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...

Full description

Saved in:
Bibliographic Details
Published in:The EMBO journal 1991-12, Vol.10 (13), p.4351-4360
Main Authors: Burhans, W.C., Vassilev, L.T., Wu, J., Sogo, J.M., Nallaseth, F.S., DePamphilis, M.L.
Format: Article
Language:English
Subjects:
Animals
Biological and medical sciences
Blotting, Southern
Cricetinae
Cricetulus
DNA - ultrastructure
DNA Replication
DNA, Single-Stranded - analysis
Emetine - pharmacology
Fundamental and applied biological sciences. Psychology
Histones - metabolism
Microscopy, Electron
Molecular and cellular biology
Molecular genetics
Nucleosomes
Protein Synthesis Inhibitors
Replication
RNA - analysis
Citations: Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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.
ISSN:0261-4189
1460-2075
DOI:10.1002/j.1460-2075.1991.tb05013.x