On the Size of the DNA in the Mammalian Chromosome

Lett, J. T.; Klucis, E.; Sun, Chenglin

doi:10.1016/s0006-3495(70)86300-7

Cited by 145 publications

(37 citation statements)

References 17 publications

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“…3). The plateau for control cells represents the maximum M-rs that can be reliably observed for single-stranded DNA (26,27 sponding to the period of the plateaus, the profiles were bimodal for GM637, but such fine structure in profiles was less clear for the other cell types. One component of the bimodal peak for GM637 was similar in size to parental DNA and the other was smaller ( Figs.…”

Section: Methodsmentioning

confidence: 97%

Postreplication repair: questions of its definition and possible alteration in xeroderma pigmentosum cell strains.

Park¹,

Cleaver²

1979

Proc. Natl. Acad. Sci. U.S.A.

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DNA synthesis in normal cells and in excision-defective and variant xeroderma pigmentosum cells was investigated after irradiation with ultraviolet light. The sizes of DNA synthesized during brief pulses of [3Hlthymidine 1-2 hr after irradiation were decreased, the xeroderma pigmentosum variant showing the smallest molecular weight. Once DNA replication in mammalian cells is disturbed in various ways by agents that damage DNA. These disturbances include a decline and recovery in the rate of DNA synthesis (1-4), a decrease in the size of newly synthesized DNA (5, 6), altered linkage of newly synthesized fragments to higher molecular weight DNA (5,6), and a decrease in the number of actively synthesizing replicons (7). Changes in the size of newly synthesized DNA have been interpreted in terms of a model, "postreplication repair," originally proposed for prokaryotes (8). According to this model, DNA damage (e.g., pyrimidine dimers produced by UV light) interrupts DNA chain growth, which then resumes beyond the damaged site, leaving a gap opposite the damage that can be filled by recombination or de novo synthesis.We studied the kinetics of DNA synthesis in UV-damaged normal and xeroderma pigmentosum (XP) cells, using both excision-repair-deficient and variant forms of XP.(9, 10), to investigate the rate at which short fragments of DNA synthesized in irradiated cells increased in size with time after pulse-labeling. We assumed that this would allow us to (i) observe the increase in size of DNA chains initially blocked by damaged sites, (ii) define the rate of this process, (iii) identify defects in various XP cells, and (iv) delineate sites of action of caffeine in inhibiting DNA replication. This experimental approach has been used widely to observe postreplication repair in mammalian cells (5,6,(10)(11)(12)(13)(14).On the basis of our results we present an alternative to the prokaryotic postreplication repair model of Rupp and Howard-Flanders (8) and explain a wider range of DNA replication phenomena in UV-damaged mammalian cells. MATERIALS AND METHODSCell Growth and Labeling. Primary and simian virus 40 (SV40)-transformed human cells were grown in Eagle's minimal essential medium with 15% fetal calf serum. Cell strains with normal excision repair were GM498 (primary skin fibroblasts) and GM637 (SV40-transformed skin fibroblasts). XP cell strains were an excision-defective XP12RO (SV40-transformed group A) and an untransformed XP variant, XP4BE.Cells were grown in 60-mm petri dishes in [14C]dThd (0.01 1uCi/ml, 64 mCi/mmol; 1 Ci = 3.7 X 1010 becquerels) until required for use, at which time the medium was replaced with nonradioactive medium for 5-20 hr. Cells were then irradiated with 0-10 J of 254-nm UV light per m2 at an incident dose rate of 1.3 J/(m2.s), grown for 0-2 hr, labeled for 10 min with[3H]dThd (10 ACi/ml, 50 Ci/mmol), and "chased" in nonradioactive medium containing 0.1 mM dThd and 10,gM deoxycytidine. The 3H to 14C ratio remained unchanged (within 20%) during this period, indicating that l...

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Section: Methodsmentioning

confidence: 97%

Postreplication repair: questions of its definition and possible alteration in xeroderma pigmentosum cell strains.

Park¹,

Cleaver²

1979

Proc. Natl. Acad. Sci. U.S.A.

