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Structural analysis of pulse-labeled DNA molecules

One approach is to label the newly synthesized DNA in an asynchronous population of DNA molecules for short periods of time (called pulse-labeling), isolate completed DNA molecules and then monitor the appearance of radioactive label in particular restriction fragments. Since the molecules are not replicating synchronously, some of the DNA molecules will be completed during a short pulse label, and the others will incorporate the radiolabel internally. As shown in Fig. 6.2, those DNA molecules that completed replication during the short pulse label will have radioactive label in the restriction fragment containing the terminus of replication. When the replicating molecules are labeled for a longer time (longer pulse), the completed DNA molecules will have radioactive label not only in restriction fragments containing the terminus, but also in adjacent fragments. The origin of replication will only be labeled with the pulse time is extended to the period required for complete synthesis of the DNA molecule. Thus in this procedure, when asynchronously replicating molecules are labeled for a series of pulse periods of increasing length and completed DNA molecules examined at the end of each pulse, the the terminus of replication will be labeled at the earliest time points, whereas those containing an origin will be labeled last.

 

 

Figure 6.2.Distribution of radioactivity in pulse-labeled daughter DNA molecules at different times of synthesis. The thin black horizontal lines represent unlabeled, replicating DNA molecules at various stages of completion. The origin (ori) is at the left and the terminus (term) is at the right. The thick gray lines are radioactively labeled portions of the replicating DNA molecules. After a 5 min pulse, some of the DNA molecules shown at time 0 have completed synthesis. These are collected, digested with a restriction endonuclease at the sites marked by vertical lines. Note that the restriction fragments containing the terminus of replication will have radioactive label after a short pulse. In the third panel, the results of a longer pulse labeling period is shown. The labeling time has been long enough for molecules that initiated synthesis after addition of the radioactive label to complete synthesis. These latter molecules will have label in restriction fragments containing the origin. Hence in this protocol, label appears in restriction fragments containing the origin only at longer pulse periods.

 

This concept came from an approach used by Dintzis to measure the direction of protein synthesis in the mid-1960’s (see Chapter V in Part Three). It can be confusing, so let’s try an analogy. Imagine that 20 students are writing essays using word processors set to use black letters. They are all at different stages of completing their papers, and they are not revising or editing their essays – just writing them from start to finish. At a defined time, all the word processors are switched to using red letters. As each student completes their paper, they print them out and turn them in. Essays by students who were almost finished when the font color was switched will have red text only at the end. Essays by students who were half-way through their essay when the the color was switched will have red text for the last half. Those by students who were just beginning their essays when the letter color was switched will have red text throughout, including the beginning. The switch of letters to red is analogous to the pulse labeling of DNA molecules with radioactivity. Just as the essays completed shortly after the color change will red text only at the end, so DNA molecules that finish replication during a short pulse label will have radioactive label at their terminus.

Once restriction enzymes had been discovered in the early 1970's, Dana and Nathans realized that they could use them to divide the mammalian polyoma virus, simian virus 40 (SV40) into discrete fragments. They used the following pulse-labeling procedure to identify the origin and termini of viral DNA synthesis. Monkey cells growing in culture were infected with SV40 and then pulse-labeled with [3H]thymidine for 5, 10 and 15 min. Completed viral DNA molecules were isolated, digested with restriction endonucleases from H. influenza(a mixture of HindII and HindIII), separated on a polyacrylamide gel, and the amount of [3H] incorporated into the DNA was determined. To normalize for the different sizes and base compositions of the restriction fragments, the [3H] counts were divided by the amount of [32P] in the same restriction fragments from DNA uniformly labeled with [32P]phosphate. As discussed above, when the length of the pulse-label is shorter than the time required to the complete synthesis of the DNA molecule, the label will first appear in the fragments closer to the terminus. As the pulse-labeled (newly synthesized) DNA appears in completed DNA molecules, a gradient of label was observed across the completed molecules, as shown in the following figures and table adapted from their paper.

 

Figure 6.3 Restriction map of SV40

 

Table 6.1. Appearance of radiolabel into restriction fragments of completed SV40 DNA molecules. The relative amount of pulse label from each restriction fragment is given below (the relative amount of pulse label is the 3H/32P ratio of each fragment, corrected for thymidine content and normalized to 1 for fragment A).

 

 

Relative amount of pulse label

 

 

Fragment

5 min

10 min

15 min

 

A

1.0

1.0

1.0

 

B

3.9

3.0

2.3

 

C

0

0.75

0.75

 

D

0.92

0.86

1.1

 

E

1.8

2.0

1.7

 

F

4.0

3.1

2.4

 

G

5.4

4.2

2.6

 

H

1.7

2.5

2.0

 

I

2.7

3.0

2.2

 

J

4.9

3.7

2.6

 

K

2.4

2.9

1.9

 

When the data on amount of pulse label in the Table is viewed with the restriction map of SV40, a clear pattern is seen. The temporal order of synthesis correlates well with the physical orders of the fragments along the chromosome. As Danna and Nathans expressed it, "there is a consistent gradient of labeling, indicating a specific order of synthesis of different parts of the SV40 DNA molecule. Since newly completed molecules were analyzed, fragments with the lowest amount of pulse label (C and D) are from that part of the DNA synthesized first. Fragments with the highest amount (G and J) are from that part of the DNA synthesized last."

Not only do these data identify the region of the chromosome with the origin (fragments C and D) and the the region with the terminus (fragments G and J), but one can also see that replication is bidirectional from that origin. The gradient of labeling is about the same on both "halves" of the SV40 genome (going either clockwise or counterclockwise from the C-D region), indicating bidirectional replication, with approximately equal rates of synthesis for the two forks.

Figure 6.4.A.

A plot of the relative amount of pulse label as a function of distance from the A-C junction illustrates the similarity in the gradient of labeling in the two halves of the molecule.

 

Fig. 6.4.B.

 

Question 6.2: What would the pattern be for unidirectional replication?

Question 6.3: What would be the pattern if there were two origins, say in fragments E and H, with bidirectional replication from each?