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3-Nitrobenzanthrone (3-nitro-7H-benz[d,e]anthracen-7-one, 3-NBA) is a powerful nurtagen and a suspected human carcinogen existing in diesel exhaust and airborne particulates. Recently, one of the major presumed metabolites of 3-NBA, 3-aminobenzanthrone (3-ABA), was detected in human urine samples. Here we analyzed DNA adducts fon-ned in 3-NBA-exposed human hepatoma HepG2 cells by a P-32-postlabeling/thin layer chromatography (TLC) method and a P-32-postlabeling/polyacrylamide gel electrophoresis (PAGE) method. With HepG2 cells exposed to 3-NBA (0.36-36.4 mu M) for 3h, we obtained three spots or bands corresponding to adducted nucleotides. Two were assigned as 2-(2 '-deoxyadenosin-N-6-yl)-3-aminobenzanthrone-3 '-phosphate (dA3 ' p-N-6-C2-ABA) and 2-(2 '-deoxyguanosin-N-2-yl)-3-aminobenzanthrone-3 '-phosphate (dG3 ' p-N-2-C2-ABA), with identical mobilities to those of synthetic standards on PAGE analysis. The chemical structure of the substance corresponding to the other spot or band could not be identified. Quantitative analyses revealed that the major adduct was dA3 ' p-N-6-C2-ABA and its relative adduct labeling (RAL) value at 36.4 mu M of 3-NBA was 200.8 +/- 86.1/10(8) nucleotide. (c) 2007 Elsevier B.V. All rights reserved.

Sister chromatid cohesion, formed by the cohesin protein complex, is essential for chromosome segregation. In order for cohesion to be established, the cohesin subunit SMC3 needs to be acetylated by a homolog of the ESCO1/Eco1 acetyltransferases, the enzymatic mechanism of which has remained unknown. Here we report the crystal structure of the ESCO1 acetyltransferase domain in complex with acetyl-coenzyme A, and show by SAXS that ESCO1 is a dimer in solution. The structure reveals an active site that lacks a potential catalytic base side chain. However, mutation of glutamate 789, a surface residue that is close to the automodification target lysine 803, strongly reduces autoacetylation of ESCO1. Moreover, budding yeast Smc3 mutated at the conserved residue D114, adjacent to the cohesion-activating acetylation site K112,K113, cannot be acetylated in vivo. This indicates that ESCO1 controls cohesion through substrate-assisted catalysis. Thus, this study discloses a key mechanism for cohesion establishment.

The SUMO ligase Mms21, which is a subunit of the Smc5/6 complex, is required for DNA repair. Here we present results showing that Mms21 was phosophorylated during S-phase in a manner dependent on the DNA damage kinase Mec1. Phosphorylation of Mms21 occurred in unchallenged cells, but was more abundant in the presence of DNA damaging agents. Mass spectrometry identified five phosphorylated serines organized in two regions of Mms21, and two C-terminal serines, S260 and S261, formed part of a Mec1/Tel1 consensus motif. Nonphosphorylatable substitutions of the C-terminal serines, inactivation of Mec1 or removal of the Mms21 C-terminus all abolished Mms21 phosphorylation. Additionally, strains carrying Mms21 phosphoablative alleles displayed reduced SUMO ligase activity, sensitivity to MMS and an increased rate of chromosome loss in the presence of MMS. We propose that one function of S260 S261 phosphorylation is to positively regulate the SUMO ligase activity of Mms21 and thereby promote genomic stability.

