Reverse gyrase is a type I DNA topoisomerase able to positively supercoil DNA and is found in thermophiic archaebacteria and eubacteria. The gene coding for this protein was cloned from Sulfolobus acidocaldarius DSM 639. Analysis of the 1247-amino acid sequence and comparison of it with available sequence data suggest that reverse gyrase is constituted of two distinct domains: (i) a C-terminal domain of -630 amino acids clearly related to eubacterial topoisomerase I (Escherichia coli topA and topB gene products) and to Saccharomyces cerevisiae top3; (ii) an N-terminal domain without any similarity to other known topoisomerases but containing several helicase motifs, including an ATP-binding site. These results are consistent with those from our previous mechanistic studies of reverse gyrase and suggest a model in which positive supercoiling is driven by the concerted action of helicase and topoisomerase in the same polypeptide: this constitutes an example of a composite gene formed by a helicase domain and a topoisomerase domain.It is today well established that topoisomerases play a crucial role in DNA structure and function (1, 2). These enzymes seem to act in two ways: (i) they are able to solve the topological problems intrinsic to the DNA double helix during replication, transcription, recombination, or chromatin condensation and decondensation; (ii) some topoisomerases have exploited the circular or pseudocircular (chromatin loops) structure of the genetic material to introduce stress into DNA by means of supercoiling. The enzymes specialized in the production of superhelical turns in a circular DNA were named "gyrases" (3, 4). Among these, the best characterized enzyme is the eubacterial gyrase, a type II topoisomerase that uses the energy of ATP hydrolysis to maintain the in vivo level of negative supercoiling of the bacterial chromosome (5). Several years ago, another kind of DNA supercoiling activity was discovered in hyperthermophilic archaebacteria (6) and was called "reverse gyrase." We have shown that reverse gyrase is widely distributed in various archaebacterial families and also in thermophilic eubacteria (7,8). This enzyme has the specific ability to catalyze in vitro positive supercoiling of a closed circular DNA at high temperature (9). Surprisingly, reverse gyrase turned out to be a type I topoisomerase (9) and is the only topoisomerase I that depends on ATP and can catalyze DNA supercoiling. The biological function of reverse gyrase is still unclear, but we found that positive supercoiling occurs in vivo (10). The enzyme from Sulfolobus acidocaldarius is composed of a single polypeptide of apparent molecular mass of -130,000 (11). Mechanistic studies (12) indicated that reverse gyrase transiently cleaves a single DNA strand, forming a covalent link with the 5' end of the broken strand, as described for eubacterial topoisomerase I (13). In addition, we have shown that stoichiometric binding of the enzyme changes the DNA conformation, presumably by unwinding the double helix (12). We ...

UVA (320 -400 nm) radiation constitutes >90% of the environmentally relevant solar UV radiation, and it has been proposed to have a role in skin cancer and aging. Because of the popularity of UVA tanning beds and prolonged periods of sunbathing, the potential deleterious effect of UVA has emerged as a source of concern for public health. Although generally accepted, the impact of DNA damage on the cytotoxic, mutagenic, and carcinogenic effect of UVA radiation remains unclear. In the present study, we investigated the sensitivity of a panel of yeast mutants affected in the processing of DNA damage to the lethal and mutagenic effect of UVA radiation. The data show that none of the major DNA repair pathways, such as base excision repair, nucleotide excision repair, homologous recombination, and postreplication repair, efficiently protect yeast from the lethal action of UVA radiation. In contrast, the results show that the Ogg1 DNA glycosylase efficiently prevents UVA-induced mutagenesis, suggesting the formation of oxidized guanine residues. Furthermore, sequence analysis of UVA-induced canavanine-resistant mutations reveals a bias in favor of GC3 TA events when compared with spontaneous or H2O2-, UVC-, and ␥-ray-induced canavanine-resistant mutations in the WT strain. Taken together, our data point out a major role of oxidative DNA damage, mostly 7,8-dihydro-8-oxoguanine, in the genotoxicity of UVA radiation in the yeast Saccharomyces cerevisiae. Therefore, the capacity of skin cells to repair 7,8-dihydro-8-oxoguanine may be a key parameter in the mutagenic and carcinogenic effect of UVA radiation in humans.base excision repair ͉ OGG1 ͉ mutagenesis ͉ DNA photolesions U VA radiation has been proposed to have a role in skin cancer and aging (1). UVA (320-400 nm) constitutes Ͼ90% of the environmentally relevant solar UV radiation. Because of the popularity of the high-intensity UVA tanning equipment and the widespread use of efficient UVB-absorbing sunscreens blocking erythema and often accompanied by prolonged periods of sunbathing, the human exposure to UVA have increased significantly in the last decades. The deleterious effect of UVA has, as a consequence, recently emerged as a source of concern for public health (2, 3).Although very complex, the biological effect of UVA radiation is at least partially related to DNA damage. Despite it being well established that UVA can damage DNA, to date, the nature of the lesions underlying its mutagenic and carcinogenic effects remains controversial. Unlike UVB photons, which are directly absorbed by DNA and cause the formation of cis-syn cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts, the UVA component of solar radiation is poorly absorbed by DNA. Rather, UVA radiation excites other endogenous chromophores, generating reactive oxygen species, some of which are possibly involved in the outcome of photocarcinogenesis (4, 5). It is generally accepted that UVA damages DNA, either through the generation of singlet oxygen ( 1 O 2 ) or by a type...