pair of adjacent origins continues until the forks meet karyotic DNA replication forks has come from studies of . origin of bidirectional DNA replication. Replication Origins for the Duplication of Telomeric DNA and to the initiation of DNA synthesis at bidirectional replication forks [18,20,21]. continue along a chromosome until they meet forks coming from adjacent origins, or they reach. Multiple Origins and Bidirectional replication in eukaryotes The DNA duplex in a eukaryotic The replication forks of adjacent bubbles fuse where they meet.
In cells such as yeast, loss of nucleotides during replication is balanced by addition of nucleo- tides by telomerase. In humans, however, telomerase is turned off in most somatic cells early in development, so that chromosomes become shorter with increasing rounds of replication.
Consider one round of rep- lication in a human somatic cell. Which one of the following statements correctly describes the status of the two daughter chromosomes relative to the parent chromosome?
One daughter chromosome will be shorter at one end; the other daugh- ter chromosome will be normal at both ends. One daughter chromosome will be shorter at both ends; the other daugh- ter chromosome will be normal at both ends.
One daughter chromosome will be shorter at both ends; the other daugh- ter chromosome will be shorter at only one end. Both daughter chromosomes will be shorter at one end, which is the same end in the two chromosomes.
Both daughter chromosomes will be shorter at one end, which is the opposite end in the two chromosomes. Both daughter chromosomes will be shorter at both ends. Identify the four replication bubbles in Figure 5— Indicate the approxi- mate locations of the origins at which each replication bubble was initi- ated, and label the replication forks 1 through 8 from left to right across the figure.
Estimate how long it will take until forks 4 and 5 collide with each other. How long will it take until forks 7 and 8 collide? Each nucleotide in DNA occupies 0. For this problem disregard the nucleosomes evi- dent in Figure 5—10 and assume that the DNA is fully extended.
Recall that the human genome comprises a total of 6. By breaking open the 0.
You overlay the DNA with a photographic emulsion, and expose it for 3 to 6 months, a procedure known as autoradiography. Because the emulsion is sensitive to radioactive emissions, the 3H-labeled DNA shows up as tracks of silver grains. Because the stretching collapses replication bubbles, the daugh- ter duplexes lie side by side and cannot be distinguished from each other.
You pretreat the cells to synchronize them at the beginning of S phase.
In other words, do human origins fire in clusters in adherence with the yeast model? Multiple pause sites due to the local chromatin structure in yeast mutants deficient for the Rrm3p helicase suggest that fork obstruction might be multimodal in rRNA genes 20 In human rRNA genes, the programmed fork barrier and its position are conserved In contrast to the case in Saccharomyces cerevisiae, forks traveling in both directions are blocked and a significant proportion of forks traverse the boundary unaffected.
Recently, a more complex organization of human rRNA genes was described, revealing a fundamental divergence from the standard model of clusters of highly repetitive, nearly identical rRNA gene units 6.
DNA Replication Origin Plasticity and Perturbed Fork Progression in Human Inverted Repeats
A significant proportion of the genes are organized into palindromes interspersed among the tandem repeats, yielding a mosaic of noncanonical and canonical rRNA genes. Palindromes are acknowledged sources of genomic instability, which has been proposed to result from problematic DNA replication, at least in yeast 1825 While most rRNA gene replication studies have been carried out in yeast, rRNA gene palindromes do not exist in this organism.
Tetrahymena thermophila does contain rRNA gene palindromes that undergo amplification through multiple initiation events on the same fragment of DNA and activation of a fork barrier at the center of the palindrome Therefore, an account of where origins fire from and how forks travel in the context of human rRNA gene palindromes would provide clues as to how the stability of the locus is maintained in this species.
Molecular combing 327 produces sufficiently large DNA molecules on which multiple active origins and progressing forks can be monitored at the level of a single molecule 21931 Since the original order of genes and flanking nontranscribed spacers is preserved during the combing process, replication can be attributed to either canonical or noncanonical rRNA genes, a prerequisite if differences between the programs of these two classes of human rRNA genes are to be delineated.
No significant initiation differences, both in origin spacing and in timing, were observed in relation to the underlying rRNA gene physical structure. These results suggest that while initiation mechanisms in humans are amenable to rRNA gene rearrangement, replication forks are sensitive to rRNA gene palindromes, and that these inverted repeats represent obstacles to fork progression. One day prior to labeling, cells were seeded in a T75 flask. Cells were then harvested, and genomic DNA was extracted and combed as previously described Hybridization and fluorescence revelation.
The antibodies used for fluorescence detection of probe and replication signals were as follows: Image acquisition and analysis.
Fluorescent signals were captured with a Zeiss Axioplan 2 microscope equipped with a Photometrics HQ charge-coupled device camera using SmartCapture software Digital Scientific. Probe and replication signals were measured with Cartographix Institut Pasteur. Analyses were performed on DNAs from two separate labeling experiments.