Trinucleotide repeats can form secondary structures, whose inappropriate repair or replication can lead to repeat expansions. There are multiple loci within the human genome where expansion of trinucleotide repeats leads to disease. Although it is known that expanded repeats accumulate double-strand breaks (DSBs), it is not known which DSB repair pathways act on such lesions and whether inaccurate DSB repair pathways contribute to repeat expansions. Using Saccharomyces cerevisiae, we found that CAG/CTG tracts of 70 or 155 repeats exhibited significantly elevated levels of breakage and expansions in strains lacking MRE11, implicating the Mre11/Rad50/Xrs2 complex in repairing lesions at structure-forming repeats. About two-thirds of the expansions that occurred in the absence of MRE11 were dependent on RAD52, implicating aberrant homologous recombination as a mechanism for generating expansions. Expansions were also elevated in a sae2 deletion background and these were not dependent on RAD52, supporting an additional role for Mre11 in facilitating Sae2-dependent hairpin processing at the repeat. Mre11 nuclease activity and Tel1-dependent checkpoint functions were largely dispensable for repeat maintenance. In addition, we found that intact homologous recombination and nonhomologous end-joining pathways of DSB repair are needed to prevent repeat fragility and that both pathways also protect against repeat instability. We conclude that failure of principal DSB repair pathways to repair breaks that occur within the repeats can result in the accumulation of atypical intermediates, whose aberrant resolution will then lead to CAG expansions, contractions, and repeat-mediated chromosomal fragility.

The histone chaperone Rtt106 binds histone H3 acetylated at lysine 56 (H3K56ac) and facilitates nucleosome assembly during several molecular processes. Both the structural basis of this modificationspecific recognition and how this recognition informs Rtt106 function are presently unclear. Guided by our crystal structure of Rtt106, we identified two regions on its double-pleckstrin homology domain architecture that mediated histone binding. When histone binding was compromised, Rtt106 localized properly to chromatin but failed to deliver H3K56ac, leading to replication and silencing defects. By mutating analogous regions in the structurally homologous chromatin-reorganizer Pob3, we revealed a conserved histone-binding function for a basic patch found on both proteins. In contrast, a loop connecting two β-strands was required for histone binding by Rtt106 but was dispensable for Pob3 function. Unlike Rtt106, Pob3 histone binding was modification-independent, implicating the loop of Rtt106 in H3K56ac-specific recognition in vivo. Our studies described the structural origins of Rtt106 function, identified a conserved histone-binding surface, and defined a critical role for Rtt106:H3K56ac-binding specificity in silencing and replication-coupled nucleosome turnover.histone acetylation | yFACT | CAF-1 | Sir | Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the histone chaperone Rtt106 binds newly synthesized histone proteins and mediates their delivery into chromatin during transcription, replication, and silencing. Rtt106 is also recruited to histone gene regulatory regions by the HIR histone chaperone complex to ensure S-phase-specific expression. Here we showed that this Rtt106:HIR complex included Asf1 and histone proteins. Mutations in Rtt106 that reduced histone binding reduced Rtt106 enrichment at histone genes, leading to their increased transcription. Deletion of the chromatin boundary element Yta7 led to increased Rtt106:H3 binding, increased Rtt106 enrichment at histone gene regulatory regions, and decreased histone gene transcription at the HTA1-HTB1 locus. These results suggested a unique regulatory mechanism in which Rtt106 sensed the level of histone proteins to maintain the proper level of histone gene transcription. The role of these histone chaperones and Yta7 differed markedly among the histone gene loci, including the two H3-H4 histone gene pairs. Defects in silencing in rtt106 mutants could be partially accounted for by Rtt106-mediated changes in histone gene repression. These studies suggested that feedback mediated by histone chaperone complexes plays a pivotal role in regulating histone gene transcription. Cell cycle-regulated transcription of the canonical histone genes is a hallmark of eukaryotic organisms. During S phase, a coordinated burst of histone gene transcription is required to double the level of histone proteins to package newly replicated DNA into chromatin (13,22). The tight coupling of both the timing and the level of histone gene expression with DNA synthesis is critical for cell viability. Mutations that perturb DNA replication lead to altered histone gene transcription (3, 23). Conversely, misregulation of the timing and/or level of histone gene expression leads to genomic instability and cell cycle defects (23,43). Consequently, cells have evolved a complex regulatory mechanism to repress histone gene transcription outside S phase and to promote a precise level of transcription during S phase.The Saccharomyces cerevisiae genome contains two nonallelic copies of each canonical histone gene which are organized in head-to-head pairs of H2A-H2B (HTA1-HTB1 and HTA2-HTB2) and H3-H4 (HHT1-HHF1 and HHT2-HHF2). Nucleosomes, the fundamental units of chromatin structure, contain two H2A/H2B dimers, one H3/H4 tetramer, and ϳ147 bp of DNA wrapped around the outer surface (37). Repression of histone transcription outside S phase is maintained by the HIR H3/H4 histone-protein chaperone complex, referred to here as the HIR complex (Hir1, Hir2, Hir3, and Hpc2). The HIR complex localizes to negative (NEG) sequence elements within the regulatory regions of HTA1-HTB1, 45,59). Although HTA2-HTB2 transcription is S-phase specific, its promoter does not contain NEG elements, and the factors involved in its HIRindependent repression are unknown (45). A recent study implicated two additional H3/H4 chaperones, Rtt106 ...

Methods for altering the sequence of endogenous Drosophila melanogaster genes remain labor-intensive. We have tested a relatively simple strategy that enables the introduction of engineered mutations in the vicinity of existing P-elements. This method was used to generate useful alleles of the roX1 gene, which produces a noncoding RNA involved in dosage compensation. The desired change was first introduced into a genomic clone of roX1 and transgenic flies were generated that carry this sequence in a P-element. Targeted transposition was then used to move the P-element into roX1. Remobilization of the targeted insertion produced large numbers of offspring carrying chromosomes that had precisely introduced the engineered sequences into roX1. We postulate that this occurred by gap repair, using the P-element on the sister chromatid as template. This strategy was used to introduce six MS2 loops into the roX1 gene (roX1MS2-6), enabling detection of roX1 RNA by a MCP-GFP fusion protein in embryos. The roX1MS2-6 remains under the control of the authentic promoter and within the correct genomic context, features expected to contribute to normal roX1 function. The ability to replace relatively large blocks of sequence suggests that this method will be of general use.