Structural basis for poor uracil excision from hairpin DNA

Abstract

Two-dimensional NMR and molecular dynamics simulations have been used to determine the three-dimensional structures of two hairpin DNA structures: d-CTAGAG GATCCUTTTGGATCCT (abbreviated as U1-hairpin) and d-CTAGAGGATCCTTUTGGATCCT (abbreviated as U3-hairpin). The 1 H resonances of both of these hairpin structures have been assigned almost completely. NMR restrained molecular dynamics and energy minimization procedures have been used to describe the three-dimensional structures of these hairpins. This study and concurrent NMR structural studies on two other d-CTAGAGGA TCCTUTTGGATCCT (abbreviated as U2-hairpin) and d-CTAGAGGATCCTTTUGGATCCT (abbreviated as U4-hairpin) have shed light upon various interactions reported between Echerichia coli uracil DNA glycosylase (UDG) and uracil-containing DNA. The backbone torsion angles, which partially influence the local conformation of U12 and U14 in U1 and U3-hairpins, respectively, are probably locked in the trans conformation as in the case of U 13 in the U2-hairpin. Such a stretched-out backbone conformation in the vicinity of U 12 and U 14 is thought to be the reason why the K m value is poor for U1-and U3-hairpins as it is for the U2-hairpin. Furthermore, the bases U 12 and U 14 in both U1-and U3-hairpins adopt an anti conformation, in contrast with the base conformation of U 13 in the U2-hairpin, which adopts a syn conformation. The clear discrepancy observed in the U-base orientation with respect to the sugar moieties could explain why the V max value is 10-to 20-fold higher for the U1-and U3-hairpins compared with the U2-hairpin. Taken together, these observations support our interpretation that the unfavourable backbone results in a poor K m value, whereas the unfavourable nucleotide conformation results in a poor V max value. These two parameters therefore make the U1-and U3-hairpins better substrates for UDG compared with the U2-hairpin, as reported earlier [Kumar, N.

A rapid reaction analysis of uracil DNA glycosylase indicates an active mechanism of base flipping

“…We therefore conclude that the large differences in the rates of base opening are due to differences in the protein–nucleic acid interface. This is supported by previous studies that have observed large K M effects when the target uracil is placed within the unusual DNA structure of a hairpin (40,41). The logical conclusion therefore is that protein–DNA interactions are critical in uracil eversion that requires active engagement of the UNG enzyme with the DNA.…”

Toward a Detailed Understanding of Base Excision Repair Enzymes: Transition State and Mechanistic Analyses of N-Glycoside Hydrolysis and N-Glycoside Transfer

“…Cytosine deamination in DNA to uracil occurs 100−500 times per human cell per day, generating G:U mismatches, which become transition mutations to A:T if the damaged DNA is replicated . Misincorporation of dUTP can also occur, generating A:U mismatches during DNA replication. , Apart from TS analysis of eUDG, the catalytic mechanisms of eUDG, hUDG, and hsvUDG have been elucidated in detail by NMR, ,,− X-ray crystallography, ,,,,− Raman spectroscopy, mutational analysis, ,,,,,− inhibitor studies, ,,, and computation. , These studies have addressed how UDG searches DNA for uracil residues, the conformational changes involved in flipping the target residue into the active site, plus studies of the chemical steps. Many of the mechanistic findings have been reviewed in detail , and are outlined briefly below.…”

NMR Structures of Damaged DNA

“…To understand the modulation of UDG activity by local DNA conformation, a series of NMR studies have determined the structure of dU-containing hairpins (Chart ), observing similar stem structures but with marked conformational differences at the loop region. − The RMD structure of the U4 hairpin ( 52d ) (PDB code 1QE7) shows the uracil base stacked between the 3‘- and 5‘-flanking residues and B I -DNA values for the sugar−phosphate backbone angles at the loop region . Conversely, in the refined structure of the U2 ( 52b ) hairpin (PDB code 1DGO) where UDG uracil incision is retarded the most, dU and its 3‘-flanking residue adopt syn conformations around the glycosidic torsion angle and the α, γ, and ζ backbone dihedral angles of the loop assume the unusual trans conformation .…”

“…Conversely, in the refined structure of the U2 ( 52b ) hairpin (PDB code 1DGO) where UDG uracil incision is retarded the most, dU and its 3‘-flanking residue adopt syn conformations around the glycosidic torsion angle and the α, γ, and ζ backbone dihedral angles of the loop assume the unusual trans conformation . RMD structures of the U1 ( 52a ) and U3 ( 52c ) (PDB codes 1II1 and 1IDX, respectively), that are incised by UDG somewhat more efficiently than the U2 hairpin, show all loop residues in anti coformations and a trans conformation for backbone dihedral angles of the dU residue . On the basis of these observations and of X-ray data available for E. coli UDG, the authors proposed that a stretched out backbone conformation of the dU nucleotide reduces the affinity of the scissile residue for UDG.…”