The repair of DNA double-strand breaks (DSBs) is critical for maintaining genome stability. Eukaryotic cells repair DSBs using both non-homologous end joining (NHEJ) and homologous recombination (HR). How chromatin structure is altered in response to DSBs and how such alterations influence DSB repair processes are important questions. In vertebrates, phosphorylation of the histone variant H2A.X (γ-H2A) occurs rapidly after formation of DSBs 1 , spreads over megabase chromatin domains, and is required for stable accumulation of DNA repair proteins at DNA damage foci 2 . In Saccharomyces cerevisiae, phosphorylation of the two major H2A species is also signaled by DSB formation, spreading ∼40 Kb in either direction from a DSB 3 . Here we show that near a DSB γ-H2A is followed by loss of histones H2B and H3 and increased sensitivity of chromatin to digestion by micrococcal nuclease. However, γ-H2A and nucleosome loss occur independently of one another. The DNA damage sensor MRX (Mre11-Rad50-Xrs2) 4 is required for histone eviction, which additionally depends on the ATP-dependent nucleosome-remodeling complex, INO80 5 . The repair protein Rad51 6 shows delayed recruitment to a DSB in the absence of histone loss, suggesting that MRX-dependent nucleosome remodeling regulates the accessibility of factors with direct roles in DNA damage repair by HR. We propose that MRX regulates two pathways of chromatin changes, including nucleosome displacement, required for efficient recruitment of HR proteins, and γ-H2A, which modulates checkpoint responses to DNA damage 2 .To elucidate the chromatin pathways leading to DSB repair in Saccharomyces cerevisiae, we employed a MATα haploid strain that lacks HMR and HML donor sequences and carries a galactose-inducible HO gene 7 . In this strain, HO endonuclease introduces a DSB at MAT that can only be repaired by NHEJ, although the major HR proteins are recruited to the break site 6 . We analyzed chromatin structure along 12-20 Kb encompassing the DSB by chromatin immunoprecipitation (ChIP) followed by real-time PCR, which provided sensitive measurement of the kinetics and spatial distribution of chromatin changes and recruitment of repair proteins around the break site.Budding yeast H2A is phosphorylated on serine 129 by the ATM/ATR homologs Tel1/ Mec1 8 . In agreement with a recent report 3 , we found that γ-H2A accumulated rapidly and extensively on either side of the DSB, and that γ-H2A levels were lower close to the DSB relative to 6 Kb distant (Figure 1a; Supplementary Figure 3a). These latter results suggested a loss in nucleosome integrity near the DSB. The nucleosome consists of 146 bp of DNA wrapped ∼two times around a histone octamer comprising an (H3/H4) 2 tetramer and two H2A/H2B dimers. To determine if nucleosome stability was altered at the DSB, we performed ChIP in strains expressing either Flag-H2B or Flag-H3. The levels of both histones decreased 60-90 min after HO induction and were reduced three-fold by 120 min (Figure 1a) loss of both histones suggests that ...

Covalent modifications of the histone N tails play important roles in eukaryotic gene expression. Histone acetylation, in particular, is required for the activation of a subset of eukaryotic genes through the targeted recruitment of histone acetyltransferases. We have reported that a histone C tail modification, ubiquitylation of H2B, is required for optimal expression of several inducible yeast genes, consistent with a role in transcriptional activation. H2B was shown to be ubiquitylated and then deubiquitylated at the GAL1 core promoter following galactose induction. We now show that the Rad6 protein, which catalyzes monoubiquitylation of H2B, is transiently associated with the GAL1 promoter upon gene activation, and that the period of its association temporally overlaps with the period of H2B ubiquitylation. Rad6 promoter association depends on the Gal4 activator and the Rad6-associated E3 ligase, Bre1, but is independent of the histone acetyltransferase, Gcn5. The SAGA complex, which contains a ubiquitin protease that targets H2B for deubiquitylation, is recruited to the GAL1 promoter in the absence of H2B ubiquitylation. The data suggest that Rad6 and SAGA function independently during galactose induction, and that the staged recruitment of these two factors to the GAL1 promoter regulates the ubiquitylation and deubiquitylation of H2B. We additionally show that both Rad6 and ubiquitylated H2B are absent from two regions of transcriptionally silent chromatin but present at genes that are actively transcribed. Thus, like histone H3 lysine 4 and lysine 79 methylation, two modifications that it regulates, Rad6-directed H2B ubiquitylation defines regions of active chromatin.

