Genes 2013, 4(2), 198-225; doi:10.3390/genes4020198
Article

Trapping DNA Replication Origins from the Human Genome

1,2,* email, 2,3email and 2,4email
Received: 25 March 2013; in revised form: 5 April 2013 / Accepted: 9 April 2013 / Published: 17 April 2013
(This article belongs to the Special Issue DNA Replication)
View Full-Text   |   Download PDF [1285 KB, uploaded 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
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Export to BibTeX |
EndNote


MDPI and ACS Style

Eki, T.; Murakami, Y.; Hanaoka, F. Trapping DNA Replication Origins from the Human Genome. Genes 2013, 4, 198-225.

AMA Style

Eki T, Murakami Y, Hanaoka F. Trapping DNA Replication Origins from the Human Genome. Genes. 2013; 4(2):198-225.

Chicago/Turabian Style

Eki, Toshihiko; Murakami, Yasufumi; Hanaoka, Fumio. 2013. "Trapping DNA Replication Origins from the Human Genome." Genes 4, no. 2: 198-225.


Genes EISSN 2073-4425 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert