Abstract
The size of supercoiled, topologically constrained DNA domains within the squamous carcinoma cell line SQ-20B were determined by direct comparison with a panel of irradiated supercoiled plasmid DNAs. Loss of supercoiling in plasmids was determined by gel electrophoresis and in cells by nucleoid flow cytometry. Comparison of dose-response data for plasmid relaxation with that obtained from SQ-20B cells enabled a direct estimation of supercoil target size in these cells. Plasmids pUCD9P (3.9 kbp), pXT-1 (10.1 kbp), pdBPV-MMT- neo (14.6 kbp), pRK290 (20.0 kbp), and R6K (38 kbp) were used and analyzed under the same exposure conditions as nucleoid DNA. Two sizes of topologically closed domains were found in nucleoids of 0.51 ± 0.17Mbp and 1.34 ± 0.3 Mbp. In an attempt to relate these large-scale organizations of DNA with function, cells were exposed to the DNA topoisomerase II inhibitor, VP16 and the G1/S cell cycle blocking agent mimosine. A 1 h exposure to VP16 was effective in reducing DNA synthesis which was associated with a parallel increase in nucleoid supercoiling. Addition of the G1 > S inhibitor mimosine enhanced both responses. It is concluded that chromosomes and interphase nuclei are organized into at least two sizes of topologically constrained domains of DNA which may have functional relevance to the control and execution of DNA synthesis.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 1051-1058 |
| Number of pages | 8 |
| Journal | DNA and Cell Biology |
| Volume | 16 |
| Issue number | 9 |
| State | Published - 1997 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Molecular Biology
- Genetics
- Cell Biology
Cite this
Topologically constrained domains of supercoiled DNA in eukaryotic cells. / Khodarev, Nikolai N.; Narayana, Ashwatha; Constantinou, Andreas; Vaughan, Andrew T M.
In: DNA and Cell Biology, Vol. 16, No. 9, 1997, p. 1051-1058.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Topologically constrained domains of supercoiled DNA in eukaryotic cells
AU - Khodarev, Nikolai N.
AU - Narayana, Ashwatha
AU - Constantinou, Andreas
AU - Vaughan, Andrew T M
PY - 1997
Y1 - 1997
N2 - The size of supercoiled, topologically constrained DNA domains within the squamous carcinoma cell line SQ-20B were determined by direct comparison with a panel of irradiated supercoiled plasmid DNAs. Loss of supercoiling in plasmids was determined by gel electrophoresis and in cells by nucleoid flow cytometry. Comparison of dose-response data for plasmid relaxation with that obtained from SQ-20B cells enabled a direct estimation of supercoil target size in these cells. Plasmids pUCD9P (3.9 kbp), pXT-1 (10.1 kbp), pdBPV-MMT- neo (14.6 kbp), pRK290 (20.0 kbp), and R6K (38 kbp) were used and analyzed under the same exposure conditions as nucleoid DNA. Two sizes of topologically closed domains were found in nucleoids of 0.51 ± 0.17Mbp and 1.34 ± 0.3 Mbp. In an attempt to relate these large-scale organizations of DNA with function, cells were exposed to the DNA topoisomerase II inhibitor, VP16 and the G1/S cell cycle blocking agent mimosine. A 1 h exposure to VP16 was effective in reducing DNA synthesis which was associated with a parallel increase in nucleoid supercoiling. Addition of the G1 > S inhibitor mimosine enhanced both responses. It is concluded that chromosomes and interphase nuclei are organized into at least two sizes of topologically constrained domains of DNA which may have functional relevance to the control and execution of DNA synthesis.
AB - The size of supercoiled, topologically constrained DNA domains within the squamous carcinoma cell line SQ-20B were determined by direct comparison with a panel of irradiated supercoiled plasmid DNAs. Loss of supercoiling in plasmids was determined by gel electrophoresis and in cells by nucleoid flow cytometry. Comparison of dose-response data for plasmid relaxation with that obtained from SQ-20B cells enabled a direct estimation of supercoil target size in these cells. Plasmids pUCD9P (3.9 kbp), pXT-1 (10.1 kbp), pdBPV-MMT- neo (14.6 kbp), pRK290 (20.0 kbp), and R6K (38 kbp) were used and analyzed under the same exposure conditions as nucleoid DNA. Two sizes of topologically closed domains were found in nucleoids of 0.51 ± 0.17Mbp and 1.34 ± 0.3 Mbp. In an attempt to relate these large-scale organizations of DNA with function, cells were exposed to the DNA topoisomerase II inhibitor, VP16 and the G1/S cell cycle blocking agent mimosine. A 1 h exposure to VP16 was effective in reducing DNA synthesis which was associated with a parallel increase in nucleoid supercoiling. Addition of the G1 > S inhibitor mimosine enhanced both responses. It is concluded that chromosomes and interphase nuclei are organized into at least two sizes of topologically constrained domains of DNA which may have functional relevance to the control and execution of DNA synthesis.
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UR - http://www.scopus.com/inward/citedby.url?scp=0031414573&partnerID=8YFLogxK
M3 - Article
C2 - 9324307
AN - SCOPUS:0031414573
VL - 16
SP - 1051
EP - 1058
JO - DNA and Cell Biology
JF - DNA and Cell Biology
SN - 1044-5498
IS - 9
ER -