Document Detail

Plasmids bearing mammalian DNA-replication origin-enriched (ors) fragments initiate semiconservative replication in a cell-free system.
MedLine Citation:
PMID:  1657184     Owner:  NLM     Status:  MEDLINE    
Four plasmids containing monkey (CV-1) origin-enriched sequences (ors), which we have previously shown to replicate autonomously in CV-1, COS-7 and HeLa cells (Frappier and Zannis-Hadjopoulos (1987) Proc. Natl. Acad. Sci. USA 84, 6668-6672), were found to replicate in an in vitro replication system using HeLa cell extracts. De novo site-specific initiation of replication on plasmids required the presence of an ors sequence, soluble low-salt cytosolic extract, poly(ethylene glycol), a solution containing the four standard deoxyribonucleoside triphosphates and an ATP regenerating system. The major reaction products migrated as relaxed circular and linear plasmid DNAs, both in the presence and absence of high-salt nuclear extracts. Inclusion of high-salt nuclear extract was required to obtain closed circular supercoiled molecules. Replicative intermediates migrating slower than form II and topoisomers migrating between forms II and I were also included among the replication products. Replication of the ors plasmids was not inhibited by ddTTP, an inhibitor of DNA polymerase beta and gamma, and was sensitive to aphidicolin indicating that DNA polymerase alpha and/or delta was responsible for DNA synthesis. Origin mapping experiments showed that early in the in vitro replication reaction, incorporation of nucleotides occurs preferentially at ors-containing fragments, indicating ors specific initiation of replication. In contrast, the limited incorporation of nucleotides into pBR322, was not site specific. The observed synthesis was semiconservative and appeared to be bidirectional.
C E Pearson; L Frappier; M Zannis-Hadjopoulos
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Publication Detail:
Type:  Comparative Study; Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Biochimica et biophysica acta     Volume:  1090     ISSN:  0006-3002     ISO Abbreviation:  Biochim. Biophys. Acta     Publication Date:  1991 Oct 
Date Detail:
Created Date:  1991-11-22     Completed Date:  1991-11-22     Revised Date:  2007-11-15    
Medline Journal Info:
Nlm Unique ID:  0217513     Medline TA:  Biochim Biophys Acta     Country:  NETHERLANDS    
Other Details:
Languages:  eng     Pagination:  156-66     Citation Subset:  IM    
McGill Cancer Centre, Department of Medicine, McGill University, Montreal, Canada.
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MeSH Terms
Aphidicolin / pharmacology
Cell-Free System / metabolism
DNA Replication* / drug effects
DNA Restriction Enzymes
Hela Cells
Plasmids* / drug effects
Thymine Nucleotides / pharmacology
Reg. No./Substance:
0/Dideoxynucleotides; 0/Thymine Nucleotides; 38966-21-1/Aphidicolin; 611-60-9/2',3'-dideoxythymidine triphosphate; EC 3.1.21.-/DNA Restriction Enzymes

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