PDBsum entry 2fcc

Hydrolase

PDB id: 2fcc

Contents

Protein chains

137 a.a. *

DNA/RNA

Ligands

SO4 ×2

GOL ×2

Waters ×183

* Residue conservation analysis

PDB id:

2fcc

Name:

Hydrolase

Title:

Crystal structure of t4 pyrimidine dimer glycosylase (t4-pdg) covalently complexed with a DNA substrate containing abasic site

Structure:

DNA (5'-d( Cp Cp Ap Gp Gp Ap (Ped)p Gp Ap Ap Gp Cp C)-3'). Chain: c, e. Fragment: ds oligonucleotide containing ap site. Engineered: yes. DNA (5'-d( Gp Gp Cp (Bru)p (Bru)p Cp Ap (Bru)p Cp Cp (Bru) p Gp G)-3'). Chain: d, f. Engineered: yes. Endonuclease v.

Source:

Synthetic: yes. Other_details: chemically synthesized.. Enterobacteria phage t4. Organism_taxid: 10665. Gene: denv. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Trimer (from PQS)

Resolution:

2.30Å R-factor: 0.249 R-free: 0.274

Authors:

G.Golan,D.O.Zharkov,A.S.Fernandes,M.L.Dodson,A.K.Mccullough, A.P.Grollman,R.S.Lloyd,G.Shoham

Key ref:

G.Golan et al. (2006). Structure of T4 pyrimidine dimer glycosylase in a reduced imine covalent complex with abasic site-containing DNA. J Mol Biol, 362, 241-258. PubMed id: 16916523 DOI: 10.1016/j.jmb.2006.06.059

Date:

12-Dec-05 Release date: 03-Oct-06

Protein chains

P04418  (END5_BPT4) -  Endonuclease V from Enterobacteria phage T4

Seq: 138 a.a.

Struc: 137 a.a.

DNA/RNA chains

C-C-A-G-G-A-PED-G-A-A-G-C-C 13 bases

G-G-C-BRU-BRU-C-A-BRU-C-C-BRU-G-G 13 bases

C-C-A-G-G-A-PED-G-A-A-G-C-C 13 bases

G-G-C-BRU-BRU-C-A-BRU-C-C-BRU-G-G 13 bases

Enzyme reactions

• Enzyme class 2: E.C.3.2.2.17 - deoxyribodipyrimidine endonucleosidase.
• Reaction: Cleaves the N-glycosidic bond between the 5'-pyrimidine residue in cyclobutadipyrimidine (in DNA) and the corresponding deoxy-D-ribose residue.
• Enzyme class 3: E.C.4.2.99.18 - DNA-(apurinic or apyrimidinic site) lyase.
• Reaction: 2'-deoxyribonucleotide-(2'-deoxyribose 5'-phosphate)- 2'-deoxyribonucleotide-DNA = a 3'-end 2'-deoxyribonucleotide-(2,3- dehydro-2,3-deoxyribose 5'-phosphate)-DNA + a 5'-end 5'-phospho- 2'-deoxyribonucleoside-DNA + H⁺

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

DOI no: 10.1016/j.jmb.2006.06.059

J Mol Biol 362:241-258 (2006)

PubMed id: 16916523

Structure of T4 pyrimidine dimer glycosylase in a reduced imine covalent complex with abasic site-containing DNA.

G.Golan, D.O.Zharkov, A.P.Grollman, M.L.Dodson, A.K.McCullough, R.S.Lloyd, G.Shoham.

ABSTRACT

The base excision repair (BER) pathway for ultraviolet light (UV)-induced cyclobutane pyrimidine dimers is initiated by DNA glycosylases that also possess abasic (AP) site lyase activity. The prototypical enzyme known to catalyze these reactions is the T4 pyrimidine dimer glycosylase (T4-Pdg). The fundamental chemical reactions and the critical amino acids that lead to both glycosyl and phosphodiester bond scission are known. Catalysis proceeds via a protonated imine covalent intermediate between the alpha-amino group of the N-terminal threonine residue and the C1' of the deoxyribose sugar of the 5' pyrimidine at the dimer site. This covalent complex can be trapped as an irreversible, reduced cross-linked DNA-protein complex by incubation with a strong reducing agent. This active site trapping reaction is equally efficient on DNA substrates containing pyrimidine dimers or AP sites. Herein, we report the co-crystal structure of T4-Pdg as a reduced covalent complex with an AP site-containing duplex oligodeoxynucleotide. This high-resolution structure reveals essential precatalytic and catalytic features, including flipping of the nucleotide opposite the AP site, a sharp kink (approximately 66 degrees ) in the DNA at the dimer site and the covalent bond linking the enzyme to the DNA. Superposition of this structure with a previously published co-crystal structure of a catalytically incompetent mutant of T4-Pdg with cyclobutane dimer-containing DNA reveals new insights into the structural requirements and the mechanisms involved in DNA bending, nucleotide flipping and catalytic reaction.

Selected figure(s)

Figure 2.
Figure 2. A schematic presentation of T4-Pdg interactions with the covalently bound DNA oligonucleotide. Nucleotides are numbered beginning from the AP site (position 0, dRbl), positive numbers towards the 5′-end, with superscript in parentheses for the complementary strand. A^(0) is the flipped-out base opposite the AP site. Hydrogen bonds are shown as arrows pointing towards their respective acceptors. Arg22, Glu23 and Arg26 are inserted into the intrahelical void. In addition to hydrogen bonds, van der Waals contacts are formed with nearby DNA residues. Bromo-uracil bases are shown here as the structurally equivalent thymine bases, for clarity.

Figure 3.
Figure 3. The conformation of the DNA duplex in the T4-Pdg/DNA complex. (a) A stereoview of the AP-site containing DNA 13-mer in the covalent complex. The protein and solvent molecules are not shown for clarity. The DNA is viewed into the kink (rotated about 90° along the DNA axis from Figure 1(a)) focusing on the intrahelical void formed as a result of the binding. The strand containing the AP site is shown in cyan (dRbl in yellow), while the opposite strand is shown in pink (flipped-out Ade^(0) in blue). Note the normal base stacking away from the lesion, except for the last two bases at the bottom, where dimerization contacts take place. (b) A similar stereoview of the DNA from the non-covalent complex.^33 The presentation and orientation are identical to (a) to allow a meaningful comparison of the two DNA conformations.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2006, 362, 241-258) copyright 2006.

Figures were selected by an automated process.