Similarities and Differences between Cyclobutane Pyrimidine Dimer Photolyase and (6-4) Photolyase as Revealed by Resonance Raman Spectroscopy

The cyclobutane pyrimidine dimer (CPD) and (6-4) photoproduct, two major types of DNA damage caused by UV light, are repaired under illumination with near UV-visible light by CPD and (6-4) photolyases, respectively. To understand the mechanism of DNA repair, we examined the resonance Raman spectra of complexes between damaged DNA and the neutral semiquinoid and oxidized forms of (6-4) and CPD photolyases. The marker band for a neutral semiquinoid flavin and band I of the oxidized flavin, which are derived from the vibrations of the benzene ring of FAD, were shifted to lower frequencies upon binding of damaged DNA by CPD photolyase but not by (6-4) photolyase, indicating that CPD interacts with the benzene ring of FAD directly but that the (6-4) photoproduct does not. Bands II and VII of the oxidized flavin and the 1398/1391 cm-1 bands of the neutral semiquinoid flavin, which may reflect the bending of U-shaped FAD, were altered upon substrate binding, suggesting that CPD and the (6-4) photoproduct interact with the adenine ring of FAD. When substrate was bound, there was an upshifted 1528 cm-1 band of the neutral semiquinoid flavin in CPD photolyase, indicating a weakened hydrogen bond at N(5)-H of FAD, and band X seemed to be downshifted in (6-4) photolyase, indicating a weakened hydrogen bond at N(3)-H of FAD. These Raman spectra led us to conclude that the two photolyases have different electron transfer mechanisms as well as different hydrogen bonding environments, which account for the higher redox potential of CPD photolyase. The cyclobutane pyrimidine dimer (CPD) and (6-4) photoproduct, two major types of DNA damage caused by UV light, are repaired under illumination with near UV-visible light by CPD and (6-4) photolyases, respectively. To understand the mechanism of DNA repair, we examined the resonance Raman spectra of complexes between damaged DNA and the neutral semiquinoid and oxidized forms of (6-4) and CPD photolyases. The marker band for a neutral semiquinoid flavin and band I of the oxidized flavin, which are derived from the vibrations of the benzene ring of FAD, were shifted to lower frequencies upon binding of damaged DNA by CPD photolyase but not by (6-4) photolyase, indicating that CPD interacts with the benzene ring of FAD directly but that the (6-4) photoproduct does not. Bands II and VII of the oxidized flavin and the 1398/1391 cm-1 bands of the neutral semiquinoid flavin, which may reflect the bending of U-shaped FAD, were altered upon substrate binding, suggesting that CPD and the (6-4) photoproduct interact with the adenine ring of FAD. When substrate was bound, there was an upshifted 1528 cm-1 band of the neutral semiquinoid flavin in CPD photolyase, indicating a weakened hydrogen bond at N(5)-H of FAD, and band X seemed to be downshifted in (6-4) photolyase, indicating a weakened hydrogen bond at N(3)-H of FAD. These Raman spectra led us to conclude that the two photolyases have different electron transfer mechanisms as well as different hydrogen bonding environments, which account for the higher redox potential of CPD photolyase. Irradiation of organisms with ultraviolet light causes damage to cellular DNA by inducing dimer formation between adjacent pyrimidine bases. This DNA damage causes mutation and cell death and can lead to cancer (1Lindahl T. Nature. 1993; 362: 709-715Crossref PubMed Scopus (4378) Google Scholar). The cyclobutane pyrimidine dimer (CPD) 4The abbreviations used are: CPD, cyclobutane pyrimidine dimer; FADox, oxidized FAD; FADH°, neutral semiquinoid FAD; FADH-, anionic fully reduced FAD. and (6-4) photoproduct (Fig. 1) account for most of this DNA damage, and these are repaired by CPD and (6-4) photolyases, respectively, under illumination by near UV-visible light (2Sancar A. Biochemistry. 