuss the use of photochemicaldehalogenation of 5-halouracil-containing DNA to probe the structureof DNA. Hydrogen abstraction by the resultant uracil-5-yl radicals isatom-specific and highly dependent on the structure of the DNA,suggesting that this photochemical approach could be applied as aprobe of DNA conformations in living cells.

Figure 1. Structure-dependent atom-specific hydrogen abstraction fromvarious important DNA local structures by the uracilyl radical gener-ated from 5-halouracil upon irradiation with UV light.

[*] Dr. Y. Xu, Prof. H. SugiyamaDepartment of Chemistry, Graduate School of ScienceKyoto University, Kitashirakawa-OiwakechoSakyo, Kyoto, 606-8502 (Japan)andSORST (Japan) Science and Technology CorporationFax: (+81)757-533-670E-mail: [email protected]

H. Sugiyama and Y. XuMinireviews

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box-binding protein induces significant bending of the DNAwhich is believed to be essential for the initiation of tran-scription.[16–22] For these reasons, investigation of local DNAconformational changes associated with biological events isessential for understanding the function of DNA.

Methods such as X-ray crystal structure analysis[23] andNMR spectroscopy[24,25] provide structural information withatomic resolution about DNAwith defined sequences. Otherspectroscopic methods such as circular dichroism and Ramanspectroscopy can also be used to study DNA structures. Thesemethods deal with model systems of DNA, such as syntheticoligonucleotides or homopolymers; however, they report theaverage conformation of the entire sample and cannot beused to pinpoint local conformational differences. Moreover,these approaches cannot be applied directly to probe localDNA structures inside living cells. Although various chemicalprobes and antibodies have been developed, these methodsusually require the isolation of DNA from the nucleus.

Light can be made to permeate into a cell by selecting asuitable wavelength and by focusing on a certain locus withinthe cell. Because conformational changes in DNA are likelyto occur in a short period within the living cell, a method thatuses the DNA photoreaction and is capable of fixing the DNAconformation in vivo would be very useful. The products ofthe photochemical reaction should reflect the DNA localstructure and provide information at sequence resolution onthe conformational change of the DNA during the irradiation.In this Minireview, we describe the photoreactivities of 5-halouracil in the five characteristic local DNA structures: theA, B, and Z forms, the protein-induced DNA kinks, and theG-quartet form (Figure 1). Hydrogen abstraction from DNAby the uracil-5-yl radical generated upon UV irradiation ofthe 5-halouracil residue is atom-specific and highly dependenton the structure of the DNA. This photochemical method,which reflects DNA structure, could be applied as a uniqueprobe of DNA conformations in living cells.

2. Competitive 1’- and 2’a-Hydrogen Abstraction bythe Uracilyl Radical in B-Form DNA

Thymine (T) in DNA can be replaced with 5-halouracil(BrU or IU), and the resultant 5-halouracil-substituted DNAremains functional in vivo. Replacement of thymine in DNAby 5-halouracil enhances the photosensitivity of the cell with

respect to DNA–protein cross-linking, breakage of DNAstrands, and the creation of alkali-labile sites through theformation of uracilyl radicals upon irradiation.[26–29] There-fore, we reasoned that the photoreactivity of 5-halouracilcould be used to probe the local conformation of DNA. Westarted our project to elucidate the structure of DNA lesionsby careful analysis of the products of photoirradiated 5-halouracil-containing hexanucleotides. Photoirradiation ofthe self-complementary B-form duplex d(GCABrUGC)2 andd(GCAIUGC)2 resulted in the release of adenine togetherwith the formation of deoxyribonolactone 1 as a C1’ oxidationproduct and erythrose-containing hexameric product 2 as aC2’ oxidation product (Scheme 1).[27–29] By using a stereospe-cifically C2’-deuterated oligonucleotide of the deoxyribosemoiety, we demonstrated that the formation of the C2’ oxi-dation product 2 occurs by a rate-limiting abstraction of the2’a-hydrogen atom of the deoxyribose moiety by the uracilylradical.[29] The results clearly indicate that the uracilyl radicalcompetitively abstracts the 1’- and 2’a-hydrogen atoms of thedeoxyribose residue of adenine on the 5’ side.

