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Molecular Cell Biology > Molecular cell biology uwindsor lecture 4 > Flashcards

Molecular cell biology uwindsor lecture 4 Flashcards

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1
Q

What underlies DNA replication and DNA repair?

A

Base pairing

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2
Q

What is the model for DNA replication?

A

Semi-conservative replication. Daughter strands are formed from parent strands.

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3
Q

Where are new nucleotides added on DNA?

A

3’ OH end. DNA polymerase catalyzes the formation of phosphodiester bonds.

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4
Q

Where does the energy for the formation of phosphodiester bonds come from?

A

Hydrolysis of incoming nucleotide.

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5
Q

What is the direction of DNA replication?

A

5’-3’

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6
Q

What does the breaking of the phosphate bond on the nucleotide yield?

A

Pyrophosphate and energy.

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7
Q

What are the different levels of specificity in DNA replication?

A

1) Incoming nucleotide has to make hydrogen bond with nucleotide in template.
2) When hydrogen bonding occurs (correct at least), DNA pol tightens grip around DNA and incoming nucleotide to faciliate reaction of covalent association with 3’ end of primer strand.

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8
Q

What are the three mechanisms that ensure accuracy of DNA replication?

A

1) Base-pairing of correct nucleotide which allows for DNA pol. tightening-before covalent attachment of nucleotide.
2) 3’-5’ (opposite direction to DNA replication) exonuclease activity of DNA pol.
3) Mismatch repair

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9
Q

What happens when an incorrect base is applied, in what concerns the exonuclease activity of DNA polymerase?

A

The primary function of DNA polymerase is its 5’-3’ nucleotide addition. Sometimes, the wrong base is added. This causes incorrect base pairing, stalls the polymerase and alters the conformation at critical 3’ end of primer. Because of the stalling, 3’-5’ exonuclease activity begins to work; chews up DNA from 3’ end to 5’ end and removes the recently incorporated nucleotide. This generates a new 3’ end.

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10
Q

What can be said about the activity of DNA polymerase and its exonuclease function?

A

The editing activity and polymerase activity map to different places on the enzyme. There is competition between both activities but polymerase generally wins out.

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11
Q

Where was the exonuclease activity first discovered?

A

Bacteriophage T7. Mutant for this would accumulate mutations in DNA.

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12
Q

Is the DNA replication fork symmetrical or asymmetrical?

A

Asymmetrical.

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13
Q

What is the cause of this assymetric behaviour of the fork?

A

Leading and lagging strand synthesis.

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14
Q

What experiment led to the discovery of a lagging strand?

A

Okazaki experiment.

1) Brief pulse of H3-thymidine to label DNA
2) Alkali denaturation
3) Sucrose gradient to separate DNA into fractions
4) Measure radioactivity in each fraction.

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15
Q

What were the different models Okazaki proposed before running the experiment?

A

Model with lagging strand and model without.

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16
Q

How did Okazaki discover DNA ligase?

A

Made temperature sensitive mutants for E. coli ligase gene-lethal at restrictive temperature.
Predicted many small fragments would be made, (along with large fragment from leading strand), then applied same method as before.

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17
Q

How did Okazaki determine that short DNA fragments contain RNA?

A

Performed the same experiment as before but, added RNAse to reaction with labelled okazaki fragments.
If RNA was present, the fragments should be smaller.
Instead of using radioactive dNTP, used raadioactive uridine, which only incorporates in RNA, incorporates into fast migrating peak.

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18
Q

How does lagging strand synthesis occur?

A

Primase adds an RNA primer, then DNA polymerase adds nucleotides in the 5’ to 3’ direction. The old RNA primer is displaced by DNA polymerase and then ligase fuses the fragments.

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19
Q

How does DNA ligase join the Okazaki fragments together?

A

Energy for reaction requires ATP. DNA ligase takes ATP and attaches AMP to 5’ end. This catalyzes phosphodiester bond formation.

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20
Q

What are the major components of the DNA replication machinery?

A

Mcm DNA helicase which opens up the protein helix.
Single stranded DNA binding protein (RPA) which stabilizes ssDNA (of particular importance on the lagging strand).
PCNA sliding clamp-encircles DNA and keeps DNA polymerase from falling off.
DNA polymerase.

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21
Q

Where does Mcm helicase open up DNA double helix?

A

In front of the replication fork.

22
Q

How does mcm helicase open up DNA?

A

Latches on to ssDNA, at 5’ end, and with the use of ATP, displaces other strand at the replication fork as it moves.

23
Q

What is helicase made up of?

A

mcm helicase is made up of six identical subunits which form a ring to surround ssDNA.

24
Q

What is the energy source for mcm helicase?

A

ATP

25
Q

How do single-stranded DNA binding proteins (RPA) work?

A

ssDNA becomes exposed as a result of helicase activity.
If ssDNA is left alone, will try to make secondary structure by finding homologous sequences.
RPA acts by cooperatively binding (i.e. multiple bind together), to stabilize ssDNA.

26
Q

What is the 3D structure of RPA?

A

Two domains, A and B.

27
Q

What is the 3D structure of PCNA?

A

Two different subunits that surround DNA.

28
Q

How does PCNA work?