1994; 33: 2-9Crossref PubMed Scopus (570) Google Scholar, 3Todo T. Takemori H. Ryo H. Ihara M. Matsunaga T. Nikaido O. Sato K. Nomura T. Nature. 1993; 361: 371-374Crossref PubMed Scopus (260) Google Scholar). Similar association constants of ∼10-9 m were identified for CPD and the (6-4) photoproduct in the complex of the corresponding photolyase, respectively, whereas those for the undamaged DNA were >4 orders of magnitude lower or not detectable (4Sancar A. Chem. Rev. 2003; 103: 2203-2237Crossref PubMed Scopus (1042) Google Scholar, 5Zhao X.D. Liu J.Q. Hsu D.S. Zhao S.Y. Taylor J.S. Sancar A. J. Biol. Chem. 1997; 272: 32580-32590Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 6Hitomi K. Kim S.T. Iwai S. Harima N. Otoshi E. Ikenaga M. Todo T. J. Biol. Chem. 1997; 272: 32591-32598Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Both photolyases contain FAD as an essential catalytic cofactor (7Sancar G.B. Jorns M.S. Payne G. Fluke D.J. Rupert C.S. Sancar A. J. Biol. Chem. 1987; 262: 492-498Abstract Full Text PDF PubMed Google Scholar, 8Cichon M.K. Arnold S. Carell T. Angew. Chem. Int. Ed. Engl. 2002; 41: 767-770Crossref PubMed Google Scholar). The amino acid sequences of (6-4) and CPD photolyases, especially within the FAD-binding region, are closely related, suggesting that these two enzymes share similar structure and reaction mechanisms (9Todo T. Ryo H. Yamamoto K. Toh H. Inui T. Ayaki H. Nomura T. Ikenaga M. Science. 1996; 272: 109-112Crossref PubMed Scopus (231) Google Scholar); however, the quantum yield for photorecovery is significantly lower for (6-4) photolyase than for CPD photolyase (4Sancar A. Chem. Rev. 2003; 103: 2203-2237Crossref PubMed Scopus (1042) Google Scholar, 6Hitomi K. Kim S.T. Iwai S. Harima N. Otoshi E. Ikenaga M. Todo T. J. Biol. Chem. 1997; 272: 32591-32598Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 10Kim S.T. Malhotra K. Smith C.A. Taylor J.S. Sancar A. J. Biol. Chem. 1994; 269: 8535-8540Abstract Full Text PDF PubMed Google Scholar). The isoalloxazine ring of FAD in a photolyase can exist in one of three possible states: oxidized (FADox), neutral semiquinoid (FADH°), and anionic fully reduced (FADH-) forms (Fig. 2). FADH- is considered to be the active form of flavin in photolyase. The first step of the repair process is the specific recognition of CPD and the (6-4) photoproduct by CPD photolyase (2Sancar A. Biochemistry. 1994; 33: 2-9Crossref PubMed Scopus (570) Google Scholar) and (6-4) photolyase (9Todo T. Ryo H. Yamamoto K. Toh H. Inui T. Ayaki H. Nomura T. Ikenaga M. Science. 1996; 272: 109-112Crossref PubMed Scopus (231) Google Scholar), respectively. After photoexcitation of FADH- by near UV-visible light, an electron is transferred to the damaged DNA, leaving the flavin in the neutral semiquinoid form (FADH°). Following bond cleavage of the DNA dimer, the electron is transferred back to FADH° to restore the active state, FADH- (4Sancar A. Chem. Rev. 2003; 103: 2203-2237Crossref PubMed Scopus (1042) Google Scholar). In the case of the (6-4) photoproduct, it has been proposed that the bound substrate is converted to a four-member oxetane ring intermediate (10Kim S.T. Malhotra K. Smith C.A. Taylor J.S. Sancar A. J. Biol. Chem. 1994; 269: 8535-8540Abstract Full Text PDF PubMed Google Scholar) catalyzed by two conserved histidines in the active site (11Hitomi K. Nakamura H. Kim S.T. 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Scopus Google Scholar), have that the between between FAD and the DNA substrate was by of the Biochemistry. 