Spatial distribution of DNA loop attachment and replicational sites in the nuclear matrix.

Biochemical fractionation was combined with high resolution electron microscopic autoradiography to study the localization in rat liver nuclear matrix of attached DNA fragments, in vivo replicated DNA, and in vitro synthesized DNA. In particular, we determined the distribution of these DNA components with the peripheral nuclear lamina versus more internally localized structural elements of isolated nuclear matrix. Autoradiography demonstrated that the bulk of in vivo newly replicated DNA associated with the nuclear matrix (71%) was found within internal matrix regions. A similar interior localization was observed in isolated nuclei and in situ in whole liver tissue. Likewise, isolated nuclear lamina contained only a small amount (12%) of the total matrix-bound, newly replicated DNA. The structural localization of matrix-bound DNA fragments was examined following long-term in vivo labeling of the DNA. The radioactive DNA fragments were found predominantly within interior regions of the matrix structure (77%), and isolated nuclear lamina contained less than 15% of the total nuclear matrix-associated DNA. Most of the endogenous DNA template sites for the replicative enzyme DNA polymerase alpha (approximately 70%) were also sequestered within interior regions of the matrix. In contrast, a majority of the endogenous DNA template sites for DNA polymerase beta (a presumptive repair enzyme) were closely associated with the peripheral nuclear lamina. A similar spatial distribution for both polymerase activities was measured in isolated nuclei before matrix fractionation. Furthermore, isolated nuclear lamina contained only a small proportion of total matrix-bound DNA polymerase alpha endogenous and exogenous template activities (3-12%), but a considerable amount of the corresponding beta polymerase activities (47-52%). Our results support the hypothesis that DNA loops are both anchored and replicated at nuclear matrix-bound sites that are predominantly but not exclusively associated with interior components of the matrix structure. Our results also suggest that the sites of nuclear DNA polymerase beta-driven DNA synthesis are uniquely sequestered within the characteristic peripheral heterochromatin shell and associated nuclear envelope structure, where they may potentially participate in DNA repair and/or replicative functions.

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Differential inhibition of rat liver DNA polymerases in vitro by direct-acting carcinogens and the protective effect of a thiol reducing agent

Biochemical Journal ◽

10.1042/bj1930985 ◽

1981 ◽

Vol 193 (3) ◽

pp. 985-990 ◽

Cited By ~ 7

Author(s):

J Y Chan ◽

F F Becker

Keyword(s):

Dna Polymerase ◽

Rat Liver ◽

Reducing Agent ◽

Gamma Activity ◽

Repair Enzyme ◽

Dna Polymerase Alpha ◽

Polymerase Beta ◽

Direct Acting

The direct-acting carcinogens acetoxyacetylaminofluorene, methylnitrosourea, and N-methyl-N'-nitro-N-nitrosoguanidine were tested for their ability to inhibit rat liver DNA polymerase-alpha, -beta, and -gamma activity in vitro. DNA polymerase-alpha was the most sensitive, polymerase-beta was the most resistant, and polymerase-gamma exhibited an intermediate response. When the reactions were reassayed in the presence and absence of dithiothreitol, a thiol reducing agent, it was shown that the inhibition by carcinogens was generally reversible with increasing dithiothreitol, except that polymerase-beta recovered only 80-90% of control values. These and binding data suggest that DNA polymerase-beta, the putative repair enzyme, is highly resistant to carcinogen damage. This resistance may contribute to the retention of normal function and fidelity of the repair enzyme during carcinogen exposure in vivo and to a normal cellular repair.

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Enhanced processivity of nuclear matrix bound DNA polymerase .alpha. from regenerating rat liver

Biochemistry ◽

10.1021/bi00392a020 ◽

1987 ◽

Vol 26 (18) ◽

pp. 5710-5718 ◽

Cited By ~ 18

Author(s):

Ross A. Tubo ◽

Alberto M. Martelli ◽

Ronald Berezney

Keyword(s):

Dna Polymerase ◽

Rat Liver ◽

Nuclear Matrix ◽

Dna Polymerase Alpha ◽

Regenerating Rat Liver ◽

Matrix Bound

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Involvement of deoxyribonucleic acid polymerase β in nuclear deoxyribonucleic acid synthesis

Biochemical Journal ◽

10.1042/bj1730309 ◽

1978 ◽

Vol 173 (1) ◽

pp. 309-314 ◽

Cited By ~ 12

Author(s):

T R Butt ◽

W M Wood ◽

E L McKay ◽

R L P Adams

Keyword(s):