In contrast to the behavior of d(GCABrUGC)2 uponphotoirradiation whereby C1’ and C2’ oxidation productswere efficiently formed, hexanucleotides that lack the ABrUsequence, such as 5’-d(GCGBrUCG)-3’/5’-d(CGACGC)-3’,showed very poor photoreactivity. On the basis of theseresults, we initially proposed the mechanism that sequence-specific electron transfer occurs from the adjacent adeninemoiety on the 5’ side to BrU, to form the BrU anion radical inthe duplex structure. Formation of a uracilyl anion radicaleliminates the Br anion to generate a uracilyl radical thatabstracts the C1’-hydrogen atom of adenosine, and subse-quent oxidation of the C1’ radical by the cation radical ofadenine produces ribonolactone through a C1’ cation. Thistype of selectivity was confirmed by others.[30] However, asguanine is a better electron-donating base the reactivity wascalled a “contrathermodynamic reaction”.[30] Recently, BrUwas used as a probe of excess electron transfer.[31] Weinvestigated the electron transfer along DNA by usinguniformly 5-halouracil-substituted DNA.[32] By PCR, weprepared 450-base-pair DNA fragments in which all thymineresidues were substituted with BrU or IU. The DNA fragmentswere irradiated with monochromatic UV light (l= 302 nm)and then analyzed on sequencing gels. Surprisingly, specificcleavage at 5’-(G/C)AAXUXU-3’ and 5’-(G/C)AXUXU-3’sequences in both BrU- and IU-containing DNA fragments

Hiroshi Sugiyama, born in 1956, receivedhis PhD in 1984 with Teruo Matuura atKyoto University. After postdoctoral studiesat the University of Virginia with Sidney M.Hecht, he returned to Kyoto University in1986 as assistant professor and becameassociate professor in 1993. In 1996, hejoined the Institute of Biomaterials andBioengineering at Tokyo Medical and DentalUniversity. Since 2003 he is a professor ofchemical biology at Kyoto University. Hishonors include the Nippon IBM award andthe Chemical Society of Japan Award forCreative Work.

Yan Xu studied chemistry at Liaoning Uni-versity of Petroleum and Chemical Technol-ogy (China). In 2004, he completed hisPhD under the guidance of Prof. HiroshiSugiyama at Tokyo Medical and DentalUniversity, where he studied non-naturalnucleic acids and small molecules to stabi-lize Z-form DNA and photochemical meth-ods of probing G-quartet structures.Currently, he is a Japan Science andTechnology Fellow in the group of Prof.Sugiyama at Kyoto University, studying thestructure and function of DNA by a chem-ical approach.

DNA StructuresAngewandte

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was observed only after heat treatment. Analysis of theoligonucleotide products by HPLC indicated that the majorproducts were ribonolactone-containing octamers. Whenoligonucleotides were irradiated in H2

18O, incorporation of18O atoms into ribonolactone residues from H2

18O wasobserved, indicating that H2O is the source of the carbonyloxygen center of the ribonolactone. From these observations,we proposed a possible mechanism for the efficient photo-reactions at 5’-GAAXUXU-3’ sequences (see Figure 2). Theinitial electron transfer would occur from guanine to theelectron-deficient XUXU step through stacked AA andA moieties.[33] Release of the halide ion from the XUXU anionradical generates uracilyl radicals to abstract the hydrogenatom at C1’ from the adjacent adenine moiety of the XUXUstep. One-electron oxidation of the C1’ radical of deoxyade-nine (dA) by the cation radical of guanine gives rise to aC1’ cation and regeneration of guanine. The intervening

adenine bases between guanine and the XUXU step areconsidered to act as a bridge between the electron donor andacceptor for charge separation after electron transfer fromguanine to the XUXU step, thus preventing rapid electronback transfer. Previously observed ABrU sequence specificitycan be explained by the same role of adenine.

3. Selective 1’-Hydrogen Abstraction in A-Form DNA

Analysis of the photolysates of the DNA–RNA hybrid 5’-d(CGAIUGC)-3’/5’-r(GCAUCG)-3’, which adopts the A-type structure, indicated that the selective 1’ oxidationproduct 1 was obtained as a major product with release offree adenine (Figure 3a).[34] Similar results were obtained forBrU-containing DNA–RNA hybrids and for DNA oligom-ers.[28,29,34] 1H NMR NOE experiments with 5’-d(CGAUGC)-