2001; PubMed Scopus Google Scholar) and by and electron resonance S. G. E. A. K. J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). the structure of CPD photolyase with a DNA A. T. Carell T. Science. PubMed Scopus Google Scholar) a of between the cofactor and the dimer (Fig. a by a and Sancar A. S. A. PubMed Scopus Google Scholar). Raman spectra have that in the hydrogen bonding of the FAD cofactor in CPD photolyase substrate binding M. O. K. S. J. Chem. 2003; Scopus Google Scholar) to the redox potential of the FAD cofactor J. Chem. PubMed Scopus Google Scholar) and it in the active FADH- in the but the of the hydrogen bonding have not been for (6-4) photolyase, a structure is not the between the two photolyases that the between the (6-4) photoproduct and the FAD cofactor may be similar to that between CPD and FAD in CPD photolyase, the quantum yield for (6-4) photolyase is lower than that for CPD photolyase Raman is a for the between the flavin cofactor and in in Scholar, of Raman Scholar). To and the between the damaged DNA and the FAD cofactor in (6-4) and CPD photolyases, we first a to of active the resonance Raman spectra for complexes of the neutral semiquinoid and oxidized forms with damaged that CPD interacts with the benzene ring and the adenine ring of the FAD whereas the (6-4) photoproduct interacts with the adenine ring of the FAD a may different of electron transfer for the two In the Raman spectra different in the hydrogen bonding for the two of (6-4) photolyase or CPD photolyase was at the and of the E. with the was to of in a and at to The was to to for and by The was at in m and and was from the by at for The was a and the was with m and m the was to a and with a of m of E. of (6-4) photolyase and of CPD photolyase. The was not from the To the fully oxidized from the (6-4) photolyase was to for and to a the of CPD photolyase by is the neutral semiquinoid FAD cofactor was by the which CPD photolyase was with oxidized FAD were to the by Jorns M.S. Biochemistry. PubMed Scopus Google Scholar), that the was from the and a was used to FAD. The was in m was to the CPD photolyase. The of the to the was by and the form was identified by the The of the oxidized form of the was the FAD at T. Kim S.T. K. Otoshi E. Inui T. H. H. E. Toh H. Ikenaga M. 1997; PubMed Scopus Google Scholar), and that of the neutral semiquinoid form was from the at Jorns M.S. Biochemistry. PubMed Scopus Google Scholar). substrate was by of or for with UV light a UV The was in a cell that was the UV by with of was by the in the band at and the of an band of the (6-4) photoproduct at J. Biol. Chem. Full Text PDF PubMed Google Scholar). of the (6-4) and CPD photolyases was a the for CPD photolyase M.S. Sancar G.B. Sancar A. Biochemistry. PubMed Scopus Google Scholar). a complex of photolyase and in and was in a cell and with a light at The UV-visible of the was as a of and Raman spectra of were with a UV-visible at Raman spectra were with a with a The were and from a for the semiquinoid and oxidized respectively. The at the was was with Raman were with and the of the of the well Raman bands was the semiquinoid the were an of in m the oxidized the was the but the was and was for CPD photolyase. of or was to the were with a Raman cell of and were with The in the was by a The resonance Raman spectra of oxidized CPD photolyase were by the Raman band for in the in and are whereas the Raman were by most band the in frequencies were for dimer repair by (6-4) and CPD photolyases in the of are in and UV damage reduced the band of undamaged DNA and in the of a band for the (6-4) of the damaged DNA complex with light in the of (6-4) or CPD photolyase in the of the band for the (6-4) photolyase, the of the the enzymes used have are of with DNA most of the (6-4) and CPD photolyases were in the neutral semiquinoid form with FADH° (Fig. and The spectra for the fully oxidized (6-4) and CPD photolyases with are in and respectively. are the spectra of the complexes with and The of FADH° and and in CPD photolyase were