SUMMARY The mitochondrion is the primary source of reactive oxygen species (ROS) in eukaryotic cells. With the aid of a novel mitochondrial matrix-targeted superoxide indicator, here we show that individual mitochondria undergo spontaneous bursts of superoxide generation, termed “superoxide flashes”. Superoxide flashes occur randomly in space and time, exhibit all-or-none properties, and reflect elementary events of superoxide production within single mitochondria across a wide diversity of cells. Individual flashes are triggered by transient openings of the mitochondrial permeability transition pore (mPTP) and are fueled by electron transfer complexes-dependent superoxide production. While decreased during cardiac hypoxia/anoxia, a flurry of superoxide flash activity contributes to the destructive rebound ROS burst observed during early reoxygenation after anoxia. The discovery of superoxide flashes reveals a novel mechanism for quantal ROS production by individual mitochondria and substantiates the central role of mPTP in oxidative stress related pathology in addition to its well-known role in apoptosis.

Although CDKN2A is the most frequent high-risk melanoma susceptibility gene, the underlying genetic factors for most melanoma-prone families remain unknown. Using whole exome sequencing, we identified a rare variant that arose as a founder mutation in the telomere shelterin POT1 gene (g.7:124493086 C>T, Ser270Asn) in five unrelated melanoma-prone families from Romagna, Italy. Carriers of this variant had increased telomere length and elevated fragile telomeres suggesting that this variant perturbs telomere maintenance. Two additional rare POT1 variants were identified in all cases sequenced in two other Italian families, yielding a frequency of POT1 variants comparable to that of CDKN2A mutations in this population. These variants were not found in public databases or in 2,038 genotyped Italian controls. We also identified two rare recurrent POT1 variants in American and French familial melanoma cases. Our findings suggest that POT1 is a major susceptibility gene for familial melanoma in several populations.

BLM encodes a member of the highly conserved RecQ DNA helicase family, which is essential for the maintenance of genome stability. Homozygous inactivation of BLM gives rise to the cancer predisposition disorder Bloom's syndrome. A common feature of many RecQ helicase mutants is a hyperrecombination phenotype. In Bloom's syndrome, this phenotype manifests as an elevated frequency of sister chromatid exchanges and interhomologue recombination. We have shown previously that BLM, together with its evolutionarily conserved binding partner topoisomerase III␣ (hTOPO III␣), can process recombination intermediates that contain double Holliday junctions into noncrossover products by a mechanism termed dissolution. Here we show that a recently identified third component of the human BLM͞hTOPO III␣ complex, BLAP75͞ RMI1, promotes dissolution catalyzed by hTOPO III␣. This activity of BLAP75͞RMI1 is specific for dissolution catalyzed by hTOPO III␣ because it has no effect in reactions containing either Escherichia coli Top1 or Top3, both of which can also catalyze dissolution in a BLM-dependent manner. We present evidence that BLAP75͞RMI1 acts by recruiting hTOPO III␣ to double Holliday junctions. Implications of the conserved ability of type IA topoisomerases to catalyze dissolution and how the evolution of factors such as BLAP75͞RMI1 might confer specificity on the execution of this process are discussed.Bloom's syndrome ͉ Holliday junction dissolution ͉ topoisomerase III ͉ sister chromatid exchanges T he RecQ family of DNA helicases is essential for the maintenance of genome stability (1). The human genome contains five RecQ helicase genes. Mutations in three of these genes give rise to clinically defined cancer predisposition disorders (2). One of these disorders is Bloom's syndrome (BS), which is caused by biallelic mutations in the BLM gene (3). The BLM protein is a 3Ј-5Ј DNA helicase that processes a broad range of structurally diverse DNA substrates (4-7). These substrates include DNA structures that arise during homologous recombination, such as D-loops and Holliday junctions (5, 6). These structures are of particular relevance to the BS phenotype because BS cells display elevated levels of homologous recombination (8). This hyperrecombination phenotype is also a feature of Saccharomyces cerevisiae and Schizosaccharomyces pombe mutants defective in their respective BLM orthologs, SGS1 and rqh1 ϩ (9-11). In the case of BS cells, recombination events are particularly apparent between sister chromatids, and such recombination events are termed sister chromatid exchanges (SCEs) (8). These exchanges arise primarily as a consequence of crossing-over during the processing of recombination intermediates (12).BLM exists in a complex with topoisomerase III␣ (hTOPO III␣), a type IA topoisomerase (13,14). This complex is evolutionarily conserved, and functional and͞or physical interactions between RecQ helicases and type IA topoisomerases have also been demonstrated in bacteria and yeast (9, 15-17). Two type IA topoisomerases ar...