Friedreich's ataxia is the most common inherited ataxia. Ninety‐six percent of patients are homozygous for GAA trinucleotide repeat expansions in the first intron of the frataxin gene. The remaining cases are compound heterozygotes for a GAA expansion and a frataxin point mutation. We report here the identification of 10 novel frataxin point mutations, and the detection of a previously described mutation (G130V) in two additional families. Most truncating mutations were in exon 1. All missense mutations were in the last three exons coding for the mature frataxin protein. The clinical features of 25 patients with identified frataxin point mutations were compared with those of 196 patients homozygous for the GAA expansion. A similar phenotype resulted from truncating mutations and from missense mutations in the carboxy‐terminal half of mature frataxin, suggesting that they cause a comparable loss of function. In contrast, the only two missense mutations located in the amino‐terminal half of mature frataxin (D122Y and G130V) cause an atypical and milder clinical presentation (early‐onset spastic gait with slow disease progression, absence of dysarthria, retained or brisk tendon reflexes, and mild or no cerebellar ataxia), suggesting that they only partially affect frataxin function. The incidence of optic disk pallor was higher in compound heterozygotes than in expansion homozygotes, which might correlate with a very low residual level of normal frataxin produced from the expanded allele. Ann Neurol 1999;45:200–206

Sir1p is one of four SIR (silent information regulator) proteins required for silencing the cryptic mating-type locus HMRa in the budding yeast Saccharomyces cerevisiae. A Sir1p interaction with Orc1p, the largest subunit of the origin recognition complex (ORC), is critical for Sir1p's ability to bind HMRa and function in the formation of silent chromatin. Here we show that a discrete domain within Sir1p, the ORC interaction region (OIR), was necessary and sufficient for a Sir1p-ORC interaction. The OIR contains the originally defined silencer recognition-defective region as well as additional amino acids. In addition, a Sir1p-Sir4p interaction required a larger region of Sir1p that included the OIR. Amino acid substitutions causing defects in either a Sir1p-Orc1p or a Sir1p-Sir4p interaction reduced HMRa silencing and Sir1p binding to HMRa in chromatin. These data support a model in which Sir1p's association with HMRa is mediated by separable Sir1p-ORC and Sir1p-Sir4p interactions requiring a common Sir1p domain, and they indicate that a Sir1p-ORC interaction is restricted to silencers, at least in part, through interactions with Sir4p.

The origin recognition complex (ORC) marks chromosomal positions as replication origins and is essential for replication initiation. At a few loci, the ORC functions in heterochromatin formation. We show that the ORC's two roles at the heterochromatic HMRa locus in Saccharomyces cerevisiae were regulated by differences in the ORC's interaction with its target site. At HMRa, a strong ORC-DNA interaction inhibited and delayed replication initiation but promoted heterochromatin formation, whereas a weak ORC-DNA interaction allowed for increased and earlier replication initiation but reduced heterochromatin formation. Therefore, the ORC's interaction with its target site could modulate ORC activity within a heterochromatin domain in vivo.Supplemental material is available at http://www.genesdev.org. The origin recognition complex (ORC) is essential for genome replication in eukaryotes and functions by binding to specific chromosomal positions and recruiting additional proteins essential for origin firing (Bell 2002). Because each chromosome requires the activity of many replication origins for its duplication, ORCs function at hundreds of positions distributed throughout the genome. Although ORCs function at all origins, individual origins vary in activity. Some initiate replication efficiently and early during S phase, whereas others initiate in only a small fraction of cell cycles and later during S phase (Friedman et al. 1997;Yamashita et al. 1997;Polomienko et al. 2001). In budding yeast, a small subset of origins, termed silencers, is associated with the formation of a specialized chromatin structure that represses transcription . Significantly, a role for the ORC in repressive chromatin is conserved in metazoans (Pak et al. 1997).Silencing of HMRa in Saccharomyces cerevisiae is a form of transcription repression requiring a specialized chromatin structure called silent chromatin that is similar to heterochromatin (Pillus and Grunstein 1995;Rusche et al. 2003). Like heterochromatin, silent chromatin causes gene-independent, position-dependent transcription repression. Both heterochromatin and silent chromatin contain relatively hypoacetylated nucleosomes as well as specialized nonhistone chromatinbinding proteins that help assemble a repressive chromatin domain encompassing many kilobase pairs of DNA. In addition, silent chromatin, like many forms of heterochromatin, is replicated late during S phase (Raghuraman et al. 2001).DNA elements bound by sequence-specific DNAbinding proteins target specialized chromatin structures to specific chromosomal domains. At HMRa, the critical DNA element is the 150-bp HMR-E silencer that contains a binding site for the ORC (A-element) as well as a single binding site for each of the abundant nuclear proteins, Rap1p and Abf1p (Loo and Rine 1995). Together, silencer-binding proteins nucleate the assembly of silenced chromatin by direct physical interactions with nonhistone chromatin-binding proteins called Sirs, which, in turn, recruit additional Sir proteins that modify and bin...