significantly shifted upon binding of the as a from the DNA of the (6-4) photoproduct J. Biol. Chem. Full Text PDF PubMed Google Scholar). The of this as well as the frequencies of bands and Raman not the of an of DNA substrate in this This is in with M. O. K. S. J. Chem. 2003; Scopus Google Scholar, M.S. Biochemistry. PubMed Scopus Google Scholar). In substrate binding the spectra of (6-4) photolyase. The and in and are to the of These are from the spectra for (Fig. and In with these we and as the Raman to the neutral semiquinoid and oxidized (Fig. Raman of with DNA resonance Raman spectra of the neutral semiquinoid (6-4) and CPD photolyases are in and the spectra for the with undamaged DNA and with DNA are in the spectra between the and the DNA complex but not in the spectra between the and the DNA complex These were in and that the structure and of the FAD cofactor in photolyases upon binding of a specific substrate and that undamaged DNA does not interact with the active site in photolyase. 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Chem. Scopus Google Scholar). and the of the are to form a hydrogen bond of the of the FAD cofactor in CPD photolyase M. O. K. S. J. Chem. 2003; Scopus Google Scholar). the lower of band II in (6-4) and CPD photolyases reflect a U-shaped of the FAD of band II upon substrate binding was identified for enzymes (Fig. This the of the adenine ring from the isoalloxazine of FAD as a of the hydrogen bonding between adenine and the in FAD by structure of CPD photolyase that N(5)-H a hydrogen bond to an that is conserved in (6-4) photolyase. that the Raman band at 1528 cm-1 in CPD photolyase was upshifted upon substrate binding, whereas that at cm-1 in (6-4) photolyase was This band is a marker for hydrogen bonding at N(5)-H in the neutral semiquinoid flavin E. A. G. J. Raman 2001; Scopus Google Scholar), and it has been used in a Raman of T. Biochemistry. PubMed Scopus Google Scholar). the of the cm-1 band that the hydrogen bond at N(5)-H of FADH° in CPD photolyase is weakened the is bound to This Raman is by the band at cm-1 in CPD photolyase, which (Fig. and hydrogen bonding may be at N(3)-H in FADH° and X of the oxidized flavin at which and N(3)-H seemed to be downshifted in (6-4) photolyase (Fig. and indicating that the hydrogen bond be weakened by substrate The of bands and in enzymes (Fig. and may reflect the of the hydrogen bonding or vibrations of I by substrate the the Raman bands of FADH° at and cm-1 in (6-4) photolyase the of the band at cm-1 in CPD photolyase. The lower of band X that the hydrogen bond at N(3)-H in CPD photolyase is than that in (6-4) the hydrogen bond at N(3)-H in (6-4) photolyase may be weakened upon substrate for the structure of CPD photolyase that I is in the of the DNA A. T. Carell T. Science. 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Chem. 1996; Scopus Google Scholar), and the FAD in the two redox have been to the substrate to Raman In the of CPD of the DNA the has been to be of the redox of the FAD cofactor by oxidized and fully reduced CPD photolyases J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). the between substrate and FAD cofactor in this were to in the fully reduced this we UV-visible and resonance Raman spectra of the neutral semiquinoid and oxidized (6-4) and CPD photolyases as well as upon substrate that a may between CPD and I of FAD in CPD photolyase but that this does not for (6-4) photolyase, indicating that the electron transfer mechanisms in these two enzymes may be hydrogen bond may be between DNA and the adenine ring of FAD in enzymes upon substrate The substrate may the hydrogen bond at N(5)-H in CPD photolyase and at N(3)-H in (6-4) photolyase. The the redox potential of FAD in CPD photolyase. These the structure of the complex and the mechanism by which substrate binding for of the