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Chapter 4 : Reversal of Base Damage Caused by UV Radiation
Category: Microbial Genetics and Molecular Biology
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This chapter considers several examples of the direct reversal of DNA damage, all of which are catalyzed by single polypeptide enzymes. Both cyclobutane pyrimidine dimers (CPD) and (6-4) pyrimidine-pyrimidone photoproducts [(6-4)PP] constitute quantitatively and qualitatively important sources of base damage following the exposure of cells to UV radiation at wavelengths near the absorption maximum of DNA. The chapter discusses a specific DNA repair mode called enzymatic photoreactivation (EPR), or simply photoreactivation. Enzymes that catalyze EPR of CPD in DNA are referred to as pyrimidine dimer-DNA photolyases (PD-DNA photolyase), pyrimidine dimerdeoxyribodipyrimidine photolyases, or pyrimidine dimerphotoreactivating enzymes. The prefix “pyrimidine dimer” is added to distinguish these enzymes from those that catalyze the repair of (6-4)PP by an essentially identical mechanism. These are called (6-4) photoproduct-DNA photolyases ((6-4)PP-DNA photolyase). The chapter describes distribution, properties, structural studies and mechanism of action of PD-DNA photolyase. It also describes PD-DNA photolyase from other organisms and its therapeutic use for protection against sunlight. Next, it explains mechanism of action and C-terminal region of (6-4)PP-DNA photolyase. In addition, the phylogenetic relationships between PD-DNA photolyases, (6-4)PP-DNA photolyases, and blue-light receptor proteins have been suggested, and the following primary subfamilies of proteins have been defined: (i) PD-DNA photolyase proteins; (ii) plant CRY and plant and animal (6-4)PP-DNA photolyase proteins; and (iii) animal CRY proteins. Other covered topics are repair of thymine dimers by a deoxyribozyme, photoreactivation of RNA, and reversal of spore photoproduct in DNA.
Schematic illustration of the enzyme-catalyzed monomerization of pyrimidine dimers by DNA photolyase (photoreactivating enzyme), an example of DNA repair by the reversal of base damage. The gold square and triangle represent the two noncovalently bound chromophores required for catalytic activity in all DNA photolyases.
Schematic illustration of the measurement of thymine-containing pyrimidine dimers (T<>T, T <>C, C<>C) in DNA. DNA radiolabeled in thymine (T) is exposed to UV radiation to produce pyrimidine dimers, some of which contain radiolabeled thymine. The DNA is hydrolyzed in strong acid at high temperature, resulting in the preservation of structurally intact radiolabeled thymine monomers and radiolabeled thymine-containing pyrimidine dimers. These species can be resolved, and the number of thymine-containing pyrimidine dimers can be quantitated by thin-layer chromatography. The thymine dimer content of the DNA is then expressed as the fraction of the total radioactivity in thymine present as thymine-containing pyrimidine dimers (see Fig. 4–3 ).
Loss of thymine-containing pyrimidine dimers from acid-precipitable DNA after incubation of UV-irradiated DNA with DNA photolyase in the presence of photoreactivating light.
Structures of chromophores found in pyrimidine dimer-DNA photolyases. The folate class of DNA photolyases contains FADH- and 5,10-MTHF with appended polyglutamate residues. The deazaflavin class of DNA photolyases contains FADH- and 8-HDF. (Adapted from reference 108 .)
The absorption (ε) and absolute-action (εΦ) spectra of DNA photolyases of the folate class (E. coli DNA photolyase) and deazaflavin class (A. nidulans DNA photolyase). The gold lines show the spectra of the holoenzymes. The black lines are the spectra of the enzyme with just FADH-. Hence, the shape and maximum wavelengths of the absorption and action spectra in the range of 300- to 500-nm light are determined primarily by the second chromophore (5,10-MTHF in the folate class and 8-HDF in the deazaflavin class). (Adapted from reference 108 .)
