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Trapping DNA Replication Origins from the Human Genome
Molecular Genetics Laboratory, Division of Bioscience and Biotechnology, Department of Environmental and Life Sciences, Toyohashi University of Technology, 1-1 Hibarigaoka, Tempaku-cho, Toyohashi, Aichi 441-8580, Japan
Cellular Physiology Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
Department of Biological Science and Technology, Faculty of Industrial Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan
Faculty of Science, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588, Japan
* Author to whom correspondence should be addressed.
Received: 25 March 2013; in revised form: 5 April 2013 / Accepted: 9 April 2013 / Published: 17 April 2013
Abstract: Synthesis of chromosomal DNA is initiated from multiple origins of replication in higher eukaryotes; however, little is known about these origins’ structures. We isolated the origin-derived nascent DNAs from a human repair-deficient cell line by blocking the replication forks near the origins using two different origin-trapping methods (i.e., UV- or chemical crosslinker-treatment and cell synchronization in early S phase using DNA replication inhibitors). Single-stranded DNAs (of 0.5–3 kb) that accumulated after such treatments were labeled with bromodeoxyuridine (BrdU). BrdU-labeled DNA was immunopurified after fractionation by alkaline sucrose density gradient centrifugation and cloned by complementary-strand synthesis and PCR amplification. Competitive PCR revealed an increased abundance of DNA derived from known replication origins (c-myc and lamin B2 genes) in the nascent DNA fractions from the UV-treated or crosslinked cells. Nucleotide sequences of 85 and 208 kb were obtained from the two libraries (I and II) prepared from the UV-treated log-phase cells and early S phase arrested cells, respectively. The libraries differed from each other in their G+C composition and replication-related motif contents, suggesting that differences existed between the origin fragments isolated by the two different origin-trapping methods. The replication activities for seven out of 12 putative origin loci from the early-S phase cells were shown by competitive PCR. We mapped 117 (library I) and 172 (library II) putative origin loci to the human genome; approximately 60% and 50% of these loci were assigned to the G-band and intragenic regions, respectively. Analyses of the flanking sequences of the mapped loci suggested that the putative origin loci tended to associate with genes (including conserved sites) and DNase I hypersensitive sites; however, poor correlations were found between such loci and the CpG islands, transcription start sites, and K27-acetylated histone H3 peaks.
Keywords: DNA replication; replication origins; sequence analysis; human genome; competitive PCR
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MDPI and ACS Style
Eki, T.; Murakami, Y.; Hanaoka, F. Trapping DNA Replication Origins from the Human Genome. Genes 2013, 4, 198-225.
Eki T, Murakami Y, Hanaoka F. Trapping DNA Replication Origins from the Human Genome. Genes. 2013; 4(2):198-225.
Eki, Toshihiko; Murakami, Yasufumi; Hanaoka, Fumio. 2013. "Trapping DNA Replication Origins from the Human Genome." Genes 4, no. 2: 198-225.