Chapter 25: Prokaryotic DNA Replication

Question 1

What does the semiconservative DNA replication mean?

Answer 1

DNA replication is semiconservative, that is, one strand of the newly synthesized DNA double helix is from parent molecule (template) and the other strand is newly synthesized and complementary to the template.

Question 2

In 1958, What was demonstrated by Meselson and Stahl? What methods did they use in their experiment?

Answer 2

The semiconservative DNA replication

Question 3

Where does Dna synthesis occur?

Answer 3

The branch points in a replication eye, at which DNA synthesis occurs, are called replication forks.

Question 4

In which direction does DNA polymerase catalyze the synthesis of DNA chain?

Answer 4

newly synthesizing DNA strand is only extended in the 5’ to 3’ direction.

Question 5

DNA replication occurs unidirectionally or bidirectionally in a cell?

Answer 5

bidirectionally

Question 6

How are the incoming nucleotides are selected

Answer 6

The incoming nucleotides are selected by their ability to form Watson–Crick base pairs with the template DNA so that the newly synthesized DNA strand forms a double helix with the template strand.

Question 7

What does semidiscontinuous replication mean? Which strand is synthesized continuously and which strand is synthesized discontinuously?

Answer 7

Replication is semidiscontinuous: The leading strand is synthesized continuously while the lagging strand is synthesized as RNA-primed Okazaki fragments that are later joined.

Question 8

leading vs lagging strand?

Answer 8

Leading strand: continuously synthesized strand that extends toward the replication fork.
Lagging strand: discontinuously synthesized strand that extends in the opposite direction of replication fork movement to form Okazaki fragments.

The newly synthesized DNA strand that extends 5′ → 3′ in the direction of replication fork movement, the leading strand, is continuously synthesized in its 5′ → 3′ direction as the replication fork advances. The other new strand, the lagging strand, is also synthesized in its 5′ → 3′ direction. However, it can only be made discontinuously, as Okazaki fragments, as single-stranded parental DNA becomes newly exposed at the replication fork. The Okazaki fragments are later covalently joined together by the enzyme DNA ligase.

Question 9

DNA is replicated by enzymes known as? What 2 things do these require in order to synthesize DNA?

Answer 9

DNA polymerases. DNA polymerase requires a template and primers to synthesize DNA.

Question 10

Radioactive labeled chromosomes from E. coli display “eyes” or “bubbles”, called?

Answer 10

θ structures. DNA replication involving θ structures is known as θ replication.

Question 11

How is DNA synthesis initiated?

Answer 11

DNA synthesis is initiated from a RNA primer

Question 12

How many priming events are required to initiate the synthesis of the leading strand and lagging strand?

Answer 12

Only one priming event is required to initiate the synthesis of the leading strand, but multiple priming events are required for lagging strand synthesis.

Question 13

The RNA primers are eventually replaced with?

Answer 13

DNA

Question 14

In E. coli RNA primers are synthesized by the enzyme called?

Answer 14

Primase

Question 15

What are the 3 processive enzymes used during replication?

Answer 15

Pol I, Pol II, and Pol III

Question 16

Why DNA polymerases are called processive enzyme?

Answer 16

DNA polymerases are processive enzymes because they catalyze addition of 20 or more nucleotides to the growing DNA chain without releasing the single-stranded template.

Question 17

What are the major activities and functions of Pol I? What is the Klenow fragment?

Answer 17

3′ → 5′ exonuclease and a 5′ → 3′ exonuclease.
5’to 3′ polymerase activity
only one with 5′ → 3′ exonuclease activity.

The 3′ → 5′ exonuclease activity allows Pol I to edit its base pairing mistakes. (proofreading)

The 5’to 3′ polymerase activity, its most important (and only essential) function is in lagging strand synthesis, in which it removes the RNA primers and replaces them with DNA.

The main duty of the 5’-3’ exonuclease activity is to remove the RNA primers at the 5’ ends of newly synthesized DNA so that the polymerase activity can fill in the resulting gaps.

Nick translation too

Question 18

The three domains of the Klenow fragment in Pol I. in which domain are the polymerase activity and 3’→5’ exonuclease activity located?

Answer 18

The Klenow fragment contains three domains: Palm, Thumb and Fingers.

The palm domain contain two separate active sites: it retains the 5’ → 3’ polymerase activity and the 3’ → 5’ exonuclease activity for removal of precoding nucleotides and proofreading, but loses its 5’ → 3’ exonuclease activity.

Question 19

How do DNA Polymerases sense watson crick base pairs?

Answer 19

via Sequence-Independent Interactions

Question 20

What are the major activities and functions of Pol II?

Answer 20

Poll II exhibits Polymerization: 5′ → 3 and Exonuclease: 3′ → 5 activity.

Pol II participates in DNA repair.

Question 21

What are the major activities and functions of Pol III?

Answer 21

Pol III is the replicase to catalyze DNA synthesis of both leading and lagging strands.

Pol III core enzyme consists of three subunits: α with polymerase activity, ε with 3’→5’ exonuclease activity, and θ.

Question 22

Components of Pol III Holoenzyme?

