At what step is the rate of DNA replication in E. coli is regulated - initiation, elongation or termination?
The following problem further illustrates the analysis of replication by pulse-labeling, using a hypothetical virus and constructed data. Consider the replication of a circular viral DNA in infected cells. The infected cells were pulse labeled with [3H] thymidine for 1, 2, 3 and 4 min; it takes 4 min for the DNA molecules to be replicated in this system (from initiation to termination). Those DNA molecules that had completed synthesis at each time point were isolated, cut with a restriction endonuclease, and assayed for radioactivity in each fragment. This restriction endonuclease cleaves the circular DNA into 6 fragments, named A, B, C, D, E, and F in a clockwise orientation around the genome. The following results were obtained; a plus (+) means the fragment was radioactively labeled, and a minus (-) means it was not labeled.
|Fragment||Time of labeling (min)|
- What restriction fragment has the origin and which has the terminus of replication?
- In which direction(s) does this viral DNA replicate?
The two-dimensional gels developed by Brewer and Fangman were used to examine the origin of replication of a DNA molecule. In this system, replicating molecules are cleaved with a restriction endonuclease and separated in two dimensions. The first dimension separates on the basis of size, and the second separates on the basis of shape (more pronounced deviations from linearity move slower in the second dimension). After blotting the DNA onto a membrane, it is probed with fragments from the replicon under study. Restriction fragment P gives the pattern shown on the left, and the adjacent fragment Q gives the pattern shown on the right. The dotted line denotes the diagonal expected if all molecules were linear. Assuming both P and Q are in the same replicon, what can you conclude about the positions of origins of replication?
Dr. Howard Cedar and his colleagues at the Hebrew University in Jerusalem have developed a replication direction assay to map origins of replication on chromosomes (Kitsberg et al., Nature 366: 588-590, 1993). Growing cells are treated with the drug emetine to inhibit lagging strand synthesis. Leading strand synthesis continues, and this newly synthesized DNA is density labeled by incorporating 5-bromodeoxyuridylate (5-bromodeoxyuridine is added to the medium). The DNA is then sheared and denatured, and the newly synthesized leading strand DNA is separated from the rest of the DNA by sedimentation equilibrium on Cs2SO4 gradients. Samples of the heavy density DNA (containing 5-bromodeoxyuridylate) are spotted onto a membrane, and equal amounts are hybridized to labeled, separated strands of restriction fragments throughout a region.
Use of this approach to map replication origins in the human b-like globin gene cluster led to results like those below. The names of the genes are given above the line, and the names of the restriction fragments are given below the line. A + means that the leading strand (with 5-bromodeoxyuridylate incorporated) hybridized preferentially to a labeled probe corresponding to the designated strand, whereas a - means that the leading strand DNA did not hybridize to the designated probe. The genes are transcribed from left to right in this diagram, so the "top" strand reads the same as the mRNA in the coding regions (our convention is "nontemplate") and the "bottom" strand (abbreviated "bot") is complementary to the top strand ("template” or "antisense" strand).
e Gg Ag yh d b
A B C D E F G H I J K L M
+ + + + + + + + + + + - - top
- - - - - - - - - - - + + bot
- Which restriction fragment(s) contain(s) the origin of replication?
- Is replication from this origin uni- or bi-directional?
- Explain how the data led you to your answers to a and b.
- What direction is the replication fork moving for fragments A through K?
- What direction is the replication fork moving for fragments L and M?
- Name a possible target enzyme that could specifically block lagging strand synthesis when inhibited.
- What cloning vector would be useful for generating the separated strands of the restriction fragments?
Let's imagine that you have isolated a new virus with a double-stranded, circular DNA that is 6000 bp long. The restriction endonuclease HhaI cleaves the DNA as shown below to generate 6 fragments.
You initially use a pulse-labeling procedure to map the origin and terminus of replication. Infected cells were first allowed to incorporate [32P] phosphate into the DNA for several hours to uniformly label the DNA, and then [3H] thymidine was added for short periods of time (pulse labels), i.e. 5, 10 and 15 min. Completed viral DNA molecules were isolated, cut with HhaI, and separated on polyacrylamide gels. The amount of [32P] and [3H] in each fragment was determined for each period of pulse label and is tabulated below. The data are corrected for thymidine content and normalized so that fragment A has a ratio of 1.
|Fragment||5 min||10 min||15 min|
- Which HhaI fragment(s) contain(s) the origin and terminus of replication?