3’/5’-r(GCAUCG)-3’ and 5’-d(CGAUGC)-3’/5’-d-(GCATCG)-3’ suggest that there is no correlationbetween the distances and the selectivity of hydrogenabstraction. To examine the origin of the selectivity ofhydrogen abstraction, a putative transition state for theprocess in a DNA duplex and a DNA–RNA hybrid wasconstructed and the conformational energy required toachieve their transition states was evaluated.[34] Newsets of parameters for the transition state of hydrogenabstraction for the AMBER force field were preparedby ab initio molecular orbital calculations of theabstraction of hydrogen from ethanol by vinyl radicals.In the DNA–RNA hybrid, the minimized energy of theC1’ transition state was the lowest, which explains theobserved selective C1’-hydrogen abstraction. The cal-culated structure of the AU step, which contains theputative transition state of C1’-hydrogen abstraction inthe DNA–RNA hybrid, is shown in Figure 3b. Contraryto the A form, for which the C1’ radical is morestabilized than that resulting from C2’-hydrogen ab-straction, the activation energy levels of the C1’ andC2’ radicals of the B form are roughly equivalent,[29]

which would explain the competitive C1’- and C2’-

Scheme 1. Competitive hydrogen abstraction from C1’ and C2’ positions in B-form DNA. X=Br, I; G=guanine, C= cytosine, A=adenine,U=uracil.

Figure 2. Proposed mechanism for the efficient photochemical reaction at 5’-(G/C)AAXUXU-3’ sequences.

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hydrogen abstraction observed. Although a more precisedescription of hydrogen abstraction would require thedynamic aspects of the nucleic acids to be accounted for,the results provide a qualitative explanation of conformation-dependent hydrogen abstraction by the uracil-5-yl radical in aDNA duplex and DNA–RNA hybrid.

In a model system using monomers, the reaction of the 2’-deoxyuridin-1’-yl radical with molecular oxygen gave theperoxyl intermediate, which ultimately led to the formation of2’-deoxyribonolactone via the C1’ cation.[35–37] The source ofoxygen of the ribonolactone in A-form DNA has not beenelucidated, although the peroxyl intermediate is possiblyinvolved in the formation of 2’-deoxyribonolactone.

4. Stereospecific 2’a-Hydroxylation in Z-Form DNA

The Z form of DNA is one of the characteristic localstructures of DNA revealed by X-ray crystallography and hasbeen extensively investigated in relation to transcription,[38,39]

the methylation of cytosine,[40] and the extent of DNAsupercoiling.[41–43] However, the biological relevance of Z-DNA is not well understood, presumably because of its shortlifetime under torsional stress caused by unwinding of DNAduring transcription. To investigate hydrogen abstraction bythe uracilyl radical in Z-DNA, we need to overcome theexperimental difficulty in obtaining stable Z-form oligonu-cleotides under physiological salt conditions. For example, theduplex deoxyoctadecamer, d(Gm5C)4A

BrU(Gm5C)4, whichhas an ABrU sequence in the middle, retains the typicalB form even in solution in 4m NaCl. To obtain stable Z-form

DNA, various modified guanine units were synthe-sized and introduced into duplex oligonucleotidesto evaluate their capacity to stabilize Z-form DNA.It was found that incorporation of 8-methyl-2’-deoxyguanosine (m8G)[44] and 8-methylguanosine(m8rG)[45] into DNA dramatically stabilized theZ form. The development of the Z-stabilizingmonomeric unit, the so-called Z stabilizer, hasallowed us to understand the photochemical reac-tion of IU in Z-DNA. We found that the iodouracil-containing Z form, 5’-d(CGCGIUGCG)-3’/5’-d(Cm8GCACm8GCG)-3’, undergoes 2’a-hydroxyl-ation of the duplex under UV irradiation (l=302 nm), as shown in Scheme 2.[46] Stereospecific2’b-hydrogen abstraction to give 2’a-hydroxylation,as in 3, was demonstrated by using a stereospecifi-cally deuterated octanucleotide in Z-form DNA.Importantly, because the 2’a-hydroxylation site inDNA can be easily hydrolyzed by ribonuclease T1,this photochemical and enzymatic hydrolysis isuseful for detecting Z-form regions in DNA(Scheme 2).