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“…Biochem. 50 (1975) were prepared in polyallomer tubes of a Beckman SW27 rotor [28]. A lysis solution containing 0.5 M NaOH, 0.02 M EDTA, and 0.1 % Nonidet P40 was layered on top of the gradients followed by 0.5 ml of sample containing 2 x lo5 cells [22].…”

Section: Alkaline Sucrose Gradientsmentioning

confidence: 99%

Discontinuous DNA Replication in Mouse P‐815 Cells

Gautschi

Clarkson

1975

European Journal of Biochemistry

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The length of newly synthesized DNA strands from mouse P-815 cells was analyzed after denaturation both by electrophoresis and by sedimentation in alkaline sucrose gradients.[3H]-Thymidine pulses of 2-8 min at 37 "C predominantly label molecules of 20-60 S. With 30-s pulses at 2 5 T , all the [3H]thymidine appears in short DNA strands of 50-200 nucleotides. Thus, DNA strand elongation occurs discontinuously via Okazaki fragments at both the 5' end and the 3' end.In dodecylsulfate lysates, only 10% of the Okazaki fragments are found as single-stranded molecules. About 90 are resistant to hydrolysis by the single-strand-specific nuclease S, and band in isopycnic gradients at the buoyant density of double-stranded DNA. No evidence for ribonucleotides at the 5' end of Okazaki fragments was obtained either in isopycnic CsCl or Cs2S04 gradients or after incubation with polynucleotide kinase and [Y-~~PIATP.Semiconservative replication of Escherichia coli DNA and T4 phage DNA is discontinuous and involves the 5'-3' synthesis of 9-S precursor molecules, Okazaki fragments, on both daughter strands at each replication fork [l -31. Similar replication intermediates of various sizes have been described in other bacteria and phages as well as in eukaryotes (see [4]). These intermediates also occur during replication of Simian virus 40 (SV40) [5] and polyoma virus DNA [6,7] ; they are 3 -5 S long.Recently, "primer" RNA molecules covalently attached to nascent Okazaki fragments have been demonstrated in E. coli [3] and in isolated nuclei synthesizing polyoma DNA [6,8]. Evidence for similar RNA-DNA copolymers was also found in Physarum polycephafum [9,10] and in an established cell line of human lymphocytes [ll].The reports on replication intermediates in mammalian cells [lo, 12-20], however, vary with respect to estimations of size, lifetime, and buoyant density. Structure also became a point of interest when, as in previous reports [15,19,21], newly synthesized DNA was recovered from lysates as single-stranded molecules without using specific conditions for DNA denaturation,In order to define different characteristics of DNA replication in the same system, we studied mouse Their fiber autoradiographic studies suggest that nuclear DNA of mammalian cells is composed of tandemly joined sections (15 to 60 pm) which are replicated bidirectionally from one origin [24]. The two growing points in each replication unit form forklike structures which are displaced from the origin at an estimated rate of about 1 pm/min. Units adjacent to each other replicate predominantly synchronously. These replication units will be referred to in this paper as "replicons". (Note that this definition of the term "replicon" is not identical to the original one for bacterial systems [25]. One should emphasize that in mammalian cells the timing of operation of individual "replicons" is programmed [26].) In HeLa cells we found replication intermediates [22] with sizes corresponding on the average to about half the length of autoradiographically visualized ...

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“…There has always been a need to develop fast and sensitive methods for monitoring DNA damage, particularly in mammalian systems. Over the years, a number of biochemical methods based on alkaline sucrose gradients (Lett, 1970), alkaline elution (Kohn 1976), alkaline unwinding (Rydberg, 1980), alkaline gel electrophoresis (Freeman 1986), and the single-cell gel electrophoresis (SCGE) (Singh et al 1988) have been developed for monitoring the DNA single strand breaks (Chaubey et al 2001). Single cell gel electrophoresis (SCGE) assay, or comet test is a very simple, rapid, and sensitive technique for measuring the DNA damage (Singh et al 1988;Fairbairn et al 1995;Demir et al 2011;Demir 2012;Demir and Kaya 2013;Demir et al 2014).…”

Section: Introductionmentioning

confidence: 99%