Dna Synthesis ◽

Dna Polymerase ◽

Deoxyribonucleic Acid ◽

Nuclear Dna ◽

Dna Polymerase Beta ◽

Cell Nuclei ◽

High Molecular Weight Dna ◽

Dna Polymerase Alpha ◽

Polymerase Beta

The effects on DNA synthesis in vitro in mouse L929-cell nuclei of differential extraction of DNA polymerases alpha and beta were studied. Removal of all measurable DNA polymerase alpha and 20% of DNA polymerase beta leads to a 40% fall in the replicative DNA synthesis. Removal of 70% of DNA polymerase beta inhibits replicative synthesis by 80%. In all cases the nuclear DNA synthesis is sensitive to N-ethylmaleimide and aCTP (arabinosylcytosine triphosphate), though less so than DNA polymerase alpha. Addition of deoxyribonuclease I to the nuclear incubation leads to synthesis of high-molecular-weight DNA in a repair reaction. This occurs equally in nuclei from non-growing or S-phase cells. The former nuclei lack DNA polymerase alpha and the reaction reflects the sensitivity of DNA polymerase beta to inhibiton by N-ethylmaleimide and aCTP.

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Binding of the DNA polymerase .alpha.-DNA primase complex to the nuclear matrix in HeLa cells

Biochemistry ◽

10.1021/bi00392a004 ◽

1987 ◽

Vol 26 (18) ◽

pp. 5600-5607 ◽

Cited By ~ 21

Author(s):

James M. Collins ◽

Annie K. Chu

Keyword(s):

Dna Polymerase ◽

Hela Cells ◽

Nuclear Matrix ◽

Dna Polymerase Alpha ◽

Dna Primase ◽

Primase Complex

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Evidence that POB1, a Saccharomyces cerevisiae protein that binds to DNA polymerase alpha, acts in DNA metabolism in vivo

Molecular and Cellular Biology ◽

10.1128/mcb.12.12.5724-5735.1992 ◽

1992 ◽

Vol 12 (12) ◽

pp. 5724-5735

Author(s):

J Miles ◽

T Formosa

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Replication ◽

Dna Polymerase ◽

Yeast Cells ◽

Chromosome Loss ◽

Dna Metabolism ◽

Checkpoint Gene ◽

Dna Polymerase Alpha ◽

In Cells

Potential DNA replication accessory factors from the yeast Saccharomyces cerevisiae have previously been identified by their ability to bind to DNA polymerase alpha protein affinity matrices (J. Miles and T. Formosa, Proc. Natl. Acad. Sci. USA 89:1276-1280, 1992). We have now used genetic methods to characterize the gene encoding one of these DNA polymerase alpha-binding proteins (POB1) to determine whether it plays a role in DNA replication in vivo. We find that yeast cells lacking POB1 are viable but display a constellation of phenotypes indicating defective DNA metabolism. Populations of cells lacking POB1 accumulate abnormally high numbers of enlarged large-budded cells with a single nucleus at the neck of the bud. The average DNA content in a population of cells lacking POB1 is shifted toward the G2 value. These two phenotypes indicate that while the bulk of DNA replication is completed without POB1, mitosis is delayed. Deleting POB1 also causes elevated levels of both chromosome loss and genetic recombination, enhances the temperature sensitivity of cells with mutant DNA polymerase alpha genes, causes increased sensitivity to UV radiation in cells lacking a functional RAD9 checkpoint gene, and causes an increased probability of death in cells carrying a mutation in the MEC1 checkpoint gene. The sequence of the POB1 gene indicates that it is identical to the CTF4 (CHL15) gene identified previously in screens for mutations that diminish the fidelity of chromosome transmission. These phenotypes are consistent with defective DNA metabolism in cells lacking POB1 and strongly suggest that this DNA polymerase alpha-binding protein plays a role in accurately duplicating the genome in vivo.

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Involvement of proliferating cell nuclear antigen (cyclin) in DNA replication in living cells

Molecular and Cellular Biology ◽

10.1128/mcb.9.1.57-66.1989 ◽

1989 ◽

Vol 9 (1) ◽

pp. 57-66

Author(s):

M Zuber ◽

E M Tan ◽

M Ryoji

Keyword(s):

