Emetine allows identification of origins of mammalian DNA replication by imbalanced DNA synthesis, not through conservative nucleosome segregation.
In the presence of emetine, an inhibitor of protein synthesis, nascent DNA on forward arms of replication forks in hamster cell lines containing either single or amplified copies of the DHFR gene region was enriched 5- to 7-fold over nascent DNA on retrograde arms. This forward arm bias was observed on both sides of the specific origin of bidirectional DNA replication located 17 kb downstream of the hamster DHFR gene (OBR-1), consistent with at least 85% of replication forks within this region emanating from OBR-1. However, the replication fork asymmetry induced by emetine does not result from conservative nucleosome segregation, as previously believed, but from preferentially inhibiting Okazaki fragment synthesis on retrograde arms of forks to produce 'imbalanced DNA synthesis'. Three lines of evidence support this conclusion. First, the bias existed in long nascent DNA strands prior to nuclease digestion of non-nucleosomal DNA. Second, the fraction of RNA-primed Okazaki fragments was rapidly diminished. Third, electron microscopic analysis of SV40 DNA replicating in the presence of emetine revealed forks with single-stranded DNA on one arm, and nucleosomes randomly distributed to both arms. Thus, as with cycloheximide, nucleosome segregation in the presence of emetine was distributive.
Burhans
W C
WC
Department of Cell and Developmental Biology, Roche Institute of Molecular Biology, Roche Research Center, Nutley, NJ 07110.
Vassilev
L T
LT
Wu
J
J
Sogo
J M
JM
Nallaseth
F S
FS
DePamphilis
M L
ML
D000818:Animals; D015139:Blotting, Southern; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000648:ultrastructure; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000032:analysis; D004640:Emetine / Q000494:pharmacology*; D006657:Histones / Q000378:metabolism; D008854:Microscopy, Electron; D009707:Nucleosomes; D011500:Protein Synthesis Inhibitors; D012313:RNA / Q000032:analysis
D004277:DNA, Single-Stranded; D006657:Histones; D009707:Nucleosomes; D011500:Protein Synthesis Inhibitors; D012313:RNA; D004247:DNA; D004640:Emetine