Premium
Fine mapping and functional activity of the adenosine deaminase origin in murine embryonic fibroblasts
Author(s) -
Sibani Sahar,
Rampakakis Emmanouil,
Di Paola Domenic,
ZannisHadjopoulos Maria
Publication year - 2008
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.21662
Subject(s) - biology , dna replication , adenosine deaminase , microbiology and biotechnology , embryonic stem cell , hypoxanthine guanine phosphoribosyltransferase , cell culture , dna , genetics , gene , enzyme , biochemistry , mutant
DNA replication initiates at origins within the genome. The late‐firing murine adenosine deaminase (mAdA) origin is located within a 2 kb fragment of DNA, making it difficult to examine by realtime technology. In this study, fine mapping of the mAdA region by measuring the abundance of nascent strand DNA identified two origins, mAdA‐1 and mAdA‐C, located 397 bp apart from each other. Both origins conferred autonomous replication to plasmids transfected in murine embryonic fibroblasts (MEFs), and exhibited similar activities in vivo and in vitro. Furthermore, both were able to recruit the DNA replication initiator proteins Cdc6 and Ku in vitro, similar to other bona fide replication origins. When tested in a murine Ku80 −/− cell line, both origins exhibited replication activities comparable to those observed in wildtype cells, as did the hypoxanthine‐guanine phosphoribosyltransferase (HPRT) and c‐ myc origins. This contrasts with previously published studies using Ku80‐deficient human cells lines and suggests differences in the mechanism of initiation of DNA replication between the murine and human systems. J. Cell. Biochem. 104: 773–784, 2008. © 2008 Wiley‐Liss, Inc.