Molecular Biology 3

Question 1

What did Watson and Crick say in 1953 about DNA replication ?

Answer 1

“It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material” J. Watson & F. Crick (1953) Nature, 171

Question 2

What is the replisome ?

Answer 2

The replisome is a complex molecular machine that carries out replication of DNA. The replisome first unwinds double stranded DNA into two single strands. For each of the resulting single strands, a new complementary sequence of DNA is synthesized.

Question 3

What is the replication fork ?

Answer 3

The replication fork is the area where the replication of DNA will actually take place. There are two strands of DNA that are exposed once the double helix is opened. One strand is referred to as the leading strand, and the other strand is referred to as the lagging strand.

Question 4

What is a replication bubble ?

Answer 4

A replication bubble is an unwound and open region of a DNA helix where DNA replication occurs.

Question 5

What is oriC ?

Answer 5

Origin of replication in bacteria.

Question 6

What is DnaA ?

Answer 6

DnaA is a protein that activates initiation of DNA replication in bacteria. It is a replication initiation factor which promotes the unwinding of DNA at oriC. The onset of the initiation phase of DNA replication is determined by the concentration of DnaA.

Question 7

What is DnaB ?

What does its activity require ?

Answer 7

A helicase (responsible for DNA unwinding)
The helicase activity requires ATP hydrolysis.

Question 8

What is DnaC ?

Answer 8

DnaC is a loading factor for helicase DnaB. DnaB and DnaC associate with the dnaA bound origin. After loading DnaB at the origin, DnaC is released. This interaction of DnaC with DnaB requires the hydrolysis of ATP.

Question 9

What does the initiation of DNA synthesis require ?

Answer 9

A primer (5-10nt long) and a template strand –> a primer-template junction.

Question 10

In what direction does DNA replication proceed ?

What is released for every nt added ?

Answer 10

DNA synthesis proceeds in 5’ to 3’ direction
A pyrophosphate group is released for every nt added.
This pyrophosphate group is converted to 2 individual phosphate groups by pyrophosphotase.

Question 11

What are okasaki fragments ?

Where are they formed ?

Answer 11

Okazaki fragments are short, newly synthesized DNA fragments that are formed on the lagging template strand during DNA replication. They are complementary to the lagging template strand, together forming short double-stranded DNA sections

Question 12

What is special about DNA replication on the lagging strand ?

Answer 12

DNA replication on the lagging strand is discontinuous

Question 13

Completion of Okazaki fragments synthesis leaves a nick between the Okazaki fragment and the preceding RNA primer on the lagging strand. How are these processed ?

Answer 13

• DNA Pol I extends the Okazaki fragment while its 5’-3’ exonuclease activity removes the RNA primer.
• This process, called nick translation, results in movement of the nick along the lagging strand.
• DNA Pol I dissociates after extending the Okazaki fragment 10-12 nucleotides.
• DNA ligase binds to the nick.

Question 14

What are the different components of the replisome ?

Answer 14

The primosome, the DNA Pol complexes, beta-clamps, SSB tetramers, DNA helicase, RNA primers (lagging strand).

Question 15

What is DNA Pol ?

Answer 15

A DNA polymerase is an enzyme that catalyzes the polymerization of deoxyribonucleotides into DNA strand

Question 16

How many DNA Pols does E Coli have ?

What about eukaryotes ?

Answer 16

E Coli –> at least 5 DNA Pols, I-V

Eukaryotes –> typically > 15 DNA Pols

Question 17

Which E Coli DNA Pol has the highest processivity ?

What does it exist as ?

Answer 17

DNA Pol III is the subunit w/ the highest processivity, and exists as part of the Pol III holoenzyme

Question 18

What are the features of DNA polymerases ?

Answer 18

Multisubunit enzymes
Processive
Template required
Proofreading activity (3’-5’ exonuclease acitivity)
Replicative polymerases remain bound to the replication fork
Coordinate synthesis of leading and lagging strands

Question 19

What is the DNA sliding clamp ?

Answer 19

A DNA clamp, also known as a sliding clamp, is a dimeric or trimeric ring-shaped protein fold that serves as a processivity-promoting factor in DNA replication. As a critical component of the DNA Pol III holoenzyme, the clamp protein binds DNA polymerase and prevents this enzyme from dissociating from the template DNA strand.
Sliding clamp structures have been well conserved throughout evolution.

Question 20

What is the trombone model of DNA replication ?

Answer 20

This terminology refers to the fact that the lagging-strand template loops back through the replisome so that the leading and lagging strands are syhtesized in the same direction, SSBs bind to single stranded DNA to prevent it from annealing to itself.

Question 21

How many priming events are required for lagging strand synthesis ?

Answer 21

Many :
- As the helicase unwinds the DNA template, a primase synthesized a RNA primer.
- The lagging-strand polymerase completes as Okasaki fragment
This cycle is repeated several times

Question 22

How many DNA Pol loading events are required for lagging strand synthesis ?

Answer 22

Many :

• When the lagging-strand DNA Pol encounters the preceding Okasaki fragment, it releases the lagging strand
• The lagging-strand DNA Pol binds to a newly synthesized primer and begins synthesizing another Okasaki fragment

Question 23

How many subunits make up the E Coli DNA Pol III holoenzyme ?
What are their roles ?

Answer 23

Alpha = DNA Pol
Epsilon = 3'-5' exonuclease
Beta = Forms sliding clamp
Tau = enhances dimerization of core; ATPase
The rest (gamma, phi... etc) = enhance processivity; assist in replisome assembly

Question 24

What does the 3D structure of DNA Pol resemble ?

Answer 24

A right hand :

• polymerase site in palm
• bend in template at fingers
• exonuclease active site at the bottom of the hand