Kinetics of the incision of UV-irradiated DNA by the E. coli UvrABC endonuclease for nucleotide excision repair in the presence or absence of PD-DNA photolyase. (Adapted from reference 111 .)
Summary of the mechanism of enzymatic photoreactivation by PD-DNA photolyases. 1, A blue-light photon is absorbed by the MTHF photoantenna. 2, The excitation energy is then transferred to the active-site flavin (FADH−). 3, The excited flavin contributes an electron to a CPD in DNA. 4, Electronic rearrangements restore the thymine bases to their native state, and the electron is transferred back to the ground-state neutral flavin radical. (Adapted from reference 109 .)
Ribbon diagram of E. coli PD-DNA photolyase protein. Conserved residues flanking the FAD chromophore are shown. The second chromophore, the MTFH photoantenna, is located on the back side of the protein.
Solvent-accessible surface of the PD-DNA photolyase from the thermophile T. thermophilus. The surface is colored to show the positive electrostatic potential (gold) surrounding the pocket that binds thymine. The thymine base binds specifically to a pocket adjacent to the FAD cofactor (see Fig. 4–8 ). It is proposed that base flipping would expose a cis-syn CPD in DNA and facilitate binding in this pocket.
The A. nidulans PD-DNA photolyase was crystallized in complex with DNA containing a cis-syn cyclobutane thymine photodimer ( 81a ). The enzyme reactivated the DNA during exposure of the crystals to an intense X-ray beam, resulting in a crystal structure of the enzyme-product complex with unlinked 5’ and 3’ thymines. The thymines are flipped into a pocket in the enzyme-active site, approximately 7 Å from the FADH electron donor. The light-harvesting antenna 8-HDF is positioned near the N-terminal subdomain in a position analogous to the methylene tetrahydrofolate antenna of E. coli PD-DNA photolyase (see Fig. 4–8 ). (Adapted from reference 81a .)
Schematic diagram of the yeast PHR1 promoter. The nucleotide sequences of the indicated regulatory elements UES, UAS, and URS are shown. Arrows above sequences indicate palindromes. The white bar under the URS sequence is the binding site for Rph1 protein. Numbering at the top of the figure is relative to the first ATG translational start codon in the coding sequence. (Adapted from reference 120 .)
Effect of derepression of the yeast PHR1 gene on the survival of nucleotide excision repair-defective strains exposed to UV radiation. Cells that are mutant for both the RPH1 and GIS1 genes show increased survival. WT, wild type. (Adapted from reference 54 .)
The Rph1 and Gis1 proteins have amino acid sequence identity and similarity. The region near the C terminus shows almost 100% amino acid identity and contains two zinc finger motifs. Amino acids in these motifs that are thought to be involved in binding DNA are over-lined. (Adapted from reference 54 .)
Amino acid sequence comparison of DNA photolyases from E. coli (E.c.), S. enterica serovar Typhimurium (S.t.), N. crassa (N.c.), S. cerevisiae (S.c.), H. halobium (H.h), A. nidulans (A.n.), and S.griseus (S.g.). Identical amino acids in all seven polypeptides are indicated in light gold, and conservative substitutions are indicated in dark gold. Asterisks represent unrelated amino acids, and the numbers in parentheses indicate amino acid distances separating conserved domains. The first 71 amino acids of the S. cerevisiae polypeptide are not shown. (Adapted from reference 1 .)
(A) Breakage (dotted gold line) of the cyclobutane ring in a thymine-thymine CPD generates two canonical thymines. (B) However, cleavage of the C-6/C-4 bond linking two thymines in a (6–4)PP would not yield normal products. (Adapted from reference 184 .)
A factor in extracts of D. melanogaster repairs (6–4)PP in UV-irradiated plasmid DNA. UV-irradiated plasmid DNA was incubated with extract in the presence (gold line) or absence (black broken line) of fluorescent light and then treated with PDDNA photolyase to remove CPD. The plasmid DNA was then transformed into an E. coli strain defective for PD-DNA photolyase activity and nucleotide excision repair. Transforming activity was not observed when the plasmid DNA was exposed to fluorescent light in the absence of Drosophila extract (solid black line). (Adapted from reference 163 .)
