Fine mapping and functional activity of the adenosine deaminase origin in murine embryonic fibroblasts.
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.
Sibani
Sahar
S
McGill Cancer Center and Department of Biochemistry, McGill University, Montreal, Quebec, Canada H3G 1Y6.
Rampakakis
Emmanouil
E
Di Paola
Domenic
D
Zannis-Hadjopoulos
Maria
M
D000243:Adenosine Deaminase / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D034961:Antigens, Nuclear / Q000096:biosynthesis; D001483:Base Sequence; D002460:Cell Line; D002874:Chromosome Mapping / Q000379:methods; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000096:biosynthesis; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000378:metabolism; D000072200:Ku Autoantigen; D051379:Mice; D008969:Molecular Sequence Data; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D018741:Replication Origin; D020133:Reverse Transcriptase Polymerase Chain Reaction
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D016271:Proto-Oncogene Proteins c-myc; D007041:Hypoxanthine Phosphoribosyltransferase; D000243:Adenosine Deaminase; C000606509:Xrcc6 protein, human; C000606510:Xrcc6 protein, mouse; D000072200:Ku Autoantigen