title: Human origins of DNA replication selected from a library of nascent DNA.
abstract: The identification of metazoan origins of DNA replication has so far been hampered by the lack of a suitable genetic screening and by the cumbersomeness of the currently available mapping procedures. Here we describe the construction of a library of nascent DNA, representative of all cellular origin sequences, and its utilization as a screening probe for origin identification in large genomic regions. The procedure developed was successfully applied to the human 5q31.1 locus, encoding for the IL-3 and GM-CSF genes. Two novel origins were identified and subsequently characterized by competitive PCR mapping, located approximately 3.5 kb downstream of the GM-CSF gene. The two origins (GM-CSF Ori1 and Ori2) were shown to interact with different members of the DNA prereplication complex. This observation reinforces the universal paradigm that initiation of DNA replication takes place at, or in close proximity to, the binding sites of the trans-acting initiator proteins.
authors.affiliation: Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology, Padriciano, 99, 34012 Trieste, Italy.
authors.forename: Vesna
authors.initials: V
authors.lastname: Todorovic
authors.forename: Sara
authors.initials: S
authors.lastname: Giadrossi
authors.forename: Cristina
authors.initials: C
authors.lastname: Pelizon
authors.forename: Ramiro
authors.initials: R
authors.lastname: Mendoza-Maldonado
authors.forename: Hisao
authors.initials: H
authors.lastname: Masai
authors.forename: Mauro
authors.initials: M
authors.lastname: Giacca
mesh_terms: D002874:Chromosome Mapping / Q000379:methods*; D004247:DNA / Q000096:biosynthesis / Q000235:genetics*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000032:analysis / Q000235:genetics; D019143:Evolution, Molecular; D015723:Gene Library; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics; D006801:Humans; D007377:Interleukin-3 / Q000235:genetics; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods*; D018741:Replication Origin
chemical_list: D004268:DNA-Binding Proteins; D007377:Interleukin-3; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D004247:DNA