CPD photolyase

Electron donation to nucleobases is a fundamental process exploited by nature to achieve the efficient repair of UV induced lesions in DNA [27, 28]. Nature developed to this end two enzymes, CPD photolyases and (6-4) photolyases, which both inject electrons into the UV damaged DNA bases [29, 30]. Both enzymes are, in many species, including plants, essential for the repair of the UV-light induced DNA lesions depicted in Scheme 1 [31]. 

Scheme 2 Mechanism of repair of cyclobutane pyrimidine dimers (CPD) by a CPD photolyase. 8-HDF 8-hydroxy-5-deazaflavin, ET electron transfer. FADH reduced and de-protonated flavin-coenzyme...

As expected, the Z-principal axis of g is oriented perpendicular to the 71-plane of the flavin ring. Analyses of EPR and ENDOR data revealed angles of (—29 4)° and (—14 2)° between the X axis of g and the N5-H5 (or N5-D5) bond in (6 ) photolyase [38] and cyclobutane pyrimidine dimer (CPD) photolyase [28, 30], respectively. The factors that cause the reorientation of the X and Y axes of g of a neutral flavin radical in the two highly homologous cofactor binding pockets of CPD photolyase and (6 ) photolyase remain elusive. Also, the orientations of the principal axes of g relative to the molecular frame of the isoalloxazine ring of a flavin anion radical still need to be determined experimentally. 

Fig. 2 Pulsed ENDOR on a neutral flavin radical. E. coli CPD photolyase was investigated with pulsed Davies ENDOR spectroscopy at r = 80 K (for details, see [25]). Detectable protons are marked accordingly...

In recent years, a wealth of information on photolyase-mediated DNA repair and cryptochrome- and phototropin-mediated blue-light responses was obtained through the combined efforts of biologists, chemists, and physicists, from both experimental and theoretical studies. Here we have chosen recent examples of experimental work from our group to demonstrate the potential of modem EPR methods to answer mechanism-related questions and to study reactive intermediates in photo-induced electron transfer. Nevertheless, some key questions of these reactions remain to be solved. Important questions are as follows. What are the precise differences between CPD photolyase and (6 ) photolyase regarding... 

DNA Photolyase. - Ultraviolet light (> 300 nm) damages cellular DNA by the formation of thymine dimers from adjacent pyrimidine bases on the same DNA strand. The two main photoproducts are (cis,syn)-cyclobutane pyrimidine dimers (CPDs) and the (6-4) photoproduct. Such dimers may be restored to their monomeric form through the action of two photoactive (300 < X < 500 nm) damage-specific DNA repair enzymes, named CPD photolyase (also called DNA photolyase) and (6-4) photolyase. These enzymes are found in various... 

To increase spectral resolution, we have examined the EPR signal of the FAD cofactor in Escherichia coli CPD photolyase using X- and W-band CW EPR and CW ENDOR and TRIPLE at X-band. The frozen solution X-band EPR signal of the flavin cofactor had a linewidth of 1.87 mT and was centred atg = 2.0034. The linewidth decreased to 1.30 mT in deuterated buffer, consistent with a neutral flavin radical. The W-band EPR signal had a lineshape characteristic for a randomly oriented radical with a gf-matrix of axial symmetry, a simulation of which gave the principal values g = 2.0043 and = 2.0022. 

Cyclobutane pyrimidine dimer DNA-photolyases (CPD-photolyases) are monomeric DNA repair proteins ( 55 kDa) that directly repair cyclobutane containing UV-light-induced DNA lesionsJ " Another type of photolyases repairs (6-4) lesions. They will be discussed later. The CPD-photolyases are mechanistically the best understood, and three crystal structures of CPD-photolyases fi om E. colij A. nidu-lansf and T. thermophilus provide detailed information about the structural requirements for efficient genome repair. 

SCHEME 8 Reaction mechanism of CPD-photolyases (Type-I) with a methenyltetrahydrofolate (MTHF) as the second cofactor. 

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