Trapping DNA replication origins from the human genome.
Trapping DNA replication origins from the human genome.
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DOI：
10.3390/genes4020198
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发表时间：
2013-04-17
期刊：
影响因子：
3.5
通讯作者：
Hanaoka F
中科院分区：
文献类型：
--
作者：
Eki T;Murakami Y;Hanaoka F
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.
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DOI：
10.1073/pnas.94.9.4587
发表时间：
1997-04-29
影响因子：
11.1
作者：
Hansen, RS;Canfield, TK;Gartler, SM
通讯作者：
Gartler, SM
影响因子：
7.7
作者：
Dazy, Sebastien;Gandrillon, Olivier;Prioleau, Marie-Noelle
通讯作者：
Prioleau, Marie-Noelle
影响因子：
7
作者：
Cayrou, Christelle;Coulombe, Philippe;Mechali, Marcel
通讯作者：
Mechali, Marcel
影响因子：
64.5
作者：
GASSER, SM;LAEMMLI, UK
通讯作者：
LAEMMLI, UK
影响因子：
7
作者：
Costantini, M;Clay, O;Bernardi, G
通讯作者：
Bernardi, G