You are analyzing the intracellular DNA intermediates formed during the replication of the single-stranded (ss) DNA phage M13. In a replication mutant, you find accumulation of DNA molecules with the following properties: Results of a 0.7% agarose gel electrophoresis run at pH 8.0 – gel is running top to bottom, with the origin at the top of the diagram. The smudges indicate several bands with slightly different migration rates. open circle M13 closed circle M13 Left lane — markers (wild-type open circle M13 and closed circle M13) 2nd lane — DNA extracted from mutant M13 phage cells 3rd lane — DNA extracted from mutant M13 phage cells denatured 4th lane — DNA extracted from normal M13 phage cells Right lane — DNA extracted from normal M13 phage cells denatured Profiles from a 5 – 20% sucrose gradient centrifuging of the mutant M13 phage DNA The arrow on the neutral (pH 7.4) gradient shows the position of the covalently closed circular DNA. On the alkaline (pH 12.5) gradient, note two peaks from the same mutant M13 phage DNA, both of which are lighter than the original covalently closed circular DNA (the diagram is a bit misleading – the second peak is near “bottom” but that doesn’t mean it’s heavier than the 23S peak in the upper diagram). Results of a 0.7% agarose gel electrophoresis (left) and a nitrocellulose blot (right) of wild-type M13 phage DNA incubated with EcoR I, BamH I, Ava I, and Hind III Gel was run top to bottom; origin is at the top of the gel. Details on the nitrocellulose blot: After the gel was run and recorded, a nitrocellulose sheet was placed on the gel, and the DNA bands were transferred to the sheet. Separately, DNA from the mutant M13 phage was denatured, and the “smudge” portions (see the results of the first experiment) were isolated, then treated with alkaline phosphatase. After removal of the phosphatase, the DNA was labeled with polynucleotide kinase and [-32P] ATP. That now-radioactively labeled DNA was then incubated with the nitrocellulose sheet. One band showed hybridization (as shown); that is, one band had enough of a complementary sequence to the mutant DNA to allow it to anneal. How would you interpret each of these experimental results? What is the structure of the DNA intermediates in the wild-type and in the mutant phage?

You are analyzing the intracellular DNA intermediates formed during the replication of the single-stranded (ss) DNA phage M13. In a replication mutant, you find accumulation of DNA molecules with the following properties: Results of a 0.7% agarose gel electrophoresis run at pH 8.0 – gel is running top to bottom, with the origin at the top of the diagram. The smudges indicate several bands with slightly different migration rates. open circle M13 closed circle M13 Left lane — markers (wild-type open circle M13 and closed circle M13) 2nd lane — DNA extracted from mutant M13 phage cells 3rd lane — DNA extracted from mutant M13 phage cells denatured 4th lane — DNA extracted from normal M13 phage cells Right lane — DNA extracted from normal M13 phage cells denatured Profiles from a 5 – 20% sucrose gradient centrifuging of the mutant M13 phage DNA The arrow on the neutral (pH 7.4) gradient shows the position of the covalently closed circular DNA. On the alkaline (pH 12.5) gradient, note two peaks from the same mutant M13 phage DNA, both of which are lighter than the original covalently closed circular DNA (the diagram is a bit misleading – the second peak is near “bottom” but that doesn’t mean it’s heavier than the 23S peak in the upper diagram). Results of a 0.7% agarose gel electrophoresis (left) and a nitrocellulose blot (right) of wild-type M13 phage DNA incubated with EcoR I, BamH I, Ava I, and Hind III Gel was run top to bottom; origin is at the top of the gel. Details on the nitrocellulose blot: After the gel was run and recorded, a nitrocellulose sheet was placed on the gel, and the DNA bands were transferred to the sheet. Separately, DNA from the mutant M13 phage was denatured, and the “smudge” portions (see the results of the first experiment) were isolated, then treated with alkaline phosphatase. After removal of the phosphatase, the DNA was labeled with polynucleotide kinase and [-32P] ATP. That now-radioactively labeled DNA was then incubated with the nitrocellulose sheet. One band showed hybridization (as shown); that is, one band had enough of a complementary sequence to the mutant DNA to allow it to anneal. How would you interpret each of these experimental results? What is the structure of the DNA intermediates in the wild-type and in the mutant phage?

Biochemistry

6th Edition

ISBN:9781305577206

Author:Reginald H. Garrett, Charles M. Grisham

Publisher:Reginald H. Garrett, Charles M. Grisham

Chapter28: Dna Metabolism: Replication, Recombination, And Repair

Section: Chapter Questions

Problem 1P: Semiconservative or Conservative DNA Replication If 15N-Iabeled E. coli DNA has a density of 1.724...

