Uracil-DNA glycosylase–DNA substrate and product structures: Conformational strain promotes catalytic efficiency by coupled stereoelectronic effects

Enzymatic transformations of macromolecular substrates such as DNA repair enzyme/DNA transformations are commonly interpreted primarily by active-site functional-group chemistry that ignores their extensive interfaces. Yet human uracil–DNA glycosylase (UDG), an archetypical enzyme that initiates DNA base-excision repair, efficiently excises the damaged base uracil resulting from cytosine deamination even when active-site functional groups are deleted by mutagenesis. The 1.8-Å resolution substrate analogue and 2.0-Å resolution cleaved product cocrystal structures of UDG bound to double-stranded DNA suggest enzyme–DNA substrate-binding energy from the macromolecular interface is funneled into catalytic power at the active site. The architecturally stabilized closing of UDG enforces distortions of the uracil and deoxyribose in the flipped-out nucleotide substrate that are relieved by glycosylic bond cleavage in the product complex. This experimentally defined substrate stereochemistry implies the enzyme alters the orientation of three orthogonal electron orbitals to favor electron transpositions for glycosylic bond cleavage. By revealing the coupling of this anomeric effect to a delocalization of the glycosylic bond electrons into the uracil aromatic system, this structurally implicated mechanism resolves apparent paradoxes concerning the transpositions of electrons among orthogonal orbitals and the retention of catalytic efficiency despite mutational removal of active-site functional groups. These UDG/DNA structures and their implied dissociative excision chemistry suggest biology favors a chemistry for base-excision repair initiation that optimizes pathway coordination by product binding to avoid the release of cytotoxic and mutagenic intermediates. Similar excision chemistry may apply to other biological reaction pathways requiring the coordination of complex multistep chemical transformations.

Human PEX1 cloned by functional complementation on a CHO cell mutant is responsible for peroxisome-deficient Zellweger syndrome of complementation group I

The peroxisome biogenesis disorders (PBDs), including Zellweger syndrome (ZS) and neonatal adrenoleukodystrophy (NALD), are autosomal recessive diseases caused by defects in peroxisome assembly, for which at least 10 complementation groups have been reported. We have isolated a human PEX1 cDNA (HsPEX1) by functional complementation of peroxisome deficiency of a mutant Chinese hamster ovary (CHO) cell line, ZP107, transformed with peroxisome targeting signal type 1-tagged “enhanced” green fluorescent protein. This cDNA encodes a hydrophilic protein (Pex1p) comprising 1,283 amino acids, with high homology to the AAA-type ATPase family. A stable transformant of ZP107 with HsPEX1 was morphologically and biochemically restored for peroxisome biogenesis. HsPEX1 expression restored peroxisomal protein import in fibroblasts from three patients with ZS and NALD of complementation group I (CG-I), which is the highest-incidence PBD. A CG-I ZS patient (PBDE-04) possessed compound heterozygous, inactivating mutations: a missense point mutation resulting in Leu-664 → Pro and a deletion of the sequence from Gly-634 to His-690 presumably caused by missplicing (splice site mutation). Both PBDE-04 PEX1 cDNAs were defective in peroxisome-restoring activity when expressed in the patient fibroblasts as well as in ZP107 cells. These results demonstrate that PEX1 is the causative gene for CG-I peroxisomal disorders.

Functional analysis of DNA bending and unwinding by the high mobility group domain of LEF-1

LEF-1 (lymphoid enhancer-binding factor 1) is a cell type-specific member of the family of high mobility group (HMG) domain proteins that recognizes a specific nucleotide sequence in the T cell receptor (TCR) α enhancer. In this study, we extend the analysis of the DNA-binding properties of LEF-1 and examine their contributions to the regulation of gene expression. We find that LEF-1, like nonspecific HMG-domain proteins, can interact with irregular DNA structures such as four-way junctions, albeit with lower efficiency than with specific duplex DNA. We also show by a phasing analysis that the LEF-induced DNA bend is directed toward the major groove. In addition, we find that the interaction of LEF-1 with a specific binding site in circular DNA changes the linking number of DNA and unwinds the double helix. Finally, we identified two nucleotides in the LEF-1-binding site that are important for protein-induced DNA bending. Mutations of these nucleotides decrease both the extent of DNA bending and the transactivation of the TCRα enhancer by LEF-1, suggesting a contribution of protein-induced DNA bending to the function of TCRα enhancer.

Cloning and functional expression of a cDNA encoding a pheromone gland-specific acyl-CoA Δ11-desaturase of the cabbage looper moth, Trichoplusia ni

Desaturation of coenzyme-A esters of saturated fatty acids is a common feature of sex pheromone biosynthetic pathways in the Lepidoptera. The enzymes that catalyze this step share several biochemical properties with the ubiquitous acyl-CoA Δ9-desaturases of animals and fungi, suggesting a common ancestral origin. Unlike metabolic acyl-CoA Δ9-desaturases, pheromone desaturases have evolved unusual regio- and stereoselective activities that contribute to the remarkable diversity of chemical structures used as pheromones in this large taxonomic group. In this report, we describe the isolation of a cDNA encoding a pheromone gland desaturase from the cabbage looper moth, Trichoplusia ni, a species in which all unsaturated pheromone products are produced via a Δ11Z-desaturation mechanism. The largest ORF of the ≈1,250-bp cDNA encodes a 349-aa apoprotein (PDesat-Tn Δ11Z) with a predicted molecular mass of 40,240 Da. Its hydrophobicity profile is similar overall to those of rat and yeast Δ9-desaturases, suggesting conserved transmembrane topology. A 182-aa core domain delimited by conserved histidine-rich motifs implicated in iron-binding and catalysis has 72 and 58% similarity (including conservative substitutions) to acyl-CoA Δ9Z-desaturases of rat and yeast, respectively. Northern blot analysis revealed an ≈1,250-nt PDesat-Tn Δ11Z mRNA that is consistent with the spatial and temporal distribution of Δ11-desaturase enzyme activity. Genetic transformation of a desaturase-deficient strain of the yeast Saccharomyces cerevisiae with an expression plasmid encoding PDesat-Tn Δ11Z resulted in complementation of the strain’s fatty acid auxotrophy and the production of Δ11Z-unsaturated fatty acids.

Functional complementation of xeroderma pigmentosum complementation group E by replication protein A in an in vitro system.

Xeroderma pigmentosum (XP) is caused by a defect in nucleotide excision repair. Patients in the complementation group E (XP-E) have the mildest form of the disease and the highest level of residual repair activity. About 20% of the cell strains derived from XP-E patients lack a damaged DNA-binding protein (DDB) activity that binds to ultraviolet-induced (6-4) photoproducts with high affinity. We report here that cell-free extracts prepared from XP-E cell strains that either lacked or contained DDB activity were severely defective in excising DNA damage including (6-4) photoproducts. However, this excision activity defect was not restored by addition of purified DDB that, in fact, inhibited removal of (6-4) photoproducts by the human excision nuclease reconstituted from purified proteins. Extensive purification of correcting activity from HeLa cells revealed that the correcting activity is inseparable from the human replication/repair protein A [RPA (also known as human single stranded DNA binding protein, HSSB)]. Indeed, supplementing XP-E extracts with recombinant human RPA purified from Escherichia coli restored excision activity. However, no mutation was found in the genes encoding the three subunits of RPA in an XP-E (DDB-) cell line. It is concluded that RPA functionally complements XP-E extracts in vitro, but it is not genetically altered in XP-E patients.