Exam 2- DNA Replication

Question 1

Semi conservative replication

Answer 1

DNA replication takes place in a semi conservative manner

One strand of the parent double helix is conserved in each new DNA molecule

Therefore, the two daughter double stranded DNA molecules each contain one strand of the parent DNA

Question 2

What 3 observations have been made for all DNA polymerases?

Answer 2

1) Incoming bases are selected by complementary base pairing by hydrogen bonding with template strand
2) Chain growth is in the 5’ to 3’ direction, and antiparallel to the direction of the template strand
3) All DNA polymerases require a primer strand to add bases to

Question 3

Function of DNA polymerase I

Answer 3

DNA polymerase for prokaryotes

Cleaved by trypsin or subtilisin into 2 parts

The larger part (Klenow fragment) has both 5’ to 3’ polymerase (synthesis) and 3’ to 5’ exonuclease activity (proofreading)

The smaller part has 5’ to 3’ exonuclease activity (repair and removal)

Question 4

Klenow fragment

Answer 4

After DNA polymerase I is cleaved, the larger fragment which exhibits both DNA 5’ to 3’ polymerase activity (synthesis) and 3’ to 5’ exonuclease activity (proofreading)

Question 5

Function of DNA Polymerase III

Answer 5

The enzyme primarily responsible for replicative DNA synthesis of E. Coli

Alpha subunit- polymerase activity

Epsilon subunit- 3’ to 5’ exonuclease activity

It has a complex structure made up of 10 subunits which forms a “sliding clamp” that works along the strand

Question 6

Topoisomerase

Answer 6

Change the topography of a DNA molecule by cutting strands, allowing the amount of supercoiling to be adjusted and then re-ligating the strands

Question 7

Helicase

Answer 7

Catalyze the unwinding of double stranded DNA by disrupting the base-pair hydrogen bonding

Question 8

What enzymes affect the linking number

Answer 8

The linking number is the sum of Twist and Writhe

Topoisomerase and Helicase

Question 9

In replication, which strand is the leading strand?

Answer 9

DNA continuously synthesized in the 5’ to 3’ direction

Question 10

Lagging strand

Answer 10

Discontinuous synthesis of 3’ to 5’ strand

(Discontinuous because the 5’ to 3’ template must hook around the beta clamp to face the correct direction for the polymerase, exposing only a small amount of template at a time)

Question 11

How is RNA used in DNA replication?

Answer 11

RNA acts as the primer for discontinuous replication of DNA on the 3’-5’ lagging strand.

(The discontinuous polymerization about the lagging strand gives rise to “Okazaki fragments”. The Okazaki fragments have short pieces of RNA attached to them which function as primer sequences for DNA polymerase. Note these primers are synthesized by an RNA polymerase called primase.)

Question 12

What are Okazaki fragments?

Answer 12

Newly synthesized DNA fragments that are formed on the lagging strand

Question 13

Where is E. Coli DNA replication initiated?

Answer 13

OriC- the origin of replication

Question 14

Where is E. Coli DNA replication terminated?

Answer 14

Tau or ter site

A protein “Ter” binds to the tau or ter site to terminate replication by contrahelicase activity

Question 15

What are the process invites of DNA polymerases I and III?

Answer 15

DNA Polymerase I: about 20 bases in sequence before it dissociates

DNA Polymerase III: high processivity, it can replicate entire 5 million base genome of E. Coli

Question 16

Simplified overview of prokaryotic DNA replication

Not on his questions

Answer 16

1) Helicase unwinds double stranded DNA
2) DNA gyrase (Topoisomerase) relieves supercoiling
3) SSB proteins maintain single strand regions
4) Primase puts on RNA primer
5) DNA Polymerase III moves in the 5’ to 3’ direction
6) DNA polymerase III sliding clamp beta units move along leading strand template accompanied by core polymerase
7) DNA Polymerase III gamma complex periodically unclamps beta units along the lagging strand, and then reclamps them on as new pieces of primer are found for Okazaki fragment synthesis
8) On the lagging strand, DNA polymerase I exonuclease activity removes RNA primers, and polymerase activity replaces them with deoxynucleotides
9) DNA ligase seals the nicks between the corrected Okazaki fragments

Question 17

Where is DNA found in eukaryotes?

Answer 17

Mostly within the nucleus packaged in chromatin

Small amount contained in the mitochondria

Question 18

How is DNA packaged?

Answer 18

The negatively charged phosphate groups of dsDNA molecules are wrapped around octamers of positively charged proteins called histones. These bundles are called nucleosomes and contain in their centers two molecules each of H2A, H2B, H3, and H4. On the outside of each nucleosome is complexed an additional H1. These nucleosomes are connected together by linker DNA to form “beads on a string” appearance. These structures are then packaged into 30 nm fibers which are coiled and supercoiled into chromosomes

Question 19

Contrast eukaryotic and prokaryotic DNA replication

Answer 19

• Eukaryotic DNA much longer length
• Eukaryotic slower synthesis - the movement of replication forks is much slower, 1/10 to 1/20 as fast as that in E. Coli
• Eukaryotic cells use a very large number of DNA polymerase molecules in comparison to prokaryotes
• Eukaryotic cells have multiple origins of replication referred to as autonomous replication sequences (ARS); prokaryotes- 1 oriC
• Eukaryotic cell synthesis in accordance with the cell cycle in contrast to prokaryotic cells which synthesize DNA essentially continuously
• Therefore, Eukaryotes- bidirectional DNA replication from multiple origins

Question 20

Which DNA polymerase is believed to be responsible for leading strand synthesis?

Answer 20

DNA polymerase delta

Question 21

Which DNA polymerase is believed to be responsible for lagging strand synthesis?

Answer 21

DNA polymerase alpha

Question 22

What proteins are in nucleosomes,and why do they bind to DNA?

Answer 22

Nucleosomes contain positively charged histone molecules. They bind because their positive charge that attracts the negatively charged phosphate groups of DNA

Question 23

PCNA

Answer 23

“Proliferating Cell Nuclear Antigen” (PCNA) also known as cyclin

PCNA forms a clamp around the template DNA strand to dramatically increase the processivity of DNA polymerase delta (in a manner similar to the effect of the beta subunits of DNA polymerase III in prokaryotes)

Question 24

What is the arrangement of nucleosomes after DNA replication?

Answer 24

Cooperatively distributed

During replication, there is simultaneous synthesis of new histones with DNA. One daughter strand has the new histones, the other daughter strand has the original ones.

Question 25

How might nucleosomes affect eukaryotic DNA replication?

Answer 25

May be the reason for short Okazaki fragment and slow DNA polymerization in eukaryotes

After replication, there is a cooperative distribution of nucleosomes. The new histones end up along one daughter strand near each replication fork. The histones form the original nucleosomes are found in the nucleosomes reformed along the other new strand. Therefore, the original histones are conserved and the histone octamers do not dissociate completely during replication. They remain somehow associated with one strand of the replication fork. These nucleosomes may be the reason why Okazaki fragments are so short, and why DNA polymerization is so much slower in eukaryotes

Question 26

DNA replication in mitochondria

Answer 26

D-Loop Replication

Maternal inheritance