| DNA synthesis in isolated resting nuclei: evidence for protease-dependent nonreplicative nucleotide incorporation. | |
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MedLine Citation:
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PMID: 1696899 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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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. |
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Authors:
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T L Jensen; J J O'Leary |
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Publication Detail:
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Type: Journal Article; Research Support, U.S. Gov't, P.H.S. |
Journal Detail:
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Title: Experimental cell research Volume: 190 ISSN: 0014-4827 ISO Abbreviation: Exp. Cell Res. Publication Date: 1990 Sep |
Date Detail:
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Created Date: 1990-09-21 Completed Date: 1990-09-21 Revised Date: 2007-11-14 |
Medline Journal Info:
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Nlm Unique ID: 0373226 Medline TA: Exp Cell Res Country: UNITED STATES |
Other Details:
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Languages: eng Pagination: 85-90 Citation Subset: IM |
Affiliation:
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Laboratory Medicine and Pathology, University of Minnesota, Minneapolis 55455. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Animals 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 Humans Lymphocytes / metabolism Micrococcus / enzymology Serine Endopeptidases / physiology* Serine Proteinase Inhibitors / pharmacology Xenopus laevis |
| Grant Support | |
ID/Acronym/Agency:
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AG02338/AG/NIA NIH HHS; CA 39692/CA/NCI NIH HHS |
| Chemical | |
Reg. No./Substance:
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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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