Emetine allows identification of origins of mammalian DNA replication by imbalanced DNA synthesis, not through conservative nucleosome segregation.

Abstract

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.

Frequency and Fidelity of Translesion Synthesis of Site-specific N-2-Acetylaminofluorene Adducts during DNA Replication in a Human Cell Extract

“…The latter could reflect uncoupling of the first replication fork to encounter the adduct, such that repli- cation of the undamaged strand continues while replication of the damaged strand is completely or transiently blocked. Evidence exists that uncoupling can occur under some circumstances in E. coli (Koffel-Schwartz et al, 1987) 3 and during SV40 replication in vivo (Burhans et al, 1991). Alternatively, the first fork may not replicate either strand beyond the lesion, with preferential replication of the undamaged strand accomplished by the fork arriving from the other direction in the circular substrate.…”

Fine mapping of a replication origin of human DNA.

“…This failure has fostered several attempts to utilize physicochemical or biochemical approaches for the identification of origins. Analysis of leading strands polarity switch indicated the existence of an origin within 14 kb in the amplified dihydrofolate reductase (DHFR) locus of Chinese hamster cells (8,9) and, more recently, within a 2-kb stretch of the human 3-globin gene (10). PCR analysis of the relative abundance of different markers in isolated nascent DNA strands has given indications that the DHFR origin may be localized in a 2-kb region (11).…”

Specific transcription factors stimulate simian virus 40 and polyomavirus origins of DNA replication.

“…For example, the polyomavirus (Py) on functions only in those mouse cell types that can activate its enhancer (6,12,58,74). In mammalian chromosomes, active genes are replicated early during S phase while quiescent genes are replicated late (82), and initiation of replication may require specific transcription factors (76) that bind DNA sites where replication begins (11).…”