No abstract

Tuberous sclerosis is an autosomal dominant trait characterized by the development of hamartomatous growths in many organs. Renal cysts are also a frequent manifestation. Major genes for tuberous sclerosis and autosomal dominant polycystic kidney disease, TSC2 and PKD1, respectively, lie adjacent to each other at chromosome 16p13.3, suggesting a role for PKD1 in the etiology of renal cystic disease in tuberous sclerosis. We studied 27 unrelated patients with tuberous sclerosis and renal cystic disease. Clinical histories and radiographic features were reviewed, and renal function was assessed. We sought mutations at the TSC2 and PKD1 loci, using pulsed field- and conventional-gel electrophoresis and FISH. Twenty-two patients had contiguous deletions of TSC2 and PKD1. In 17 patients with constitutional deletions, cystic disease was severe, with early renal insufficiency. One patient with deletion of TSC2 and of only the 3' UTR of PKD1 had few cysts. Four patients were somatic mosaics; the severity of their cystic disease varied considerably. Mosaicism and mild cystic disease also were demonstrated in parents of 3 of the constitutionally deleted patients. Five patients without contiguous deletions had relatively mild cystic disease, 3 of whom had gross rearrangements of TSC2 and 2 in whom no mutation was identified. Significant renal cystic disease in tuberous sclerosis usually reflects mutational involvement of the PKD1 gene, and mosaicism for large deletions of TSC2 and PKD1 is a frequent phenomenon.

Repair of oxidative damage to DNA bases is essential to prevent mutations and cell death. Endonuclease III is the major DNA glycosylase activity in Escherichia coli that catalyzes the excision of pyrimidines damaged by ring opening or ring saturation, and it also possesses an associated lyase activity that incises the DNA backbone adjacent to apurinic͞apyrimidinic sites. During analysis of the area adjacent to the human tuberous sclerosis gene (TSC2) in chromosome region 16p13.3, we identified a gene, OCTS3, that encodes a 1-kb transcript. Analysis of OCTS3 cDNA clones revealed an open reading frame encoding a predicted protein of 34.3 kDa that shares extensive sequence similarity with E. coli endonuclease III and a related enzyme from Schizosaccharomyces pombe, including a conserved active site region and an iron͞sulfur domain. The product of the OCTS3 gene was therefore designated hNTH1 (human endonuclease III homolog 1). The hNTH1 protein was overexpressed in E. coli and purified to apparent homogeneity. The recombinant protein had spectral properties indicative of the presence of an iron͞sulfur cluster, and exhibited DNA glycosylase activity on double-stranded polydeoxyribonucleotides containing urea and thymine glycol residues, as well as an apurinic͞ apyrimidinic lyase activity. Our data indicate that hNTH1 is a structural and functional homolog of E. coli endonuclease III, and that this class of enzymes, for repair of oxidatively damaged pyrimidines in DNA, is highly conserved in evolution from microorganisms to human cells.Reactive oxygen species generated either as a by-product of cellular respiration or by ionizing radiation and other oxidizing agents can cause extensive damage to DNA bases and disrupt the phosphodiester backbone. Unrepaired oxygen radicalderived DNA lesions have been implicated as a causative factor in both cancer and aging (1, 2). Repair of most forms of oxidative DNA damage is mediated via the base excisionrepair pathway (3, 4), which has been well characterized in Escherichia coli. One of the key components of the base excision-repair pathway in that organism is endonuclease III, the product of the nth gene (5). The crystal structure of this iron͞sulfur protein has been determined recently at 1.85 Å resolution (6, 7).Although originally isolated as an activity that incised at certain sites of DNA damage generated by ultraviolet (UV) irradiation in a Mg 2ϩ -independent reaction (8), endonuclease III was subsequently shown to act primarily as a DNA glycosylase that hydrolyses the N-glycosyl bond linking damaged pyrimidines to the DNA backbone. Numerous different substrates for endonuclease III have been identified representing saturated, opened, or contracted pyrimidine rings, such as thymine and cytosine glycol, urea, and N-substituted urea, 5-hydroxy-5-methylhydantoin, and 5,6-dihydrothymine (9-11). The originally observed DNA nicking activity associated with endonuclease III was subsequently shown to result not from hydrolytic cleavage of phosphodiester bonds, but from ␤-e...