Amino acid sequence alignment of regions of (6–4)PP- and PD-DNA photolyases in the FAD-binding (A) and active-site (B) regions. The lighter gold boxes show conservation of amino acids, especially aromatic residues. The dark gold boxes indicate conserved histidine residues in the proposed active site of the (6–4)PP-DNA photolyases. (Adapted from reference 48 .)
Proposed mechanism for photoreactivation of (6–4)PP by Xenopus (6–4)PP-DNA photolyase. The mechanism involves two histidine residues (His354 and His358) located at the active site (see Fig. 4–17 ). His354 and His358 hydrogen bond with the N-3 of the 3’ pyrimidone and with the OH-group of the 5’ pyrimidone, respectively. His358 abstracts a proton from the OH-group (or from the protonated amino group at C-5) of the 5’ pyrimidine, and His354 protonates N-3 of the 3’ pyrimidone. Nucleophilic attack at the cationic 3’ C-4 results in the formation of an oxetane intermediate. As is the case with PD-DNA photolyases, light absorbed by a photoantenna (not shown here) generates excitation energy that is transferred to the active-site flavin (FADH). Electron transfer results in the conversion of the oxetane intermediate to native thymines and transfer of an electron back to the ground-state flavin neutral radical. (Adapted from reference 48 .)
Phylogenetic relationships between blue-light receptor proteins. Shown are photolyases and CRY proteins from N. crassa (Nc), S. cerevisiae (Sc), E. coli (Ec), S. enterica serovar Typhimurium (St), A. nidulans (An), H. halobium (Hh), S. griseus (Sg), A. thalania (At), S. alba (Sa), C. reinhardtii (Cr), M. domestica (Md), C. auratus (Ca), D. melanogaster (Dm), M. thermoautotrophicum (Mt), zebrafish (Z), H. sapiens (Hs), and X. laevis (X1). PD-DNA photolyases (CPD), (6–4)PP-DNA photolyases ( 64 ), and CRY proteins (CRY or CRT) are shown. (Adapted from reference 160 .)
Oligonucleotide constructs used to examine deoxyribozyme activity on CPD. A 35-nucleotide element consisting of 20- and 15-mer oligonucleotides linked through a T<>T dimer but lacking a connecting phosphodiester linkage was hybridized to a perfectly complementary 25-nucleotide splint (A), a “flipped” splint in which the two A residues complementary to the T<>T were deleted (B), and a “mismatched” splint in which AA was replaced by either TT or CC, generating mismatches with the dimer (IC). (Adapted from reference 13 )
Structure of SP (bottom). The structure of a CPD (top) is shown for comparison. (Adapted from reference 134 .)
A plasmid containing the Spl + gene of B. subtilis confers UV radiation resistance to spores of B. subtilis strains that are mutant for Spl +, regardless of whether they are proficient or defective (uvrA) in nucleotide excision repair. (Adapted from reference 28 .)
Proposed mechanism for the repair of SP. Two subunits of SplB protein dimerize through the formation of a reduced [4Fe-4S] center to form active SP lyase (not shown). The [4Fe-4S] center donates an electron(s) to SAM, generating methionine and a 5’-adenosyl radical (top). The latter moiety is used to abstract a proton from C-6’ of SP, leading to β-scission of the bond linking the thymines and completion of the reaction by back transfer of the proton (bottom). dRib, deoxyribose. (Adapted from reference 94 .)
Kinetic and equilibrium constants for substrate binding and photolysis by S. cerevisiae DNA photolyase
Physical, spectroscopic, and photochemical properties of DNA photolyases
Properties of multiple DNA photolyases
Conservation of selected residues in photolyases
Photolyase and cryptochrome in different kingdoms