Document Detail

DNA synthesis in isolated resting nuclei: evidence for protease-dependent nonreplicative nucleotide incorporation.
MedLine Citation:
PMID:  1696899     Owner:  NLM     Status:  MEDLINE    
We have used an in vitro assay to study the induction of DNA synthesis by cytoplasmic extracts from the actively growing cell line Molt 4 in nuclei isolated from quiescent human lymphocytes. The TTP incorporation which takes place in these nuclei has been shown to be inhibitable by serine protease inhibitors, particularly aprotinin. This DNA synthesis has also been proposed to reflect the initiation of true DNA replication; however, we find evidence that much, if not most, of this incorporation is due to nonreplicative synthesis initiated on primer templates formed by calcium-dependent activation of the nuclear chromatin substrate. The principal DNA polymerase supplied by the Molt 4 extract appears to be polymerase alpha and the results show that the activated chromatin is a substrate for purified bacterial DNA polymerases. DNA synthesis is significantly enhanced by preincubation at 37 degrees C in the presence of calcium, and the almost complete inhibition of DNA synthesis induced by extracts or bacterial polymerases in the presence of T4 ligase suggests that this chromatin activation involves calcium-dependent endonucleases. Nevertheless, DNA synthesis in the isolated nuclei, with both Molt 4 extracts and bacterial polymerases, is substantially inhibited by addition of serine protease inhibitors, with aprotinin the most potent of those tested on a molar basis. Thus, the results suggest that specific proteolytic activity is required before nicked or damaged nuclear DNA can serve as an acceptable substrate for DNA polymerase activity.
T L Jensen; J J O'Leary
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Experimental cell research     Volume:  190     ISSN:  0014-4827     ISO Abbreviation:  Exp. Cell Res.     Publication Date:  1990 Sep 
Date Detail:
Created Date:  1990-09-21     Completed Date:  1990-09-21     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  0373226     Medline TA:  Exp Cell Res     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  85-90     Citation Subset:  IM    
Laboratory Medicine and Pathology, University of Minnesota, Minneapolis 55455.
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MeSH Terms
Aprotinin / pharmacology
Calcium / physiology
Cell Extracts
Cell Nucleus / metabolism*
Cytoplasm / physiology
DNA / biosynthesis*
DNA Polymerase II / physiology
DNA Replication / drug effects,  physiology
Deoxyribonucleotides / metabolism*
Endonucleases / physiology
Enzyme Activation
Lymphocytes / metabolism
Micrococcus / enzymology
Serine Endopeptidases / physiology*
Serine Proteinase Inhibitors / pharmacology
Xenopus laevis
Grant Support
Reg. No./Substance:
0/Cell Extracts; 0/Deoxyribonucleotides; 0/Serine Proteinase Inhibitors; 7440-70-2/Calcium; 9007-49-2/DNA; 9087-70-1/Aprotinin; EC 2.7.7.-/DNA Polymerase II; EC 3.1.-/Endonucleases; EC 3.4.21.-/Serine Endopeptidases

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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