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Identification and functional analysis of a human homologue of the monkey replication origin ors8
Author(s) -
Callejo Mario,
Sibani Sahar,
Di Paola Domenic,
Price Gerald G.,
ZannisHadjopoulos Maria
Publication year - 2006
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.20868
Subject(s) - biology , microbiology and biotechnology , dna replication , eukaryotic dna replication , origin of replication , origin recognition complex , plasmid , dna , control of chromosome duplication , chromatin , genetics
We previously isolated from African green monkey (CV‐1) cells a replication origin, ors8, that is active at the onset of S‐phase. Here, its homologous sequence (hors8, accession number: DQ230978) was amplified from human cells, using the monkey‐ors8‐specific primers. Sequence alignment between the monkey and the human fragment revealed a 92% identity. Nascent DNA abundance analysis, involving quantification by real‐time PCR, indicated that hors8 is an active replication origin, as the abundance of nascent DNA from a genomic region containing it was 97‐fold higher relative to a non‐origin region in the same locus. Furthermore, the data showed that the hors8 fragment is capable of supporting the episomal replication of its plasmid, when cloned into pBlueScript (pBS), as assayed by the Dpn I resistance assay after transfection of HeLa cells. A quantitative chromatin immunoprecipitation (ChIP) assay, using antibodies against Ku, Orc2, and Cdc6, showed that these DNA replication initiator proteins were associated in vivo with the human ors8 (hors8). Finally, nascent DNA abundance experiments from human cells synchronized at different phases of the cell cycle revealed that hors8 is a late‐firing origin of DNA replication, having the highest activity 8 h after release from late G 1 . J. Cell. Biochem. 99: 1606–1615, 2006. © 2006 Wiley‐Liss, Inc.