Replication of polyoma DNA in isolated nuclei: analysis of replication fork movement

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In discontinuous polyoma DNA replication, the synthesis of Okazaki fragments is primed by RNA. During viral DNA synthesis in nuclei isolated from infected cells, 40% of the nascent short DNA fragments had the polarity of the leading strand which, in theory, could have been synthesized by a continuous mechanism. To rule out that the leading strand fragments were generated by degradation of nascent DNA, they were further characterized. DNA fragments from a segment of the genome which replication forks pass in only one direction were strand separated. The sizes of the fragments from both strands were similar, suggesting that one strand was not specifically degraded. Most important, however, the majority of the Okazaki fragments of both strands were linked to RNA at their 5' ends. For identification, the RNA was labeled at the 5' ends by [beta-32P]GTP, internally by [3H]CTP, [3H]GTP, and [3H]UTP, or at the 3' ends by 32P transfer from adjacent [32P]dTMP residues. All three kinds of labeling indicated that an equal proportion of DNA fragments from the two strands was linked to RNA primers.

Section: Downloaded Frommentioning

confidence: 99%

Discontinuous synthesis of both strands at the growing fork during polyoma DNA replication in vitro

Närkhammar-Meuth¹,

Eliasson²,

Magnusson³

1981

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“…Because a full-length fragment is labeled only after the fork has passed entirely through it, analyzing the relative amounts of labeled nucleotides incorporated into individual restriction fragments during replication (8,17) can only provide a measure of regions already replicated rather than a direct way to analyze the movement of the actual replication fork. For example, labeled fragments near the origin may be derived from early, middle, or late replicative intermediates as well as from mature form I molecules terminated during the labeling period.…”

Section: Discussionmentioning

confidence: 99%

“…As a result, analysis of label incorporated only into the fulllength restriction fragments may underestimate the late replicative intermediates and conse-quently may overestimate the proportion of the label incorporated into regions close to the replication origin. This caveat is particularly germane to analyses based on data solely from the full-length restriction fragments as a measure of de novo initiation (8,17).…”

Section: Discussionmentioning

confidence: 99%

“…Although intact intermediates of replication have been analyzed by.sedimentation, gel electrophoresis, and electron microscopy (2,4,18,19,22,26), each of these techniques can provide only limited information about fork movement through individual regions of the genome. Fork movement has been followed by incorporation of radioactive precursors into replicating DNA with subsequent analysis of the relative amount of label incorporated into regions already replicated (8,17) or by measurement of the lengths of daughter strands (25). Neither of these approaches analyzes the intact replication forks or replication intermediates.…”

mentioning

confidence: 99%

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Isolation and characterization of replication forks from discrete regions of the polyoma genome

Buckler-White¹,

Pigiet²

1982

Synthesis of polyoma DNA in nuclei isolated from virus-infected 3T6 mouse fibroblasts leads to the selective labeling of replicative intermediates. Digestion of these replicative intermediates with the restriction endonuclease HpaII resulted in three highly labeled heterogeneous species in addition to the expected full-length fragments. These three species migrated more slowly in agarose than did any of the full-length restriction fragments and were shown to represent families of replication forks by criteria of sensitivity to S1 nuclease, kinetics of labeling both in vitro and in vivo, electron microscopy, and migration behavior during agarose gel electrophoresis. Subsequent digestion with other restriction enzymes showed that the two largest of the three fork bands originated from HpaII fragments 1 and 2. These fragments flank the putative terminus located 180 degrees relative to the origin. The third fork-containing band was less labeled and was derived from fragment 3, which is juxtaposed to the replication origin on the side corresponding to late transcription. A two-dimensional gel system revealed the presence of a fourth fork band, derived from fragment 4, that was obscured by full-length fragments 1 and 2 in the single-dimension electrophoresis. Resolution of the fork families revealed multiple discrete species within the major bands, implying the existence of stops or hesitations during replication of a given region of the genome. This conclusion is consistent with the presence of multiple species upon electrophoresis of the fork bands under denaturing conditions.

“…That this could be the case in our system is highly unlikely. First, almost all polyoma DNA molecules have been shown to replicate bidirectionally from one defined origin (15). Second, our virus stock was prepared from a doubly plaque-purified virus, and the cleavage pattern generated by digestion with HpaII of polyoma form I DNA synthesized during infection with this stock was normal.…”

Section: Fraction Numbermentioning

confidence: 99%

Both strands of polyoma DNA are replicated discontinuously with ribonucleotide primers in vivo

Närkhammar-Meuth¹,

Kowalski²,

Denhardt³

1981

Nascent polyoma DNA molecules were isolated after pulse-labeling of infected murine 3T6 cells with [3H]thymidine. The extent of digestion of these DNA molecules by spleen exonuclease was increased by exposure to alkali or RNase, suggesting that ribonucleotides were present at or near the 5' terminal of the newly synthesized pieces of DNA. Intermediates shorter than 300 nucleotides were hybridized to the separated strands of restriction enzyme fragments of the polyoma genome: 2.5 to 3-fold more radioactivity was found in the strand whose synthesis is necessarily discontinuous (the lagging strand) than in the strand whose synthesis is potentially continuous (the leading strand) than in the strand whose synthesis is potentially continuous (the leading strand). Separation of the strands of [5'-32P]DNA molecules showed that the excess [3H]thymidine in lagging-strand molecules was not simply the result of an increased number of molecules. Therefore, assuming equivalent efficiencies of labeling, lagging-strand pieces must be slightly longer than those with leading-strand polarity. The presence of ribonucleotides on the 5' termini of molecules with both leading- and lagging-strand polarity was demonstrated by (i) release of 32P-ribonucleoside diphosphates upon alkaline hydrolysis of [5'-32P]DNA separated according to replication polarity and (ii) the change in the degree of self-annealing of nascent molecules upon preferential degradation of DNA molecules possessing initiator RNA moieties by spleen exonuclease. We conclude that replication of polyoma DNA in vivo occurs discontinuously on both sides of the growing fork, using RNA as the major priming mechanism.