DNA chain growth in isolated HeLa nuclei

Stephen Planck, Gerald C. Mueller

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

A new method for studying DNA chain growth has been used to investigate DNA replication in isolated nuclei. For this purpose, DNA replication was initiated in S-phase cells with bromodeoxyuridine in order to photosensitize active replicons. The nuclei were then isolated and caused to synthesize DNA in vitro with [3H]dTTP as a precursor to label growing chains. Subsequent irradiation with 313-nm light fragments the bromodeoxyuridine-DNA leaders and releases the contiguous, newly replicated DNA chains for analysis by sedimentation through alkaline sucrose gradients. This method of analysis provides data on the length of the DNA chain segments that are actually synthesized in vitro and on the fraction of these DNA chains that extends from bromodeoxyuridine-prelabeled replicons. Segments of 5 to 9 × 103 nucleotides attached to the bromodeoxyuridine leaders comprised approximately 40 to 50% of the DNA synthesized in the complete subcellular system. The remainder of the DNA consisted of short fragments of 6-13 S that were initiated in vitro, but failed to be ligated to longer strands. The latter appears to accumulate in the subcellular system as the result of a partial interruption of the native DNA replication process. Omission of the 105 000g cytoplasmic supernatant from the reaction mixture reduced the lengths of the DNA segments, synthesized in vitro and the amount of DNA that was replicated. No significant amount of repair synthesis occurred on the template DNA and the initiation of replication in new replicons was restricted in the nuclear system. The data, however, support the concept that DNA synthesis in isolated nuclei continues largely from sites that were actively replicating in the living cells and that isolated nuclei provide a system for dissection of the molecular processes involved in the replication of chromosomal DNA.

Original languageEnglish (US)
Pages (from-to)2778-2782
Number of pages5
JournalBiochemistry
Volume16
Issue number12
StatePublished - 1977
Externally publishedYes

Fingerprint

DNA
Growth
DNA Replication
Bromodeoxyuridine
Replicon
Cell Nucleus
S Phase
Sucrose
Dissection
Nucleotides
Sedimentation
Labels
In Vitro Techniques
Repair
Cells
Irradiation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Planck, S., & Mueller, G. C. (1977). DNA chain growth in isolated HeLa nuclei. Biochemistry, 16(12), 2778-2782.

DNA chain growth in isolated HeLa nuclei. / Planck, Stephen; Mueller, Gerald C.

In: Biochemistry, Vol. 16, No. 12, 1977, p. 2778-2782.

Research output: Contribution to journalArticle

Planck, S & Mueller, GC 1977, 'DNA chain growth in isolated HeLa nuclei', Biochemistry, vol. 16, no. 12, pp. 2778-2782.
Planck S, Mueller GC. DNA chain growth in isolated HeLa nuclei. Biochemistry. 1977;16(12):2778-2782.
Planck, Stephen ; Mueller, Gerald C. / DNA chain growth in isolated HeLa nuclei. In: Biochemistry. 1977 ; Vol. 16, No. 12. pp. 2778-2782.
@article{f9657486856f4693b4fe077a15782899,
title = "DNA chain growth in isolated HeLa nuclei",
abstract = "A new method for studying DNA chain growth has been used to investigate DNA replication in isolated nuclei. For this purpose, DNA replication was initiated in S-phase cells with bromodeoxyuridine in order to photosensitize active replicons. The nuclei were then isolated and caused to synthesize DNA in vitro with [3H]dTTP as a precursor to label growing chains. Subsequent irradiation with 313-nm light fragments the bromodeoxyuridine-DNA leaders and releases the contiguous, newly replicated DNA chains for analysis by sedimentation through alkaline sucrose gradients. This method of analysis provides data on the length of the DNA chain segments that are actually synthesized in vitro and on the fraction of these DNA chains that extends from bromodeoxyuridine-prelabeled replicons. Segments of 5 to 9 × 103 nucleotides attached to the bromodeoxyuridine leaders comprised approximately 40 to 50{\%} of the DNA synthesized in the complete subcellular system. The remainder of the DNA consisted of short fragments of 6-13 S that were initiated in vitro, but failed to be ligated to longer strands. The latter appears to accumulate in the subcellular system as the result of a partial interruption of the native DNA replication process. Omission of the 105 000g cytoplasmic supernatant from the reaction mixture reduced the lengths of the DNA segments, synthesized in vitro and the amount of DNA that was replicated. No significant amount of repair synthesis occurred on the template DNA and the initiation of replication in new replicons was restricted in the nuclear system. The data, however, support the concept that DNA synthesis in isolated nuclei continues largely from sites that were actively replicating in the living cells and that isolated nuclei provide a system for dissection of the molecular processes involved in the replication of chromosomal DNA.",
author = "Stephen Planck and Mueller, {Gerald C.}",
year = "1977",
language = "English (US)",
volume = "16",
pages = "2778--2782",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "12",