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open circle M13
closed circle M13

Left lane — markers (wild-type open circle M13 and closed circle M13)
2nd lane — DNA extracted from mutant M13 phage cells
3rd lane — DNA extracted from mutant M13 phage cells denatured
4th lane — DNA extracted from normal M13 phage cells
Right lane — DNA extracted from normal M13 phage cells denatured
Profiles from a 5 – 20% sucrose gradient centrifuging of the mutant M13 phage DNA

The arrow on the neutral (pH 7.4) gradient shows the position of the covalently closed circular DNA.

On the alkaline (pH 12.5) gradient, note two peaks from the same mutant M13 phage DNA, both of which are
lighter than the original covalently closed circular DNA (the diagram is a bit misleading – the second peak is near
“bottom” but that doesn’t mean it’s heavier than the 23S peak in the upper diagram).

Results of a 0.7% agarose gel electrophoresis (left) and a nitrocellulose blot (right) of wild-type M13 phage DNA incubated with EcoR I, BamH I, Ava I, and Hind III

Gel was run top to bottom; origin is at the top of the gel.

Details on the nitrocellulose blot: After the gel was run and recorded, a nitrocellulose sheet was placed on the gel, and the DNA bands were transferred to the sheet. Separately, DNA from the mutant M13 phage was denatured, and the “smudge” portions (see the results of the first experiment) were isolated, then treated with alkaline phosphatase. After removal of the phosphatase, the DNA was labeled with polynucleotide kinase and [-32P] ATP. That now-radioactively labeled DNA was then incubated with the nitrocellulose sheet. One band showed hybridization (as shown); that is, one band had enough of a complementary sequence to the mutant DNA to allow it to anneal.

How would you interpret each of these experimental results? What is the structure of the DNA intermediates in the wild-type and in the mutant phage?

2. You are analyzing the intracellular DNA intermediates formed during the replication
of the single-stranded (ss) DNA phage M13. In a replication mutant, you find
accumulation of DNA molecules with the following properties:
-
Results of a 0.7% agarose gel electrophoresis run at pH 8.0 – gel is running top to
bottom, with the origin at the top of the diagram. The smudges indicate several bands
with slightly different migration rates.
open circle M13
closed circle M13
alkaline
sucrose
gradient
bottom
Left lane markers (wild-type open circle M13 and closed circle M13)
2nd lane - DNA extracted from mutant M13 phage cells
3rd lane DNA extracted from mutant M13 phage cells denatured
4th lane - DNA extracted from normal M13 phage cells
Right lane - DNA extracted from normal M13 phage cells denatured
Profiles from a 5 – 20% sucrose gradient centrifuging of the mutant M13 phage DNA
23S
neutral
sucrose
gradient
8S
Marker
top
Position in centrifuge tube
MUTANT
WILD TYPE
The arrow on the neutral (pH 7.4) gradient shows the
position of the covalently closed circular DNA.
On the alkaline (pH 12.5) gradient, note two peaks from
the same mutant M13 phage DNA, both of which are
lighter than the original covalently closed circular DNA
(the diagram is a bit misleading - the second peak is near
"bottom" but that doesn't mean it's heavier than the 23S
peak in the upper diagram).

Results of a 0.7% agarose gel electrophoresis (left) and a nitrocellulose blot (right) of
wild-type M13 phage DNA incubated with EcoR I, BamH I, Ava I, and Hind III
Bp
2900
1100
800
600
300
Agarose Gel
Nitrocellulose
Blot
Gel was run
top to bottom;
origin is at the
top of the gel.
Details on the nitrocellulose blot: After the gel was run and recorded, a nitrocellulose
sheet was placed on the gel, and the DNA bands were transferred to the sheet.
Separately, DNA from the mutant M13 phage was denatured, and the "smudge"
portions (see the results of the first experiment) were isolated, then treated with alkaline
phosphatase. After removal of the phosphatase, the DNA was labeled with
polynucleotide kinase and [y-3²P] ATP. That now-radioactively labeled DNA was then
incubated with the nitrocellulose sheet. One band showed hybridization (as shown); that
is, one band had enough of a complementary sequence to the mutant DNA to allow it to
anneal.
How would you interpret each of these experimental results? What is the structure of
the DNA intermediates in the wild-type and in the mutant phage?

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In the gel electrophoresis: the mutant would just run the same as the open circle DNA if it simply weren't able to close its circle. The mutant contains heavier than the single-stranded DNA because what kind of structure is it forming during replication? Wild-type phage DNA does not ever form a double-strand. What happens under denaturing conditions to that structure that helps explain that lane's smudge?

In the sucrose centrifuging: what data from the gel electrophoresis support the conclusion?

In the electrophoresis/Southern blot: which end of the DNA is the 1100 bp fragment, which end does the kinase ONLY work on?

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