The origin recognition complex (ORC) is an initiator protein for DNA replication, but also effects transcriptional silencing in Saccharomyces cerevisiae and heterochromatin function in Drosophila. It is not known, however, whether any of these functions of ORC is conserved in mammals. We report the identification of a novel protein, HBO1 (histone acetyltransferase binding to ORC), that interacts with human ORC1 protein, the largest subunit of ORC. HBO1 exists as part of a multisubunit complex that possesses histone H3 and H4 acetyltransferase activities. A fraction of the relatively abundant HBO1 protein associates with ORC1 in human cell extracts. HBO1 is a member of the MYST domain family that includes S. cerevisiae Sas2p, a protein involved in control of transcriptional silencing that also has been genetically linked to ORC function. Thus the interaction between ORC and a MYST domain acetyltransferase is widely conserved. We suggest roles for ORC-mediated acetylation of chromatin in control of both DNA replication and gene expression. The origin recognition complex (ORC)1 is a key protein for the initiation of DNA replication in eukaryotes. Initially identified in Saccharomyces cerevisiae (1), ORC is a multisubunit protein composed of six polypeptides that binds to yeast replication origins in vivo and in vitro and is essential for the initiation of DNA replication (2). Homologues of S. cerevisiae ORC subunits have been identified in Schizosaccharomyces pombe, Drosophila melanogaster, Arabidopsis thaliana, Xenopus laevis, and human cells (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). Xenopus ORC is necessary for chromosome replication in egg extracts (7,8,(15)(16)(17)(18). Moreover, Drosophila ORC2 is required for chromosome replication in diploid cells and chorion gene amplification in anuploid ovarian follicle cells (6, 19). These observations suggest an evolutionary conserved role for ORC in DNA replication.In addition to its role in initiation of DNA replication in S. cerevisiae, ORC has separable functions in S and M phase (20) and an interesting function in transcriptional silencing of the mating type loci (21-27). ORC binds to specific, cis-acting silencer elements that are adjacent to the HMRa and HML␣ silent mating-type genes (21)(22)(23)(24)28). To facilitate this function, the budding yeast silencing protein Sir1p has been shown to associate with ORC via an interaction with Orc1p (29, 30). Sir1p has been proposed to stabilize a heterochromatin-like protein complex containing ORC, Rap1p, Sir2p, Sir3p, and Sir4p, which represses gene expression in a heritable manner (31).In Drosophila, transcriptional silencing and position effect variegation of gene expression occurs when chromosomal rearrangements bring genes in close proximity to heterochromatin (32). This silencing requires the participation of a structural component of heterochromatin known as HP1 (33). Interestingly, Drosophila ORC (dORC) binds to HP1, and heterozygous, recessive lethal mutations in ORC2 cause a suppression of position effect varie...

The initiation of DNA replication is tightly regulated in eukaryotic cells to ensure that the genome is precisely duplicated once and only once per cell cycle. This is accomplished by controlling the assembly of a prereplicative complex (pre-RC) which involves the sequential binding to replication origins of the origin recognition complex (ORC), Cdc6/Cdc18, Cdt1, and the minichromosome maintenance complex (Mcm2-Mcm7, or Mcm2-7). Several mechanisms of pre-RC regulation are known, including ATP utilization, cyclindependent kinase levels, protein turnover, and Cdt1 binding by geminin. Histone acetylation may also affect the initiation of DNA replication, but at present neither the enzymes nor the steps involved are known. Here, we show that Hbo1, a member of the MYST histone acetyltransferase family, is a previously unrecognized positive regulatory factor for pre-RC assembly. When Hbo1 expression was inhibited in human cells, Mcm2-7 failed to associate with chromatin even though ORC and Cdc6 loading was normal. When Xenopus egg extracts were immunodepleted of Xenopus Hbo1 (XHbo1), chromatin binding of Mcm2-7 was lost, and DNA replication was abolished. The binding of Mcm2-7 to chromatin in XHbo1-depleted extracts could be restored by the addition of recombinant Cdt1.

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Summary• Fructan is the major nonstructural carbohydrate reserve in temperate grasses. To understand regulatory mechanisms in fructan synthesis and adaptation to cold environments, the isolation, functional characterization and genetic mapping of fructosyltransferase (FT) genes in perennial ryegrass (Lolium perenne) are described.• Six cDNAs (prft1-prft6) encoding FTs were isolated from cold-treated ryegrass plants, and three were positioned on a perennial ryegrass linkage map. Recombinant proteins were produced in Pichia pastoris and enzymatic activity was characterized. Changes in carbohydrate levels and mRNA levels of FT genes during cold treatment were also analysed.• One gene encodes sucrose-sucrose 1-fructosyltransferase (1-SST), and two gene encode fructan-fructan 6G-fructosyltransferase (6G-FFT). Protein sequences for the other genes (prfts 1, 2 and 6) were similar to sucrose-fructan 6-fructosyltransferase (6-SFT). The 1-SST and prft1 genes were colocalized with an invertase gene on the ryegrass linkage map. The mRNA levels of prft1 and prft2 increased gradually during cold treatment, while those of the 1-SST and 6G-FFT genes first increased, but then decreased before increasing again during a longer period of cold treatment.• Thus at least two different patterns of gene expression have developed during the evolution of functionally diverse FT genes, which are associated in a coordinated way with fructan synthesis in a cold environment.

In addition to the well-characterized proteins that comprise the pre-replicative complex, recent studies suggest that chromatin structure plays an important role in DNA replication initiation. One of these chromatin factors is the histone acetyltransferase (HAT) Hbo1 which is unique among HAT enzymes in that it serves as a positive regulator of DNA replication. However, several of the basic properties of Hbo1 have not been previously examined, including its intrinsic catalytic activity, its molecular abundance in cells, and its pattern of expression in primary cancer cells. Here we show that recombinant Hbo1 can acetylate nucleosomal histone H4 in vitro, with a preference for lysines 5 and 12. Using semi-quantitative western blot analysis, we find that Hbo1 is approximately equimolar with the number of active replication origins in normal human fibroblasts but is an order of magnitude more abundant in both MCF7 and Saos-2 established cancer cell lines. Immunohistochemistry for Hbo1 in 11 primary human tumor types revealed strong Hbo1 protein expression in carcinomas of the testis, ovary, breast, stomach/esophagus, and bladder.