Rich and co-workers recently reported thatseveral proteins bind specifically to Z-form DNA,including the ubiquitous enzyme double-strandedRNA adenosine deaminase (ADAR1).[47, 48] The

possible biological roles of Z-DNA have come to attract muchcurrent interest. We examined the photochemical reaction ofZ-DNA induced by the binding of Za, that is, the NH2

terminus of ADAR1 which is responsible for high-affinitybinding to Z-DNA.[49] Stereospecific 2’a-hydroxylation oc-curred efficiently at the 5’ side of IU in the Z form induced byZa. The X-ray crystal structure of the Za–d(CGCGCG)2complex suggests that the deoxyribose C2’b hydrogen atomof the guanine at the 5’ side is located very close to the uracilyl

Figure 3. a) Photochemical products of hexanucleotides in A-type DNA–RNAhybrids upon irradiation with UV light. Selective 1’-hydrogen abstraction occurredin the A-like structure. X=Br, I. b) Structure of the AU step for a minimizedoctamer, showing the putative transition state for C1’-hydrogen abstraction in theDNA–RNA hybrid, and c) a close-up view of one AU step.

Scheme 2. Irradiation of d(CGCGXUGCG)/d(Cm8GCACm8GCG) with UVlight in the presence of Za (2 equiv/DNA), indicating that specific 2’b-hydrogen abstraction gives rise to 2’a-hydroxylation, followed by enzymaticdigestion of the hydroxylated product with ribonuclease T1. X=Br, I.


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radical, whereas C1’- and C2’a-hydrogen atoms are locatedvery far from the C5 position of uracil, as depicted inFigure 4.[49] The calculated structure of the GBrU step, whichcontains the putative transition state of the C2’b-hydrogenabstraction in Z-DNA, is shown in Figure 4c. These resultssuggest that Za packs tightly in Z-DNA, with the C3’-endo-sugar puckering the guanine of the duplex, and promotesspecific C2’b-hydrogen abstraction by the uracilyl radical toexclude 1’ and 2’ oxidation.

5. Intrastrand Hydrogen Abstraction from the5-Methyl Group of Thymine in Protein-InducedDNA Kinks

Over the past decade, resolution of the crystal structuresof many DNA–protein complexes has increased our under-standing of DNA–protein interactions. In some instances, thebinding of a protein induces significant conformationalchanges in DNA which are thought to play importantbiological roles.[17–22] For example, the TATA box-bindingprotein induces DNA bending, which is essential for theinitiation of transcription. To detect the DNA kink induced bythe protein, the photoreaction of 5-halouracil-containingDNA in the presence of Sso7d protein was examined. Sso7dis a small chromosomal protein from the hyperthermophilicarchaeabacterium Sulfolobus solfataricus that displays stabil-ity towards heat, acids, and other chemicals. The crystalstructure of the complex of Sso7d and d(GTAATTAC)2 wassolved at high resolution.[50] The protein binds in the minorgroove, causing a sharp bending (608) at the TpT step; thiskink results from the intercalation of the hydrophobic sidechains of Val26 and Met29. Consistent with previous obser-vations of B-form DNA, photoirradiation of d(GTAATI-UAC)2 produced 1’ and 2’ oxidation products in the absenceof Sso7d. In the presence of Sso7d, the formation of two newproducts, 4 and 5, was observed as shown in Scheme 3.[51]

These results clearly indicate that the protein-induced DNAkink causes an intrastrand hydrogen abstraction from the 5-methyl group of thymine in the same strand. Inspection of thecrystal structure indicates that the 5-methyl group in thymineis in close proximity to the uracilyl radical, whereas the T5-1’-and T5-2’-hydrogen atoms are located far from the adjacenturacilyl radical, as shown in Figure 5. We conclude that theunusual intrastrand hydrogen abstraction from T5-Me by the

Figure 4. a) Crystal structure of the Za–d(CGCGCG)2 complex and b) aclose-up view of one 5’-GC-3’ region. The carbon atom at the 5-position of uracil is shown in red, and abstracted hydrogen atoms ofguanine are shown in yellow. c) Structure of the GBrU step for aminimized Z-DNA structure showing the putative transition state ofC2’b-hydrogen abstraction and d) a close-up view of one GBrU step.

Scheme 3. Irradiation of d(GTAATIUAC)2–Sso7d with UV light. Theformation of new products was observed, indicating an intrastrandhydrogen abstraction from the 5-methyl group of thymine.




radical occurred efficiently at the observed bending site in thecrystal structure.[51] This specific intrastrand methyl hydrogenabstraction provides a powerful tool to detect directly DNAkinks in solution.

