DNA apurinic-apyrimidinic site binding and excision by endonuclease IV

Abstract

Escherichia coli endonuclease IV is an archetype for an abasic or apurinic-apyrimidinic endonuclease superfamily crucial for DNA base excision repair. Here biochemical, mutational and crystallographic characterizations reveal a three-metal ion mechanism for damage binding and incision. The 1.10-A resolution DNA-free and the 2.45-A resolution DNA-substrate complex structures capture substrate stabilization by Arg37 and reveal a distorted Zn3-ligand arrangement that reverts, after catalysis, to an ideal geometry suitable to hold rather than release cleaved DNA product. The 1.45-A resolution DNA-product complex structure shows how Tyr72 caps the active site, tunes its dielectric environment and promotes catalysis by Glu261-activated hydroxide, bound to two Zn2+ ions throughout catalysis. These structural, mutagenesis and biochemical results suggest general requirements for abasic site removal in contrast to features specific to the distinct endonuclease IV alpha-beta triose phosphate isomerase (TIM) barrel and APE1 four-layer alpha-beta folds of the apurinic-apyrimidinic endonuclease families.

Alternative Excision Repair Pathways

“…It should be noted that UVDE also recognizes AP sites, platinum G -G diadducts, uracil, dihydrouracil, and other oxidative DNA-damage products and introduces a nick immediately 5 0 to the lesions (Avery et al 1999;Kanno et al 1999). A crystal structure of UVDE derived from the thermophilic bacterium Thermus thermophilus was solved (Paspaleva et al 2007) and showed essential structural features of a TIM-barrel fold shared with that of Endo IV (Hosfield et al 1999;Garcin et al 2008). Although both enzymes share some characteristic features of a DNA-binding groove, where both the damaged bases and their opposite bases are thought to flip out of the double helix into the binding groove, the predicted residues in the structure of UVDE make a larger space for the flipped-out UV lesions than in Endo IV.…”

Uncoupling of the apyrimidinic/apurinic endonucleolytic and 3′→5′ exonucleolytic activities of the Nfo protein of Mycoplasma pneumoniae through mutation of specific amino acid residues

“…The data were qualified as '2' (,5 %), '+/2' (5-10 %), '+' (10-50 %), '++' (50-80 %) and '+++' (.80 %). The Nfo Eco counterparts of the targeted amino acid residues were found to interact with no, one or two of the three divalent cations (Zn 1 , Zn 2 and Zn 3 ) that were identified previously in Nfo Eco 's active site (Garcin et al, 2008;Hosfield et al, 1999 In addition to the damage-specific endoncleolytic activities of Nfo Mpn and Nfo Mge , we also found these proteins to possess non-specific 39A59 exonucleolytic activity. Such activity has previously also been attributed to other Nfo proteins (Back et al, 2006;Kerins et al, 2003;Liu et al, 2007).…”

“…Both Mg 2+ and Mn 2+ , however, did stimulate the proteins' activities. Similarly, the wild-type Nfo Eco protein was stimulated by Mn 2+ , indicating that this cation may be favoured over Zn 2+ at one of the three 'Zn 2+ ' sites (Garcin et al, 2008); on the basis of crystallographic and biochemical analyses, it is likely that this is the Zn 3 site (Garcin et al, 2008).…”

“…These residues include H72, H112, E147, D181, H184, H215, D228, H230 and E260 of Nfo Mpn . The Nfo Eco counterparts of these highly conserved residues have been demonstrated to be involved in the interaction with divalent cations in the active site of the protein (Garcin et al, 2008;Hosfield et al, 1999).…”

“…Most of these residues were shown to interact with divalent cations (Zn 1 , Zn 2 and Zn 3 ) in the protein's active site (Garcin et al, 2008;Hosfield et al, 1999). To establish the role of the Nfo Mpn counterparts of these conserved residues in AP site cleavage and 39A59 exonucleolytic activity, we substituted these residues as well as other highly conserved acidic residues (in particular, Glu and Asp residues) to produce a set of 13 point mutants of Nfo Mpn .…”