}

TY - JOUR

T1 - DNA chain growth in isolated HeLa nuclei

AU - Planck, Stephen

AU - Mueller, Gerald C.

PY - 1977

Y1 - 1977

N2 - A new method for studying DNA chain growth has been used to investigate DNA replication in isolated nuclei. For this purpose, DNA replication was initiated in S-phase cells with bromodeoxyuridine in order to photosensitize active replicons. The nuclei were then isolated and caused to synthesize DNA in vitro with [3H]dTTP as a precursor to label growing chains. Subsequent irradiation with 313-nm light fragments the bromodeoxyuridine-DNA leaders and releases the contiguous, newly replicated DNA chains for analysis by sedimentation through alkaline sucrose gradients. This method of analysis provides data on the length of the DNA chain segments that are actually synthesized in vitro and on the fraction of these DNA chains that extends from bromodeoxyuridine-prelabeled replicons. Segments of 5 to 9 × 103 nucleotides attached to the bromodeoxyuridine leaders comprised approximately 40 to 50% of the DNA synthesized in the complete subcellular system. The remainder of the DNA consisted of short fragments of 6-13 S that were initiated in vitro, but failed to be ligated to longer strands. The latter appears to accumulate in the subcellular system as the result of a partial interruption of the native DNA replication process. Omission of the 105 000g cytoplasmic supernatant from the reaction mixture reduced the lengths of the DNA segments, synthesized in vitro and the amount of DNA that was replicated. No significant amount of repair synthesis occurred on the template DNA and the initiation of replication in new replicons was restricted in the nuclear system. The data, however, support the concept that DNA synthesis in isolated nuclei continues largely from sites that were actively replicating in the living cells and that isolated nuclei provide a system for dissection of the molecular processes involved in the replication of chromosomal DNA.

AB - A new method for studying DNA chain growth has been used to investigate DNA replication in isolated nuclei. For this purpose, DNA replication was initiated in S-phase cells with bromodeoxyuridine in order to photosensitize active replicons. The nuclei were then isolated and caused to synthesize DNA in vitro with [3H]dTTP as a precursor to label growing chains. Subsequent irradiation with 313-nm light fragments the bromodeoxyuridine-DNA leaders and releases the contiguous, newly replicated DNA chains for analysis by sedimentation through alkaline sucrose gradients. This method of analysis provides data on the length of the DNA chain segments that are actually synthesized in vitro and on the fraction of these DNA chains that extends from bromodeoxyuridine-prelabeled replicons. Segments of 5 to 9 × 103 nucleotides attached to the bromodeoxyuridine leaders comprised approximately 40 to 50% of the DNA synthesized in the complete subcellular system. The remainder of the DNA consisted of short fragments of 6-13 S that were initiated in vitro, but failed to be ligated to longer strands. The latter appears to accumulate in the subcellular system as the result of a partial interruption of the native DNA replication process. Omission of the 105 000g cytoplasmic supernatant from the reaction mixture reduced the lengths of the DNA segments, synthesized in vitro and the amount of DNA that was replicated. No significant amount of repair synthesis occurred on the template DNA and the initiation of replication in new replicons was restricted in the nuclear system. The data, however, support the concept that DNA synthesis in isolated nuclei continues largely from sites that were actively replicating in the living cells and that isolated nuclei provide a system for dissection of the molecular processes involved in the replication of chromosomal DNA.

UR - http://www.scopus.com/inward/record.url?scp=0017387094&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0017387094&partnerID=8YFLogxK

M3 - Article

C2 - 889787

AN - SCOPUS:0017387094

VL - 16

SP - 2778

EP - 2782

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 12

ER -