The origin recognition complex (ORC) marks chromosomal sites as replication origins and is essential for replication initiation. In yeast, ORC also binds to DNA elements called silencers, where its primary function is to recruit silent information regulator (SIR) proteins to establish transcriptional silencing. Indeed, silencers function poorly as chromosomal origins. Several genetic, molecular, and biochemical studies of HMR-E have led to a model proposing that when ORC becomes limiting in the cell (such as in the orc2-1 mutant) only sites that bind ORC tightly (such as HMR-E) remain fully occupied by ORC, while lower affinity sites, including many origins, lose ORC occupancy. Since HMR-E possessed a unique non-replication function, we reasoned that other tight sites might reveal novel functions for ORC on chromosomes. Therefore, we comprehensively determined ORC “affinity” genome-wide by performing an ORC ChIP–on–chip in ORC2 and orc2-1 strains. Here we describe a novel group of orc2-1–resistant ORC–interacting chromosomal sites (ORF–ORC sites) that did not function as replication origins or silencers. Instead, ORF–ORC sites were comprised of protein-coding regions of highly transcribed metabolic genes. In contrast to the ORC–silencer paradigm, transcriptional activation promoted ORC association with these genes. Remarkably, ORF–ORC genes were enriched in proximity to origins of replication and, in several instances, were transcriptionally regulated by these origins. Taken together, these results suggest a surprising connection among ORC, replication origins, and cellular metabolism.

Silencing of the mating-type locus HMR in Saccharomyces cerevisiae requires DNA elements called silencers. To establish HMR silencing, the origin recognition complex binds the HMR-E silencer and recruits the silent information regulator (Sir)1 protein. Sir1 in turn helps establish silencing by stabilizing binding of the other Sir proteins, Sir2-4. However, silencing is semistable even in sir1⌬ cells, indicating that SIR1-independent establishment mechanisms exist. Furthermore, the requirement for SIR1 in silencing a sensitized version of HMR can be bypassed by high-copy expression of FKH1 (FKH1 hc ), a conserved forkhead transcription factor, or by deletion of the S phase cyclin CLB5 (clb5⌬). FKH1 hc caused only a modest increase in Fkh1 levels but effectively reestablished Sir2-4 chromatin at HMR as determined by Sir3-directed chromatin immunoprecipitation. In addition, FKH1 hc prolonged the cell cycle in a manner distinct from deletion of its close paralogue FKH2, and it created a cell cycle phenotype more reminiscent to that caused by a clb5⌬. Unexpectedly, and in contrast to SIR1, both FKH1 hc and clb5⌬ established silencing at HMR using the replication origins, ARS1 or ARSH4, as complete substitutes for HMR-E (HMR⌬E::ARS). HMR⌬E::ARS1 was a robust origin in CLB5 cells. However, initiation by HMR⌬E::ARS1 was reduced by clb5⌬ or FKH1 hc , whereas ARS1 at its native locus was unaffected. The CLB5-sensitivity of HMR⌬E::ARS1 did not result from formation of Sir2-4 chromatin because sir2⌬ did not rescue origin firing in clb5⌬ cells. These and other data supported a model in which FKH1 and CLB5 modulated Sir2-4 chromatin and late-origin firing through opposing regulation of a common pathway. INTRODUCTIONChromatin structures vary with genome position, creating structural heterogeneity along chromosomes that modulates every aspect of DNA metabolism (Fischle et al., 2003). Specific DNA sequence elements form the foundation for this heterogeneity. For example, certain DNA sequences act to establish centromeric chromatin required for chromosome segregation (Cleveland et al., 2003;Henikoff and Dalal, 2005). In addition, some chromatin structures, such as heterochromatin, can dominate the functional capacity of DNA sequence elements across chromosomal domains or even over an entire chromosome. Heterochromatin, for example, can delay or repress initiation by DNA replication origins and initiation of transcription from promoters (Gomez and Brockdorff, 2004;Weinreich et al., 2004;Chang et al., 2006). Thus, a competing balance exists between DNA sequence elements that perform different functions or establish different types of chromatin structures (Kamakaka, 1997;Donze and Kamakaka, 2002;Valenzuela and Kamakaka, 2006).Genetic analyses of transcriptional silencing of the HMRa locus in Saccharomyces cerevisiae reveal that perturbations in the cell cycle tip the balance between the types of chromatin that can form at a particular chromosomal domain (Laman et al., 1995;Fox and Rine, 1996;Ehrenhofer-Murray et al., 1999).HMRa sile...