TY - JOUR
T1 - A fixed site of DNA replication in eucaryotic cells
AU - Pardoll, Drew M.
AU - Vogelstein, Bert
AU - Coffey, Donald S.
N1 - Funding Information:
We thank William Lennarz, Jr. for performing the electron microscopic autoradiography, Adam Leaderman for assistance with the rat liver experiments and Joel Shaper for valuable discussions. This work was supported in part by NIH grants and a gift from Bristol Myers Company. D.P. is a Medical Scientist Trainee and is supported by an NIH grant. The electron micrographs were prepared by Ms. Carol J. Tillman through the Core Electron Microscopy Laboratory of the Johns Hopkins Population Center, with the support of a grant from the USPHS.
Copyright:
Copyright 2014 Elsevier B.V., All rights reserved.
PY - 1980/2
Y1 - 1980/2
N2 - We studied the role of the nuclear matrix (the skeletal framework of the nucleus) in DNA replication both in vivo and in a cell culture system. When regenerating rat liver or exponentially growing 3T3 fibroblasts are pulse-labeled with 3H-thymidine and nuclear matrix is subsequently isolated, the fraction of DNA remaining tightly attached to the matrix is highly enriched in newly synthesized DNA. After a 30 sec pulse labeling period and limited DNAase I digestion, the matrix DNA of 3T3 fibroblasts, which constitutes 15% of the total DNA, contains approximately 90% of the labeled newly synthesized DNA. Over 80% of this label can be chased out of the matrix DNA if the pulse is followed by a 45 min incubation with excess unlabeled thymidine. These and other kinetic studies suggest that the growing point of DNA replication is attached to the nuclear matrix. Studies measuring the size distribution of the matrix DNA also support this conclusion. Reconstitution controls and autoradiographic studies indicate that these results are not due to preferential, nonspecific binding of nascent DNA to the matrix during the extraction procedures. Electron microscopic autoradiography shows that, as with intact nuclei, sites of DNA replication are distributed throughout the nuclear matrix. A fixed site of DNA synthesis is proposed in which DNA replication complexes are anchored to the nuclear matrix and the DNA is reeled through these complexes as it is replicated.
AB - We studied the role of the nuclear matrix (the skeletal framework of the nucleus) in DNA replication both in vivo and in a cell culture system. When regenerating rat liver or exponentially growing 3T3 fibroblasts are pulse-labeled with 3H-thymidine and nuclear matrix is subsequently isolated, the fraction of DNA remaining tightly attached to the matrix is highly enriched in newly synthesized DNA. After a 30 sec pulse labeling period and limited DNAase I digestion, the matrix DNA of 3T3 fibroblasts, which constitutes 15% of the total DNA, contains approximately 90% of the labeled newly synthesized DNA. Over 80% of this label can be chased out of the matrix DNA if the pulse is followed by a 45 min incubation with excess unlabeled thymidine. These and other kinetic studies suggest that the growing point of DNA replication is attached to the nuclear matrix. Studies measuring the size distribution of the matrix DNA also support this conclusion. Reconstitution controls and autoradiographic studies indicate that these results are not due to preferential, nonspecific binding of nascent DNA to the matrix during the extraction procedures. Electron microscopic autoradiography shows that, as with intact nuclei, sites of DNA replication are distributed throughout the nuclear matrix. A fixed site of DNA synthesis is proposed in which DNA replication complexes are anchored to the nuclear matrix and the DNA is reeled through these complexes as it is replicated.
UR - http://www.scopus.com/inward/record.url?scp=0018858066&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0018858066&partnerID=8YFLogxK
U2 - 10.1016/0092-8674(80)90527-9
DO - 10.1016/0092-8674(80)90527-9
M3 - Article
C2 - 7357619
AN - SCOPUS:0018858066
SN - 0092-8674
VL - 19
SP - 527
EP - 536
JO - Cell
JF - Cell
IS - 2
ER -