3-Nitrobenzanthrone (3-NBA) is a potent mutagen and potential human carcinogen identified in diesel exhaust and ambient air particulate matter. Previously, we detected the formation of 3-NBA-derived DNA adducts in rodent tissues by 32P-postlabeling, all of which are derived from reductive metabolites of 3-NBA bound to purine bases, but structural identification of these adducts has not yet been reported. We have now prepared 3-NBA-derived DNA adduct standards for 32P-postlabeling by reacting N-acetoxy-3-aminobenzanthrone (N-Aco-ABA) with purine nucleotides. Three deoxyguanosine (dG) adducts have been characterised as N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone-3'-phosphate (dG3'p-C8-N-ABA), 2-(2'-deoxyguanosin-N2-yl)-3-aminobenzanthrone-3'-phosphate (dG3'p-N2-ABA) and 2-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone-3'-phosphate (dG3'p-C8-C2-ABA), and a deoxyadenosine (dA) adduct was characterised as 2-(2'-deoxyadenosin-N6-yl)-3-aminobenzanthrone-3'-phosphate (dA3'p-N6-ABA). 3-NBA-derived DNA adducts formed experimentally in vivo and in vitro were compared with the chemically synthesised adducts. The major 3-NBA-derived DNA adduct formed in rat lung cochromatographed with dG3'p-N2-ABA in two independent systems (thin layer and high-performance liquid chromatography). This is also the major adduct formed in tissue of rats or mice treated with 3-aminobenzanthrone (3-ABA), the major human metabolite of 3-NBA. Similarly, dG3'p-C8-N-ABA and dA3'p-N6-ABA cochromatographed with two other adducts formed in various organs of rats or mice treated either with 3-NBA or 3-ABA, whereas dG3'p-C8-C2-ABA did not cochromatograph with any of the adducts found in vivo. Utilizing different enzymatic systems in vitro, including human hepatic microsomes and cytosols, and purified and recombinant enzymes, we found that a variety of enzymes [NAD(P)H:quinone oxidoreductase, xanthine oxidase, NADPH:cytochrome P450 oxidoreductase, cytochrome P450s 1A1 and 1A2, N,O-acetyltransferases 1 and 2, sulfotransferases 1A1 and 1A2, and myeloperoxidase] are able to catalyse the formation of 2-(2'-deoxyguanosin-N2-yl)-3-aminobenzanthrone, N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone and 2-(2'-deoxyadenosin-N6-yl)-3-aminobenzanthrone in DNA, after incubation with 3-NBA and/or 3-ABA.

The cohesin complex, which is essential for sister chromatid cohesion and chromosome segregation, also inhibits resolution of sister chromatid intertwinings (SCIs) by the topoisomerase Top2. The cohesin-related Smc5/6 complex (Smc5/6) instead accumulates on chromosomes after Top2 inactivation, known to lead to a buildup of unresolved SCIs. This suggests that cohesin can influence the chromosomal association of Smc5/6 via its role in SCI protection. Using high-resolution ChIP-sequencing, we show that the localization of budding yeast Smc5/6 to duplicated chromosomes indeed depends on sister chromatid cohesion in wild-type and top2-4 cells. Smc5/6 is found to be enriched at cohesin binding sites in the centromere-proximal regions in both cell types, but also along chromosome arms when replication has occurred under Top2-inhibiting conditions. Reactivation of Top2 after replication causes Smc5/6 to dissociate from chromosome arms, supporting the assumption that Smc5/6 associates with a Top2 substrate. It is also demonstrated that the amount of Smc5/6 on chromosomes positively correlates with the level of missegregation in top2-4, and that Smc5/6 promotes segregation of short chromosomes in the mutant. Altogether, this shows that the chromosomal localization of Smc5/6 predicts the presence of the chromatid segregation-inhibiting entities which accumulate in top2-4 mutated cells. These are most likely SCIs, and our results thus indicate that, at least when Top2 is inhibited, Smc5/6 facilitates their resolution., Author Summary When cells divide, sister chromatids have to be segregated away from each other for the daughter cells to obtain a correct set of chromosomes. Using yeast as model organism, we have analyzed the function of the cohesin and the Smc5/6 complexes, which are essential for chromosome segregation. Cohesin is known to hold sister chromatid together until segregation occurs, and our results show that cohesin also controls Smc5/6, which is found to associate to linked chromatids specifically. In line with this, our analysis points to that the chromosomal localization of Smc5/6 is an indicator of the level of entanglement between sister chromatids. When Smc5/6 is non-functional, the resolution of these entanglements is shown to be inhibited, thereby preventing segregation of chromatids. Our results also indicate that DNA entanglements are maintained on chromosomes at specific sites until segregation. In summary, we uncover new functions for cohesin, in regulating when and where Smc5/6 binds to chromosomes, and for the Smc5/6 complex in facilitating the resolution of sister chromatid entanglements.