DNA fragments that function as autonomously replicating sequences (ARSs) have been isolated from Ustilago maydis. When inserted into an integrative transforming vector, the fragments increased the frequency of U. maydis transformation several-thousandfold. ARS-containing plasmids were transmitted in U. maydis as extrachromosomal elements through replication. They were maintained at a level of about 25 copies per cell but were mitotically unstable. One ARS characterized in detail, which we called UARS1, was localized to a 1.7-kilobase fragment. UARS1 contained a cluster of active sequences. This element could be reduced further into three separate subfragments, each of which retained ARS activity. The smallest one was 383 base pairs (bp) long. Although not active itself in yeast, this small fragment contained seven 8-bp direct repeats, two contiguous 30-bp direct repeats, and five 11-bp units in both orientations with sequences similar but not identical to the consensus sequence found to be crucial for ARS activity in Saccharomyces cerevisiae.Fragments have been isolated from yeast genomic DNA that supply nonreplicating plasmids with the ability to replicate autonomously in Saccharomyces cerevisiae (3,9,31). The frequency of occurrence of these genetic elements, or autonomously replicating sequences (ARSs), in the genome is in line with the number of origins of replication estimated from electron microscopy and DNA fiber autoradiography (see reference 35 for review). For several years these observations were used to support the notion that the ARS is equivalent to an origin of replication (29). Any uncertainty over this question was eliminated recently by the direct demonstration by two-dimensional gel electrophoretic analysis that the ARS element functions as a replication origin (6,20). ARSs were first identified as sequences linked to genetic markers that transformed yeast cells at high frequencies (18,31). Plasmid DNA lacking an ARS can transform yeast cells only by rare integrative recombination into the cellular genome (15). In contrast, the transformation frequency may be 103 to 104 times higher when the plasmid is carrying an ARS. This differential transformation frequency has been used as a diagnostic measure for defining ARS sequences operationally.Our interest in ARS elements stems from our efforts to establish a high-frequency transformation system in the basidiomycete fungus Ustilago maydis as a means for isolating and cloning genes. A few years ago, Banks (1) reported transformation of U. maydis to antibiotic G418 resistance by using a plasmid containing a yeast 2,um DNA origin and the bacterial resistance factor aminoglycoside phosphotransferase gene. Preliminary evidence suggested that the plasmid was maintained in transformants by autonomous replication, but the transformation frequency was low, around 10 per microgram of DNA. More recently, Leong and colleagues (34) developed a pUC12 derivative plasmid containing a bacterial phosphotransferase gene conferring resistance to the antibiotic hygromy...

The organization of eukaryotic DNA into chromatin poses a barrier to all processes that require access of enzymes and regulatory factors to their sites of action. While the majority of studies in this area have concentrated on the role of chromatin in the regulation of transcription, there has been a recent emphasis on the relationship of chromatin to DNA damage repair. In this review, we focus on the role of chromatin in nucleotide excision repair (NER) and double-strand break (DSB) repair. NER and DSB repair use very different enzymatic machineries, and these two modes of DNA damage repair are also differentially affected by chromatin. Only a small number of nucleosomes are likely to be involved in NER, while a more extensive region of chromatin is involved in DSB repair. However, a key feature of both NER and DSB repair pathways is the participation of ATP-dependent chromatin remodeling factors at various points in the repair process. We discuss recent data that have identified roles for SWI/SNF-related chromatin remodeling factors in the two repair pathways.

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Thermus strain SA-01, previously isolated from a deep (3.2 km) South African gold mine, is closely related to Thermus strains NMX2 A.1 and VI-7 (previously isolated from thermal springs in New Mexico, USA, and Portugal, respectively). Thermus strains SA-01 and NMX2 A.1 have also been shown previously to grow using nitrate, Fe(III), Mn(IV) or S(O) as terminal electron acceptors and to be capable of reducing Cr(VI), U(VI), Co(III), and the quinone-containing compound anthraquinone-2,6-disulfonate. The objectives of this study were to determine the phylogenetic positions of the three known metal-reducing Thermus strains and to determine the phylogenetic significance of metal reduction within the genus Thermus. Phylogenetic analyses of 16S rDNA sequences, BOX PCR genomic fingerprinting, and DNA-DNA reassociation analyses indicated that these strains belong to the previously described genospecies T. scotoductus. The morphologies and lipid fatty acid profiles of these metal-reducing strains are consistent with their identification as T. scotoductus; however, the T. scotoductus strains tested in this study evinced a wide intraspecies variability in some other phenotypic traits, e.g., carbon substrate utilization and pigmentation. Iron reduction occurred in all strains of T. scotoductus tested except the mixotrophic, sulfur-oxidizing strain IT-7254. Thermus strains belonging to other species did not reduce Fe(III) to Fe(II) or reduced it only poorly.

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