HPLC analysis of the photoirradiated d(GTAATIUAC)2–Sso7d complex also indicated that the protein Sso7d wasefficiently oxidized to Sso7dOH. Upon treatment with lysylendopeptidase, oxidation of Sso7dOH occurred mainly at thefragment from residues 29 to 40 (78%). Post-source-decaymass spectrometry (PSD-MS/MS) was performed to elucidatethe site of the oxidized amino acid residue in fragment 29–40(Figure 6). Fragment b2 was found in oxidized fragment 29–40 but not in the unmodified fragment, whereas fragment y11was constant in both cases. These results indicate that specificoxidation occurred at Met29 during irradiation of thed(GTAATIUAC)2–Sso7d complex. Inspection of the X-raycrystal structure suggests that residue Met29 is in closeproximity to the uracilyl radical, which is intercalated with themajor groove at the bending site. These results suggest thatthe interaction of DNA–Sso7d in solution is substantiallysimilar to its crystal structure.

6. Highly Efficient Photochemical Formation of2’-Deoxyribonolactone in the Antiparallel G Quartet

DNA tetraplexes, otherwise known as DNA quadruplexesor G quartets, are four-stranded DNA structures formed byguanine-rich sequences.[52–54] Although G quartets have so farbeen studied in vitro only, they are attracting increasingattention because of their postulated involvement in a varietyof biological processes. The DNA of human telomeresconsists of repeat units of the nucleotide sequence TTAGGG,ending in a single-stranded segment that overhangs at the endof the double-stranded DNA helix. The single-strandedrepeat units can form four-stranded G-quartet structures.[55–57]

NMR spectroscopic analysis demonstrated that a 22-mer of5’-d[AGGG(TTAGGG)3]-3’ in the solution state in thepresence of Na+ ions adopts an antiparallel G-quartetstructure in which the opposing GGG columns are antipar-allel, with one diagonal and two lateral TTA loops (Fig-ure 7).[58] In contrast, the same 22-mer adopts a completelydifferent G-quartet architecture in a crystal grown in thepresence of K+ ions, in which four core GGG units areparallel, with the three linking external loops positioned onthe exterior of the G-quartet core.[59]

To explore the structure-dependent abstraction of hydro-gen atoms in antiparallel and parallel G quartets, one of thesix thymine residues in 22-mer human telomeric DNA 5’-d(AGGGT1T2AGGGT3T4AGGGT5T6AGGG)-3’ was substi-tuted with IU to generate six types of oligodeoxynucleotide(ODNs 1–6).[60] Upon irradiation with UV light, more than60% of antiparallel ODN 4, in which T4 in the middle of the

Figure 5. a) Crystal structure of the photoirradiated Sso7d–d(GTAA-TIUAC)2 complex and b) a close-up view of the reacting region. In (a)the intercalated Met29 and Val26 moieties are shown in yellow; in (b)residues U6 and T5 of d(GTAATUAC)2 are colored blue, the carbonatom at the 5-position of U6 is red, and the putative abstractedhydrogen atoms of thymine are white.

Figure 6. Sequence of the fragment 29–40 of Sso7d. The oxidizedfragment 29–40 produced fragment b2, whereas fragment y11remained constant in both the oxidized and unmodified fragments29–40.

Figure 7. Schematic representations of the folded structures ofd[AG3(T2AG3)3]: a) the K

+-stabilized crystal structure, with TTA externalloops abutting the sides of the G quartet and parallel GGG columns;b) the Na+-stabilized solution-state structure, with one diagonal andtwo lateral TTA units.


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diagonal loop was substituted with IU, was consumed. Inmarked contrast to the photoreactivity of the antiparallelODN 4, the parallel ODNs 1–6 were not consumed (< 2%)under the same irradiation conditions. Analysis of theproducts indicated that 2’-deoxyribonolactone was efficientlyproduced with release of thymine from photoirradiatedODN 4 in the antiparallel structure (Figure 8).

The high photochemical reactivity of ODN 4 in theantiparallel G quartet and its poor photoreactivity in theparallel G quartet can be explained by comparing the loopregions of the two structures (Figure 9).[58,59] In the parallelG quartet, the adenine in the TTA external loop intercalatesbetween the two thymines to prevent hydrogen abstraction.In contrast, there is no such intercalation of the adenine basein the diagonal loop structure of the antiparallel G quartet,thereby allowing 1’-hydrogen abstraction by the uracilylradical in the loop. Furthermore, the structure determinedby NMR experiments suggests that the uracilyl radical ispositioned closer to the 1’-hydrogen atom of the adjacent T3than the other hydrogen atoms in the diagonal loop.

