The Role of Leucine 191 of Escherichia coliUracil DNA Glycosylase in the Formation of a Highly Stable Complex with the Substrate Mimic, Ugi, and in Uracil Excision from the Synthetic Substrates

Abstract

motif (DNA intercalation loop). We show that with the decrease in side chain length at position 191, the stability of the UDG-Ugi complexes regresses. Further, while the L191V and L191F mutants were as efficient as the wild type protein, the L191A and L191G mutants retained only 10 and 1% of the enzymatic activity, respectively. Importantly, however, substitution of Leu 191 with smaller side chains had no effect on the relative efficiencies of uracil excision from the single-stranded and a corresponding doublestranded substrate. Our results suggest that leucine within the HPSPLS motif is crucial for the uracil excision activity of UDG, and it contributes to the formation of a physiologically irreversible complex with Ugi. We also envisage a role for Leu 191 in stabilizing the productive enzyme-substrate complex.

Uracil in DNA – occurrence, consequences and repair

“…More recent data, however, indicate that the function of the inserting leucine side chain may be more complex than merely pushing the uracil out of the double helix. When analysing kinetic parameters of E. coli Ung L191A against single-stranded substrates, a 15-fold reduction in k cat /K m was observed compared to the wild type (Handa et al, 2001;Jiang and Stivers, 2001), which would not be expected in the absence of stabilizing base pairing. A likely explanation for this could be that the leucine side chain rather functions as a 'doorstop' to prevent return of the flipped out uracil residue, as recently suggested by Wong et al (2002).…”

Complexes of the uracil-DNA glycosylase inhibitor protein, Ugi, with Mycobacterium smegmatis and Mycobacterium tuberculosis uracil-DNA glycosylases

“…Also, as seen from the presence of free Ugi in the samples not treated with urea (Fig. 4a, lanes 1, 3 and 10), expression of Ugi from the bicistronic constructs occurred in slight molar excess to UDG (Handa et al, 2001). …”

“…The purification of complexes of Ugi with Eco-, Mtu-and MsmUDGs was carried out by a protocol described by using T7 RNA polymerase-based expression constructs in E. coli BL21 (DE3). Similarly, EcoUDG, MtuUDG and Ugi were also purified from T7 RNA polymerasebased hyperexpression constructs using standard protocols (Handa et al, 2001;. The purified proteins were estimated by Bradford's reagent using bovine serum albumin as standard (Sedmak & Grossberg, 1977).…”

“…The UDG side chains of Q63, D64, Y66, H67, Q71, S88 and S189 (numbered according to EcoUDG), and the main chain atoms of A133, S (or T)166, H187 and P190 are among the highly conserved residues in E. coli and human UDGs that establish direct or watermediated hydrogen bonds with various atoms in Ugi. In addition, another highly conserved interaction between UDG and Ugi is established through the insertion of sidechain residue L191 (or F191) into the hydrophobic pocket of Ugi, which contributes significantly to the stability of the UDG-Ugi complex (Handa et al, 2001). In Msm-and MtuUDGs, while most of these interacting residues are conserved, the equivalents of H67, Q71, A133 and S166 are represented by P, H, P and R, respectively.…”

“…In a complementary approach, we have carried out detailed mutational analysis of EcoUDG to understand the role of L191 in interaction with the hydrophobic pocket of Ugi (Handa et al, 2001). However, mutations in the side chains of UDGs that interface the b1-edge of Ugi have not yet been studied in detail (Handa et al, 2002).…”