Answer 22

Two copies of the DNA polymerase III core enzyme, one copy of five protein γ-complex (clamp loader), and a sliding clamp.

the Pol III holoenzyme.
a multi-protein complex in which a core enzyme activity is associated with additional components that enhance function.

Question 23

From which site is the DNA replication initiated in E. coli?

Answer 23

Replication of E. coli chromosome is initiated at a 245-bp region known as the oriC locus.

Question 24

DnaA function

Answer 24

DnaA protein binds to oriC and causes a segment of the DNA to melt open.

Question 25

DnaB protein function?

Answer 25

a so-called helicase, unwinds the DNA strands in both directions in an ATP-dependent manner. This unwinding causes formation of supercoils in front of the replication fork.

The function of DnaB helicase is to couple ATP hydrolysis with the unwinding of duplex DNA at the replication fork. separates the two strands of the parental DNA during its replication by translocating along the lagging strand template in the 5′ → 3′ directionThis creates 2 anti parallel DNA single strands.

Question 26

DNA gyrase (a type II topoisomerase) function?

Answer 26

relaxes the supercoils and facilitates the unwinding of DNA strands by the helicase.

Question 27

Separated DNA strands behind the advancing helicase are prevented from annealing by

Answer 27

he binding of single-stranded binding protein (SSB).

Question 28

What is the function of primosome?

Answer 28

when primase binds to the DNA helicase that initiates RNA primers on the lagging DNA strand during DNA replication.

Primase in the primosome is the enzyme catalyzing synthesis of RNA primers.

primase binds to the DNA helicase forming a complex called the primosome.A complex of two proteins, a primase and a helicase, that initiates RNA primers on the lagging DNA strand during DNA replication.

Question 29

What catalyzes the synthesis of both leading and lagging strands

Answer 29

Pol III holoenzyme
Assembly of Pol III holoenzyme ensures the synthesis of both leading and lagging strands at same rate.

Question 30

The definition of replisome.

Answer 30

The synthesis of both strands occurs in a single multiprotein particle, the Replisome: the combination of all proteins that function at the replication fork.

Question 31

trombone model?

Answer 31

In order for the replisome to move as a single unit in the 5′ → 3′ direction along the leading strand, the lagging strand template must loop around

Question 32

What promotes the processivity of Pol III? Why?

Answer 32

The sliding clamp (β subunit)

binds tightly to DNA polymerase and it keeps DNA polymerase attached to the template,keeping the Pol III holoenzyme from diffusing away.

must beinstalled around the lagging strand template about once every second

Question 33

By which enzyme are the nicks in the lagging strand sealed?

Answer 33

DNA Ligase
This enzyme seals a broken phosphodiester bond

E. coli DNA ligase uses NAD+

Question 34

By which enzyme are the RNA primers removed and the gaps filled?

Answer 34

DNA poly I

Question 35

how does termination of dna replication occur?

Answer 35

Termination of DNA replication occurs when the two forks meet and fuse, creating two separate double-stranded DNA molecules.The bound Tus protein effectively halts DNA polymerase movement.[2] Tus helps end DNA replication in prokaryotes.[2In E. coli, Tus binds to 10 closely related sites encoded in the chromosome.

Question 36

the final step of dna replication?

Answer 36

Segregation of newly replicated chromosomes must be completed before cell division can occur

After replication is complete, the two daughter DNA molecules remain linked together as catenanes.
Topoisomerase II separates the two circular DNA by breaking one double-stranded DNA molecule and
pass a second double-stranded DNA through this break

to separate two catenated DNA molecules by the action of topoisomerases II (The resulting catenated circular dsDNAs must be separated so that each can be passed to a different daughter cell. This is the job of the type II topoisomerase named topoisomerase IV,he formation and separation of catenated dsDNAs at the replication termination site.Type II topoisomerases are essential for resolving topologically entwined double-stranded DNA.

Question 37

The structure of the E. coli replication terminus.

Question 38

The resulting catenated circular dsDNAs must be separated so that each can be passed to a different daughter cell.
What are essential for resolving topologically entwined double-stranded DNA?

Answer 38

This is the job of the type II topoisomerase named topoisomerase IV

Topoisomerase IV is one of two Type II topoisomerases in bacteria, the other being DNA gyrase.

Question 39

Why can organisms keep high fidelity of replication?

Answer 39

The high fidelity of DNA replication is achieved by the regulation of dNTP levels, by the low error rate of the polymerase reaction, by the requirement for RNA primers, by 3′ → 5′ proofreading, and by DNA repair mechanisms.
The fidelity of a DNA polymerase refers to its ability to accurately replicate a template.

Question 40

the final step of dna replication?

Answer 40

Segregation of newly replicated chromosomes must be completed before cell division can occur

After replication is complete, the two daughter DNA molecules remain linked together as catenanes.
Topoisomerase II separates the two circular DNA by breaking one double-stranded DNA molecule and
pass a second double-stranded DNA through this break

to separate two catenated DNA molecules by the action of topoisomerases II (The resulting catenated circular dsDNAs must be separated so that each can be passed to a different daughter cell. This is the job of the type II topoisomerase named topoisomerase IV,he formation and separation of catenated dsDNAs at the replication termination site.Type II topoisomerases are essential for resolving topologically entwined double-stranded DNA.