Several other guanine-rich sequences have also beenproposed to function biologically through the formation of aG quartet. For instance, G quartets have been associated withsite-specific genetic recombination in immunoglobulin switchregions (IgG), the insulin gene linked polymorphic region, theretinoblastoma susceptibility gene (Rb), and the c-myconcogene.[61–64] Numerous potential G-quartet-forming se-quences have been identified in many important genes.[65] Thesequence motif required for intrastrand G quartets can bewritten as GnNm1GnNm2GnNm3Gn, where n is the number ofguanine tetrads;m1,m2, andm3 are the loop lengths; and thediagonal loop (Nm2) of antiparallel structures is common inthe sequence motif (Figure 10a). To test the efficacy of thephotochemical method, we examined the photoreactions ofthe IU-substituted IgG switch regions and the 5’ termini of theRb gene.[60] Photoirradiation was performed at l= 302 nm for5’-d(AGGGGAGCTGGGGIUAGGTGGGA)-3’ (IgG) and5’-d(CGGGGGGTTIUTGGGCGGC)-3’ (Rb). HPLC analy-sis of the photolysates indicated that the photoproducts(�90% yield) were 2’-deoxyribonolactone-containingoligomers, which were obtained as the major products with

release of free guanine or thymine. This highly efficientproduction of 2’-deoxyribonolactone strongly suggests theformation of an antiparallel G quartet with a diagonal loopfor these guanine-rich sequences. Figure 10b and c show theproposed G-quartet structure based on our photochemicalstudies, showing two stacked guanine tetrads and a diagonalfour-base loop for both IgG and Rb.

7. Summary and Outlook

Our results indicate that the hydrogen abstraction ofDNA by the uracil-5-yl radical generated from 5-halouracilunder irradiation is atom-specific and highly dependent on

Figure 8. IU in the diagonal loop of an antiparallel G quartet undergoes anextremely facile photochemical reaction to produce a 2’-deoxyribonolactoneresidue. A similar reaction does not occur in parallel G quartets.

Figure 9. Structure of photoirradiated oligonucleotide d(AGGGT-TAGGGTIUAGGGTTAGGG) (ODN 4) based on NMR experiments (Na+

form) and X-ray crystallography (K+ form) and close-up views of theloop regions. Guanine moieties are colored blue; carbon atom C5 ofuracil is pink; abstracted hydrogen atoms of thymine are cyan;thymine red, uracil yellow, and adenine green.




the conformation of the DNA (Scheme 4). Competitive 1’-and 2’a-hydrogen abstractions were observed in B-DNA,whereas selective 1’-hydrogen abstraction occurred in the A-like structure of DNA–RNA hybrids. In Z-form DNA,stereospecific 2’b-hydrogen abstraction gave rise to 2’a-hydroxylation. In protein-induced DNA kinks, photoirradia-tion caused intrastrand hydrogen abstraction from the 5-methyl group of thymine at the 5’ side. The photoreactivity ofiodouracil-containing telomeric DNA depends on the orien-tation of the G quartet: the 2’-deoxyribonolactone residue iseffectively produced only in the diagonal loop of theantiparallel G quartet. These studies have demonstrated thedetailed relationship between the DNA local structure andthe photochemical product, and show the potential of thisphotochemical method in detecting DNA structure. Irradi-ation with UV light leads to the formation of two major typesof photoadducts: cyclobutane dimers and the (6–4) photo-product in DNA.[66] Ligation-mediated polymerase chainreaction (LMPCR) is a useful technique to detect theseproducts in vivo.[67] As 5-halouracil-substituted DNA isfunctional in living cell systems such as E. coli, use of thephotochemical reactions of 5-halouracil-containing DNA

Figure 10. The generic G-quartet-forming sequence, Gn, from fourguanine-rich tracts undergoes hydrogen bonding to form tetrads,which then stack to form the G-quartet stem. a) 1, 2, and 3 form thethree loops. b,c) Schematic representation showing the K+-inducedG-quartet forms of IgG (b) and Rb (c).

Scheme 4. Photochemical products formed by hydrogen abstraction in the five different structures of 5-halouracil-containing DNA upon irradiationwith UV light.


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would provide a powerful tool to probe local DNA con-formations in vivo.

Received: June 7, 2005Published online: January 16, 2006

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