Spectral properties of the chromophoric material associated with the deoxyribonucleic acid photoreactivating enzyme isolated from baker's yeast

Minato, Sadamasa; Werbin, Harold

doi:10.1021/bi00800a025

Cited by 50 publications

(22 citation statements)

References 8 publications

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“…The S. cerevisiae photolyase was isolated from cells harboring a multicopy plasmid with the yeast PHRl gene. Previously yeast photolyase has been prepared from baker's yeast32 and by overproduction in E. coli cells.28 Two bands (60 and 58 kDa) in SDS-PAGE have been reported for purified yeast photo-lyase.28, 32 We found a single band at 57 kDa (Fig. IB), which is lower than the calculated value (66 kDa).…”

Section: Discussionmentioning

confidence: 54%

DNA PHOTOLYASE FROM THE FUNGUS NEUROSPORA CRASSA. PURIFICATION, CHARACTERIZATION AND COMPARISON WITH OTHER PHOTOLYASES

Eker

Yajima

Yasui

1994

Photochem & Photobiology

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A phr-gene from the filamentous fungus Neurospora crassa was overexpressed in Escherichia coli cells, yielding a biologically active photolyase. After purification till apparent homogeneity, the 66 kDa protein was found to contain equimolar amounts of 5,10-methenyltetrahydrofolic acid (MTHF) and FAD, classifying it as an MTHF-type photolyase. Compared to other MTHF photolyases the absorption maximum of Neurospora photolyase is shifted from ca 380 nm to 391 nm (epsilon = 34,800), while an additional shoulder is present at 465 nm. In dark-adapted enzyme the FAD chromophore is predominantly present in the oxidized form, in contrast with E. coli and Saccharomyces cerevisiae photolyase, which contain mainly semiquinone or fully reduced FAD, respectively. Preillumination or dithionite treatment converted oxidized FAD in Neurospora photolyase into the fully reduced form, with a concomitant shift of the absorption maximum from 391 to 396 nm and disappearance of the 465 nm shoulder. The action spectrum of photoreactivation coincides with the absorption spectrum of preilluminated (reduced) photolyase, extending the spectral region of MTHF-type photolyases from 380 till 396 nm. A quantum yield of 0.57 was obtained for the overall repair reaction. Comparison of spectral properties of FAD in Neurospora photolyase and the model compound lumiflavin points to an apolar microenvironment of photolyase-bound FAD. Neurospora photolyase has distinct advantages over E. coli photolyase as it is more stable and contains a full complement of chromophores.

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Section: Discussionmentioning

confidence: 54%

DNA PHOTOLYASE FROM THE FUNGUS NEUROSPORA CRASSA. PURIFICATION, CHARACTERIZATION AND COMPARISON WITH OTHER PHOTOLYASES

Eker

Yajima

Yasui

1994

Photochem & Photobiology

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“…This enzyme binds to DEAE-cellulose, as opposed to another UV-specific endonuclease that has little or no affinity to DEAE-cellulose (13). Yeast photoreactivating enzyme (EC 4.1.99.3; deoxyribocyclobutadipyrimidine pyrimidine-lyase), purified through the DNA cellulose step (14), was generously provided by H. Werbin.…”

Section: Methodsmentioning

confidence: 99%

An Endonuclease from Escherichia coli That Acts Preferentially on UV-Irradiated DNA and Is Absent from the uvrA and uvrB Mutants

Braun

Grossman

1974

Proc. Natl. Acad. Sci. U.S.A.

125

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At least two endonucleolytic activities that preferentially incise ultraviolet (UV)-irradiated DNA exist in extracts of E. coli. These two activities can be separated by phosphocellulose chromatographic fractionation. The subject of this paper is one of these activities, which elutes from phosphocellulose with 0.25 M potassium phosphate, pH 7.5. This endonucleolytic activity specific for UV-irradiated DNA is absent from partially purified extracts of uvrA and uvrB mutants, which are defective in excision of pyrimidine dimers, but is present in normal amounts in the uvrC excision-defective mutant. The enzyme binds very tightly and specifically to UV-irradiated DNA. Binding can be prevented by prior treatment of the irradiated DNA with photoreactivating enzyme. This binding activity is absent in uvrA and uvrB mutants, but present in uvrC and uvrD mutants.Excision repair of UV-induced cyclobutane-type pyrimidine dimers in Escherichia coli is currently thought to be a fourstage process (1). Initially, an endonucleolytic incision is made in the vicinity of the dimer. This is followed by exonucleolytic removal of the dimer and several adjacent nucleotides. Nucleotides are reinserted into the resultant cavity by a polymerase, using the opposite, intact strand as a template, with the continuity of the repaired region restored by polynucleotide ligase.Mutants of E. coli with markedly reduced levels of polymerase I (2) and polynucleotide ligase (3, 4) are substantially more UV-sensitive than the wild type. UvrA, uvrB, and uvrC mutants do not excise dimers (5) and presumably are defective in either the endonucleolytic or exonucleolytic steps of excision repair.Endonucleolytic activities specific for UV-irradiated DNA have been isolated and purified from Micrococcus luteus (6, 7) and E. coli infected with phage T4 (8, 9). These activities are absent from some UV-sensitive mutants (8, 10). Takagi et al., have described an activity in extracts of E. coli that specifically incises UV-irradiated DNA (11). However, since this activity was reported to be present in extracts of all studied U1-sensitive mutants, the role of this enzymatic activity in excision repair was not clear.In this paper, we describe the partial purification and some of the properties of an endonucleolytic activity, specific was prepared by methods described by Schekman et al. (12). This method involves ethidium bromide-CsCl density gradient centrifugation followed by a neutral sucrose gradient. A substantial amount of material absorbing 260-nm light, presumably unlabeled RNA, is removed in the sucrose gradient. In many of the preparations used in these experiments, the final gradient was omitted. This omission had no detectable effect on the assays described below.UV-Irradiation. The OX RFI ['H]DNA was irradiated in a stoppered quartz cuvette at 280 nm with a monochrometer described previously (6). In general, irradiation was to an average dose of 9000 ergs/mm2. The incident dose was adjusted to compensate for self-absorption.Enzymes. M. luteus UV-en...