Dna Polymerase ◽

Proliferating Cell Nuclear Antigen ◽

Living Cells ◽

Nuclear Antigen ◽

Plasmid Replication ◽

Dna Polymerase Delta ◽

Dna Polymerase Alpha ◽

Proliferating Cell

Proliferating cell nuclear antigen (PCNA) (also called cyclin) is known to stimulate the activity of DNA polymerase delta but not the other DNA polymerases in vitro. We injected a human autoimmune antibody against PCNA into unfertilized eggs of Xenopus laevis and examined the effects of this antibody on the replication of injected plasmid DNA as well as egg chromosomes. The anti-PCNA antibody inhibited plasmid replication by up to 67%, demonstrating that PCNA is involved in plasmid replication in living cells. This result further implies that DNA polymerase delta is necessary for plasmid replication in vivo. Anti-PCNA antibody alone did not block plasmid replication completely, but the residual replication was abolished by coinjection of a monoclonal antibody against DNA polymerase alpha. Anti-DNA polymerase alpha alone inhibited plasmid replication by 63%. Thus, DNA polymerase alpha is also required for plasmid replication in this system. In similar studies on the replication of egg chromosomes, the inhibition by anti-PCNA antibody was only 30%, while anti-DNA polymerase alpha antibody blocked 73% of replication. We concluded that the replication machineries of chromosomes and plasmid differ in their relative content of DNA polymerase delta. In addition, we obtained evidence through the use of phenylbutyl deoxyguanosine, an inhibitor of DNA polymerase alpha, that the structure of DNA polymerase alpha holoenzyme for chromosome replication is significantly different from that for plasmid replication.

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Association of the telomere-telomere-binding protein complex of hypotrichous ciliates with the nuclear matrix and dissociation during replication

Journal of Cell Science ◽

10.1242/jcs.114.10.1861 ◽

2001 ◽

Vol 114 (10) ◽

pp. 1861-1866 ◽

Cited By ~ 1

Author(s):

J. Postberg ◽

S.A. Juranek ◽

S. Feiler ◽

H. Kortwig ◽

F. Jonsson ◽

...

Keyword(s):

Cell Cycle ◽

Nuclear Localization ◽

Nuclear Matrix ◽

Binding Protein ◽

Specific Interaction ◽

Eukaryotic Cells ◽

Dna Molecules ◽

Telomere Binding Protein ◽

The Matrix

Telomeric interactions with the nuclear matrix have been described in a variety of eukaryotic cells and seem to be essential for specific nuclear localization. Macronuclear DNA of hypotrichous ciliates occurs in small gene-sized DNA molecules, each being terminated by telomeres. Each macronucleus contains over 10(8)individual DNA molecules. Owing to the high number of telomeres present in this nucleus it provides an excellent model to study telomere behaviour throughout the cell cycle. In this study we provide experimental evidence that the telomere-telomere-binding protein (TEBP) complex specifically interacts with components of the nuclear matrix in vivo. In the course of replication the specific interaction of the TEBP with components of the nuclear matrix is resolved and an attachment of the telomeres to the matrix no longer occurs.

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Fibroblast proliferation during myocardial development in rats is regulated by IGF-1 receptors

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1995.269.3.h943 ◽

1995 ◽

Vol 269 (3) ◽

pp. H943-H951 ◽

Cited By ~ 3

Author(s):

K. Reiss ◽

W. Cheng ◽

J. Kajstura ◽

E. H. Sonnenblick ◽

L. G. Meggs ◽

...

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

Proliferating Cell Nuclear Antigen ◽

Cardiac Fibroblasts ◽

Nuclear Antigen ◽

Dna Polymerase Alpha ◽

Myocardial Development ◽

Left And Right ◽

Proliferating Cell

To determine whether the growth of cardiac fibroblasts during development is modulated by the insulin-like growth factor (IGF)-1 receptor (IGF-1R), the expression of IGF-1, IGF-2, and IGF-1R was determined in fibroblasts from fetal and postnatal hearts. The expression of proliferating cell nuclear antigen (PCNA) and DNA polymerase-alpha was also evaluated in combination with the estimation of DNA replication. In comparison with fetal hearts, at postnatal day 21, fibroblast expression of IGF-1R mRNA, IGF-2, PCNA, and DNA polymerase-alpha was reduced by 77, 70, 80, and 86%, respectively. Moreover, IGF-1R protein decreased by 48% at 21 days. Bromodeoxyuridine labeling decreased by 88 and 89% in the left and right ventricle, respectively, at this time. Two different antisense oligodeoxynucleotides to IGF-1R reduced DNA replication by 60 and 44% in fibroblasts in culture. In addition, this intervention markedly attenuated the growth response of fibroblasts to IGF-1 or serum. In conclusion, the IGF-1R system appears to play a major role in the regulation of fibroblast growth in the heart in vivo.

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