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DNA elements modulating the KARS12 chromosomal replicator in Kluyveromyces lactis

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Abstract

Eukaryotic chromosomal DNA replication is initiated by a highly conserved set of proteins that interact with cis-acting elements on chromosomes called replicators. Despite the conservation of replication initiation proteins, replicator sequences show little similarity from species to species in the small number of organisms that have been examined. Examination of replicators in other species is likely to reveal common features of replicators. We have examined a Kluyeromyces lactis replicator, KARS12, that functions as origin of DNA replication on plasmids and in the chromosome. It contains a 50-bp region with similarity to two other K. lactis replicators, KARS101 and the pKD1 replication origin. Replacement of the 50-bp sequence with an EcoRI site completely abrogated the ability of KARS12 to support plasmid and chromosomal DNA replication origin activity, demonstrating this sequence is a common feature of K. lactis replicators and is essential for function, possibly as the initiator protein binding site. Additional sequences up to 1 kb in length are required for efficient KARS12 function. Within these sequences are a binding site for a global regulator, Abf1p, and a region of bent DNA, both of which contribute to the activity of KARS12. These elements may facilitate protein binding, protein/protein interaction and/or nucleosome positioning as has been proposed for other eukaryotic origins of DNA replication.

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Acknowledgments

We thank Flavio della Seta for the affinity purified ABF1 fraction, Giorgio Camilloni, Silvia Ciafré and the members of the Newlon laboratory group for helpful discussions throughout the study. This work was supported by MURST (Ministero Università Ricerca Scientifica Tecnologica - Progetti Ateneo), Istituto Pasteur Fondazione Cenci Bolognetti and NIH (grant number GM35679 awarded to CSN). C.M. is a PhD student on Pasteurian Sciences, Università “La Sapienza”, Roma.

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  1. Carmela Irene

    Present address: Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, Newark, NJ, 07103, USA

Authors and Affiliations

  1. Dipartimento di Biologia Cellulare e dello Sviluppo, Università “La Sapienza”, Piazzale A. Moro, 5, Roma, Italy

    Carmela Irene, Clelia Maciariello & Lucia Fabiani

  2. IBPM-CNR c/o Dipartimento Genetica e Biologia Molecolare, Università “La Sapienza”, Piazzale A. Moro, 5, Roma, Italy

    Gioacchino Micheli

  3. Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, Newark, NJ, 07103, USA

    James F. Theis & Carol S. Newlon

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  1. Carmela Irene
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  2. Clelia Maciariello
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  3. Gioacchino Micheli
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  4. James F. Theis
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  5. Carol S. Newlon
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  6. Lucia Fabiani
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Correspondence to Lucia Fabiani.

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Irene, C., Maciariello, C., Micheli, G. et al. DNA elements modulating the KARS12 chromosomal replicator in Kluyveromyces lactis . Mol Genet Genomics 277, 287–299 (2007). https://doi.org/10.1007/s00438-006-0188-7

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