A

PCNA uses ATP to bind to clamp loader, allowing PCNA to open up.
Clamp loader also associates with dsDNA.
PCNA-clamp loader complex moves along DNA until it encounters a 3’ end.
ATP hydrolysis on clamp loader allows for dissociation with PCNA and closing of PCNA.
Clamp loader is recycled and DNA polymerase associates with PCNA.

29
Q

What happens on lagging strand with DNA polymerase-PCNA complex?

A

DNA polymerase releases from the clamp when it encounters 5’ of an RNA primer.

30
Q

What does the replication machinery do at the replication fork?

A

The proteins at a replication fork cooperate to from a replication machine-replicase.
The replication fork contains a single large complex including polymerases for leading and lagging strand.
Lagging strand is looped around to that its polymerase can associate with leading strand polymerase.

31
Q

How does a strand-directed mismatch repair system remove replication errors that escape from the replication machine?

A

MSH2 and MLH1 protein complex scans newly synthesized DNA.
The complex detects mismatches through seeing bulb (from incorrect base pairing).
Then they scan and detect nicks (on newly synthesized lagging strand).
They then make another nick in the same strand.
Strand gets removed and then repaired by DNA polymerase.

32
Q

What do families with mutations in the MSH2 and MLH1 complex have a high incidence of?

A

Cancer

33
Q

What do DNA topoisomerases do?

A

Prevent DNA tangling during replication.

34
Q

How does DNA topoisomerase prevent torsional strain?

A

One end of the DNA double helix cannot rotate relative to the other end.
Type 1 DNA topoisomerase with tyrosine at the active site covalently attaches to a DNA phosphate, thereby breaking a phosphodiester linkage in one DNA strand.
The two ends of the DNA double helix can now rotate relative to each other, relieving accumulated strain.
The original phosphodiester bond energy is stored in the phosphotyrosine linkage, making the reaction reversible.
Spontaneous re-formation of the phosphodiester bond regenerates both the DNA helix and the DNA topoisomerase.

35
Q

Where does DNA synthesis begin?

A

At replication origins.

36
Q

What are the general steps of DNA synthesis starting at the replication origins?

A

Local opening of DNA helix.
RNA primer synthesis.
Leading strand DNA synthesis.
Lagging strand synthesis.

37
Q

How many origins of replication do bacteria have?

A

1

38
Q

How does one determine origins of replication in eukaryotes?

A

Oris are sequence specific.
Can radioactively incorporate H3-thymidine then add unlabelled medium to reduce levles of newly incorporated labelled DNA. Origins of replication are between the sites of radioactivity.
Can also use pulse labelling technique.

39
Q

What is the difference, in what concerns oris, between yeast and most other eukaryotes?

A

Yeast have specific, defined sequence, most others are not well defined.

40
Q

What is pulse labelling?

A

Microarray variation technique.
Place all DNA from genome on microarray.
DNA on microarray is defined.
Probe microarray with fluorescently labelled DNA from cells that are undergoing replication.
If only allowing a short pulse, some spots will be more fluorescent because double the DNA has been made in this regions.
All cells are DNA synchronized.

41
Q

How are cells replicating DNA synchronized?

A

This is accomplished by adding an inhibitor of DNA replication or by adding serum through serum starvation. This leads to cells arresting just before S-phase.
When the experiment needs to be performed, just remove the inhibitor or add serum.

42
Q

How is DNA replication initiated?

A

A large multisubunit complex binds to origins of replication-origin recognition complex (ORC).
ORC recruits other proteins to origins in G1 of cell cycle.

43
Q

How is the pre-replication complex formed?

A

ORC subunits are bound to origins constitutively.
In G1, prereplication complex proteins are brought to origins.
1) cdt1 and cdc6 bind to ORC
2) cdt1 and cdc6 recruit mcm helicase.
3) cdt1 and cdc6 are then released
4) ORC-mcm forms mature pre-RC

44
Q

How is initiation of S phase done?

A

The prereplication complex is made in G1.
At S phase, a kinase, cdk2-cyclin A phosphorylates initiator proteins; these assemble DNA polymerase complex.
DDK, another kinase, phosphorylates mcm to stimulate its activity.

45
Q

What happens to histones during DNA replication?

A

New nucleosomes are assembled behind the replication fork.

1) histones are temporarily displaced as replication proceeds.
2) new histones are added after replication fork passes
3) histone genes are highly transcribed at initiation of S phase.

46
Q

How can DNA be synthesized til the end of the lagging strand if their is a primer that blocks it?

A

Telomerase extends chromosome ends by adding telomeres.

47
Q

What is the 3D structure of telomerase?

A

Enzyme made up of RNA and protein. RNA within enzyme is specific sequence for specific species, specific to telomeres.
RNA can bind to complementary DNA at the end of the chromsome.

48
Q

How does telomerase replicate the ends of chromosomes?

A

Recognizes sequence on exposed 3’ end.
Extends 3’ end with repeat sequence.
Allows for DNA polymerase to synthesize crucial DNA portion.

49
Q

What dictates what the telomere sequence will be?

A

Sequence from RNA of telomerase.

50
Q

How are telomeres protected?

A

By telomere binding proteins called sheltrin complex.
If the ends of chromosomes are exposed, DNA repair mechanism will try to put chromosomes together.
Thus, sheltrin complex prevents chromosome fusion.

Molecular Cell Biology (9 decks)

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