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“…T h e enzyme involved ('photoreactivating enzyme', or PRE)'', which has been purified over 70,000 fold from crude extracts of baker's yeast (Muhammed. 1966; Minato and Werbin, 1971). and is specific for cyclobutyl pyrimidine dimers in double-or single-stranded sequences of a t least nine deoxyribonucleotides (R. B.…”

Section: Introductionmentioning

confidence: 99%

SUBSTRATE SPECIFICITY OF A BACTERIAL UV ENDONUCLEASE AND THE OVERLAP WITH IN VITRO PHOTOENZYMATIC REPAIR

Patrick¹,

Harm²

1973

Photochem & Photobiology

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P h u l~~~h i~m i~l r~o t i~l P l m r u b k~l u~? .Abstract -The action of an endonuclease from Micrococcus Iuieus. that operates on ultraviolet ( U V ) radiation damage, overlaps greatly with that of the yeast photoreactivating enzyme: homo and hetero cyclobutyl pyrimidine dimers in DNA are substrate for both enzymes, but pyrimidine adducts or the 'spore photoproduct' in DNA are not.As expected from this overlap, the action of the two enzymes is mutually interfering: single-strand nicks introduced by the endonuclease effectively preclude photoreactivation: conversely. formation of a photoreactivating enzyme-dimer complex can prevent nicking by the UV endonuclease. While complex formation between photoreactivating enzyme and dimers in U V-endonuclease-treated DNA is apparently normal, the light-dependent repair step either fails to occur or proceeds at a very low rate. Hence, besides the requirement of a minimum chain length for the function of the photoreactivating enzyme. there is the additional restriction on the position of the dimer in a polynucleotide strand.Finally, rough approximations of the rate constants. k , and k,. for the U V endonuclease indicate that the in uitro UV-endonuclease-dimer complex is relatively unstable, with dissociation of the complex being more probable than hydrolysis of the phosphodiester bond. 1968).Recently, the use of light flashes has allowed detailed characterization of the dark and light 371 372 M. H. PATRICK and HELGA HARM dependent reaction steps under varying experimental conditions, indicating many similarities between the action of yeast PRE in uitro and Escherichia coli PRE in uiuo and permits some general conclusions concerning the mechanisms of the photoenzymatic process (W. Harm et al., 1971).Because pyrimidine dimers are primarily responsible for the lethal effects of far UV, it is not surprising that there is a large overlap in the actions of photoenzymatic repair and ERR. The results presented in this paper show that, like the PRE, the M . luteus endonuclease recognizes and acts upon all types of cyclobutyl pyrimidine dimers in DNA but not other known UV-induced photoproducts. Furthermore, the two reactions can be mutually exclusive: single-strand nicks introduced by the endonuclease effectively preclude PR: conversely, formation of a PRE-dimer complex can prevent nicking by the UV endonuclease. This has permitted estimation of the constants associated with binding and dissociation of the endonuclease.

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Spectral properties of the chromophoric material associated with the deoxyribonucleic acid photoreactivating enzyme isolated from baker's yeast

Cited by 50 publications

References 8 publications

DNA PHOTOLYASE FROM THE FUNGUS NEUROSPORA CRASSA. PURIFICATION, CHARACTERIZATION AND COMPARISON WITH OTHER PHOTOLYASES

DNA PHOTOLYASE FROM THE FUNGUS NEUROSPORA CRASSA. PURIFICATION, CHARACTERIZATION AND COMPARISON WITH OTHER PHOTOLYASES

An Endonuclease from Escherichia coli That Acts Preferentially on UV-Irradiated DNA and Is Absent from the uvrA and uvrB Mutants

SUBSTRATE SPECIFICITY OF A BACTERIAL UV ENDONUCLEASE AND THE OVERLAP WITH IN VITRO PHOTOENZYMATIC REPAIR

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