- What is the mode (uni- or bi-directional, or other) and direction(s) of replication (i.e. clockwise and/or counterclockwise)?
- To confirm this result and map the origin and terminus more precisely, you analyzed the replicative intermediates on 2-dimensional gels. The DNA from infected cells, containing viral DNAs at all stages of synthesis, was digested with HhaI and then run initially on a gel that separates on the basis of size and then in a perpendicular direction in a gel that accentuates separations based on shape (Brewer and Fangman gels). The DNA in the gel was blotted onto a nylon membrane and hybridized with radiolabeled probes for the viral DNA fragments. The hybridization patterns obtained for HhaI fragments A, C and D are shown. The hypothetical line for linear intermediates of a fragment expanding from unit length to twice unit length is provided as a guide. How do you interpret these data, and what do you learn about the origin and terminus? Please indicate the significance of any transitions in the patterns.
- (d) You also used a replication direction assay to examine the replication origin. Virally infected cells were treated with the drug emetine to inhibit lagging strand synthesis. Leading strand synthesis continued during the drug treatment, and this newly synthesized DNA was density labeled by incorporating 5-bromodeoxyuridylate (5-bromodeoxyuridine is added to the medium). The DNA was sheared and denatured, and the newly synthesized leading strand DNA was separated from the rest of the DNA by sedimentation equilibrium on Cs2SO4 gradients. Samples of the heavy density DNA (containing 5-bromodeoxyuridylate) were spotted onto a membrane, and equal amounts are hybridized to labeled, separated strands of restriction fragments throughout the virus. To keep track of strands and orientation in this problem, lets imagine the duplex circle to have an outerstrand oriented 5' to 3' in a clockwise direction and an innerstrand oriented 5' to 3' in a counterclockwise direction, as diagrammed below.
A grid of samples of heavy density DNA (containing 5-bromodeoxyuridylate, and enriched for leading strand DNA) immobilized on the filter is shown below, with each rectangle representing an equal loading of the heavy density DNA. What will be the pattern of hybridization to the indicated strands of each of the restriction fragments?
What does this experiment tell you about the origin and terminus of replication?
Are the following statements about the function of the DnaA protein true or false?
- DnaA protein binds to 9-mer (nonamer) repeats at the origin for chromosomal replication.
- DnaA protein catalyzes the formation of the primers for leading strand synthesis at the origin.
- About 20 to 40 monomers of the DnaA protein form a large complex at the origin.
- DnaA protein melts DNA at a series of 13-mer repeats at the origin.
Consider a bacterium with a circular chromosome with one replication origin. It takes 30 min for bi-directional replication to copy its chromosome (the elongation time or C period) and 10 min from the end of DNA synthesis until the cell divides (the D period). How many replication forks are needed per chromosome to allow a culture of this bacterium to double in cell number every 20 min? Follow the molecules through a complete cell division cycle.
In many eukaryotes, actively transcribed genes are replicated early in S phase and inactive genes are replicated late. One assay to determine replication timing is in situ hybridization of cells with a gene-specific, fluorescent probe, followed by examination of the number of signals per nucleus. In diploid cells, an unreplicated gene will be seen as 2 fluorescent dots per nucleus, whereas a replicated gene will be seen as 4 dots. They look like 2 doublets, indicating that the replicated chromatids are close in the nucleus.
The types of pattern one can see at various stages of the cell cycle are shown below. Each dark dot is a fluorescent signal, the larger circle is the cell, and the smaller circle is the nucleus.
The fraction of cells in an asynchronous population with 2 dots or 4 dots is then tabulated. In an asynchronous population, the number of cells in each phase of the cell cycle is directly proportional to the length of that phase. If GENEAwere replicated 1 hr after entry into S phase, and GENEBwere replicated 1 hr before the end of S phase, what fraction of cells would show 4 dots (two doublets) for each? The length of each phase of the cell cycle is given in the figure, and the vertical arrowhead shows the time of synthesis. The time from synthesis of each gene until the beginning of G2 is shown above a horizontal line. Consider cells in M to have 4 dots (i.e., assume that the transition from 4 dots to 2 occurs at the M to G1 boundary).