Cloning and characterization of hOGG1, a human homolog of the OGG1 gene of Saccharomyces cerevisiae

Abstract

The OGG1 gene of Saccharomyces cerevisiae encodes a DNA glycosylase activity that is a functional analog of the Fpg protein from Escherichia coli and excises 7,8-dihydro-8-oxoguanine (8-oxoG) from damaged DNA. The repair of this ubiquitous kind of oxidative damage is essential to prevent mutations both in bacteria and in yeast. A human cDNA clone carrying an ORF displaying homology to the yeast protein was identified. The predicted protein has 345 amino acids and a molecular mass of 39 kDa. This protein shares a 38% sequence identity with the yeast Ogg1 protein, adding this novel human gene product to the growing family of enzymes that the repair of oxidatively damaged bases and are related to the E. coli endonuclease III. Northern blot analysis indicates that this gene, localized to chromosome 3p25, is ubiquitously expressed in human tissues. The cloned coding sequence was expressed in an E. coli strain that carried a disrupted fpg gene, the bacterial functional analog of OGG1. Cell-free extracts from these cultures displayed a specific lyase activity on duplex DNA that carried an 8-oxoG/C base pair. The products of the reaction are consistent with an enzymatic activity like the one displayed by the yeast Ogg1. Analysis of the substrate specificity reveals a very strong preference for DNA fragments harboring 8-oxoG/C base pairs. The pattern of specificity correlates well with the one found for the yeast enzyme. Moreover, when the human coding sequence was expressed in a yeast strain mutant in OGG1 it was able to complement the spontaneous mutator phenotype. These results make this novel gene (hOGG1) a strong candidate for the human homolog of the yeast OGG1 and suggest an important role of its product in the protection of the genome from the mutagenic effects of the oxidatively damaged purines.

Figure 1

Cloning of a human homolog of OGG1. (A) Schematic representation of the sources of cDNA used to assemble the complete clone. The open reading frame is indicated by an arrow. (B) Alignment of the encoded protein with the S. cerevisiae Ogg1 protein. Positions with identical residues are enclosed in black boxes.

Figure 2

Northern blot analysis of human mRNA from various tissues. The same blot was hybridized with a hOGG1 probe (Upper) or a β-actin probe (Lower).

Figure 3

Chromosome localization of the hOGG1 gene determined by FISH. (a) Example of hybridization signals of twin dots on human chromosome 3. (b) Ideogram of human chromosome 3 showing localization of the hOGG1 gene on 3p25 based on analysis of metaphases with twin dots.

Figure 4

Cleavage of the 8-oxoG/C duplex by crude extracts of E. coli fpg. The sequences of the 34-mer oligonucleotides containing a single 8-oxoG are reported in Materials and Methods. The 8-oxoG/C duplex was incubated either with cell-free extracts or purified Fpg or Ogg1 protein. The two extremities of the 8-oxoG-containing strand were ³²P-labeled, and, consequently, two oligonucleotides were generated after cleavage. The products of the reaction were analyzed by denaturing 20% PAGE containing 7 M urea. PPRD1 is the 3′ oligonucleotide [5′P-(N17)-3′-N-³²P-5′], and PPRD2 is the 5′ oligonucleotide [5′-³²P-(N15)-3′-P], which are generated by chemical cleavage of the 34-mer oligonucleotide at 8-oxoG by 1 M piperidine at 90°C for 60 min or by the Fpg protein. The product P3 corresponds to PPRD2 with a deoxyribose residue attached at its 3′ end. (A) Cell-free extracts (30 μg of protein) obtained from cultures of pPR221 (fpg) hosting the vector pTrc99A (lane 2) or pPR55 (lanes 3 and 4). For lane 4, the culture was treated with 1 mM IPTG before lysis. (B) Comparison of the products of the 8-oxoG/C incision by crude extracts from PR221 cells expressing hOgg1 (lane 6), purified Fpg protein (2 ng) (lane 7), or purified Ogg1 (5 ng) (lane 8). Lanes 1 and 5 correspond to the untreated 34-mer oligonucleotide.

Figure 5

Cleavage of DNA duplexes containing a single 8-oxoG mismatched with one of the four DNA bases by cell-free extracts of E. coli fpg cells expressing hOgg1. The sequences of the 34-mer oligonucleotides containing 8-oxoG or G at position 16 are reported in Materials and Methods. The 8-oxoG or G₁₆ containing strands were labeled and annealed with one of the four complementary sequences carrying A, T, C, or G opposite to the 8-oxoG or G₁₆. These substrates were incubated with cell-free extracts (30 μg of protein) of PR221 hosting pPR55 induced with 1 mM IPTG. The analysis of the products of the reaction was as described in the legend of Fig. 4.