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PMID: 561393 Published · ppublish English Journal Article

The origin of nascent single-stranded DNA extracted from mammalian cells.

Nucleic acids research ·Vol. 4 ·No. 6 ·1977-06-00 ·Pages 2083-97

Wanka F, Brouns RM, Aelen JM, Eygensteyn A, Eygensteyn J

Abstract

In vitro cultured bovine liver cells were labelled with radioactive thymidine and dissolved in 0.5% sodium dodecyl sulphate. Centrifugation of the lysate through sucrose gradients in a zonal rotor revealed a slowly sedimenting fraction of preferentially pulse labelled DNA. The DNA of this zone was further analysed by chromatography on hydroxy-apatite, banding in CsCl density gradients, and sedimentation in neutral and alkaline sucrose gradients. It contained besides small amounts of fragmented bulk DNA, single-stranded nascent DNA and single-stranded pre-labelled DNA which could be separated from each other by using BrdU as a density label. The density labelling also revealed small amounts of nascent-nascent DNA duplexes. The slowly sedimenting fraction was practically absent from cell lysates which were prepared in 2 M NaCl - 50 microgram/ml pronase. The results suggest that nascent single-strands and nascent-nascent duplexes are released from the forks of replicating DNA by branch migration. Pre-labelled single strands may be released by the same branch migration. Pre-labelled single strands may be released by the same mechanism, but the in vivo structure from which they originate has yet to be elucidated.

MeSH Terms
Animals Cattle Cell Line Centrifugation, Isopycnic DNA Replication DNA, Single-Stranded/biosynthesis,isolation & purification Liver/analysis Thymidine/metabolism
Chemicals
DNA, Single-Stranded Thymidine
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Wanka F
Brouns R M
Aelen J M
Eygensteyn A
Eygensteyn J
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29 references, click to expand
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Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
0305-1048
DOI
10.1093/nar/4.6.2083
Published
1977-06-00
Pages
2083-97
Language
English
Region
England
NLM ID
0411011
PMCID
PMC342545
Subset
IM
External Links
PubMed source DOI: 10.1093/nar/4.6.2083 PMC Full Text View journal details
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