Genome Replication

Question 1

Genome

Answer 1

All genetic information of an
organism

Question 2

Transcriptome

Answer 2

Set of all RNA transcripts
from a genome

Question 3

Proteome

Answer 3

Set of all proteins expressed
by an organism

Question 4

Semiconservative replication

Answer 4

In this model, the two strands of DNA unwind from each other, and each acts as a template for synthesis of a new, complementary strand. This results in two DNA molecules with one original strand and one new strand.

Question 5

Conservative replication

Answer 5

In this model, DNA replication results in one molecule that consists of both original DNA strands (identical to the original DNA molecule) and another molecule that consists of two new strands (with exactly the same sequences as the original molecule).

Question 6

Dispersive replication.

Answer 6

In the dispersive model, DNA replication results in two DNA molecules that are mixtures, or “hybrids,” of parental and daughter DNA. In this model, each individual strand is a patchwork of original and new DNA.

Question 7

topology

Answer 7

How two DNA strands are intertwined

Question 8

positive supercoil

Answer 8

Positive supercoiling of DNA occurs when the right-handed, double-helical conformation of DNA is twisted even tighter (twisted in a right-handed fashion) until the helix begins to distort and “knot.”

Question 9

Negative supercoil

Answer 9

Negative supercoiling, on the other hand, involves twisting against the helical conformation (twisting in a left-handed fashion), which preferentially underwinds and “straightens” the helix at low twisting stress, and knots the DNA into negative supercoils at high twisting stress.

Question 10

Topoisomerase

Answer 10

Enzymes that catalyze changes in the
topological state of DNA by cutting DNA strands
They relax positive and negative supercoils
Type I -> single-strand break
Type II -> double-strand break

Question 11

Gyrase

Answer 11

Enzyme that introduces negative supercoils
It relaxes and prevents overwinding during DNA
replication

Question 12

origin of replication (ori)

Answer 12

An origin of replication is a sequence of DNA at which replication is initiated on a chromosome, plasmid or virus.
Prokaryotes have only 1 ori
Eukaryotes have many ori

Question 13

replication bubble

Answer 13

an unwound and open region of a DNA helix where DNA replication occurs.
Upon the initiation of DNA replication, the DNA is `unzipped’ (by helicase) to reveal single strands at a single point of the DNA structure that resembles a bubble known as the replication bubble

Question 14

replication forks

Answer 14

DNA replication occurs in both directions in the replication bubble, leading to two replication forks forming on each replication bubble -> the place where the replication occurs.

Question 15

Replicon

Answer 15

the entire region of DNA that is independently replicated from a single origin of replication.
Bacteria have 1 ori so their whole chromosome is a replicon. Eukaryotes have multiple replicons

Question 16

OriC (E.coli)

Answer 16

OriC is 245 bp long and contains multiple recognition sites for DNA binding proteins and enzymes. Methylation of the OriC regulates initiation of replication.
OriC contains 11 copies of a palindromic sequence (GATCCTAG). Dam methylase enzyme methylates the adenines of the sequence.
When both DNA strands are methylated, oriC is active and replication starts.

Question 17

DNA adenine methylase,

Answer 17

an enzyme that adds a methyl group to the adenine of the sequence 5’-GATC-3’ in newly synthesized DNA. Immediately after DNA synthesis, the daughter strand remains unmethylated for a short time.
This is a large group of enzymes unique to prokaryotes and bacteriophages

Question 18

DNA polymerase

Answer 18

1. DNA synthesis (5’-3’ direction)
2. DNA polymerases cannot initiate synthesis of DNA de novo, they need a primer with a free 3’-OH end.
3. Proofreading error-control system. Exonuclease activity (3’-5’ direction)

Question 19

DNA polymerase I

Answer 19

contains both, 3’-5’ and 5’-3’ exonuclease activity.
It degrades the primer
with the 5’-3 exo. activity and synthesizes new
complementary DNA simultaneously

Question 20

DNA polymerase III

Answer 20

Main polymerizing enzyme.
DNA polymerase III has only 3’- 5’ exonuclease
activity. It stops synthesizing DNA when it finds
a primer

Question 21

Primase

Answer 21

a DNA-dependent RNA polymerase that
synthesizes RNA Primers of ~10 nt long

Question 22

DNA replication of the leading and lagging strand

Answer 22

The helicase unzips the double-stranded DNA for replication, making a forked structure. The primase generates short strands of RNA that bind to the single-stranded DNA to initiate DNA synthesis by the DNA polymerase. This enzyme can work only in the 5’ to 3’ direction, so it replicates the leading strand continuously. Lagging-strand replication is discontinuous, with short Okazaki fragments being formed and later linked together by ligase.

Question 23

Joining of adjacent Okazaki fragments in E. coli

Answer 23

• DNA polymerase III has only 3’- 5’ exonuclease
  activity. It stops synthesizing DNA when it finds
  a primer.
• DNA polymerase I has both, 3’- 5’ and 5’- 3’
  exonuclease activities. It degrades the primer
  with the 5’-3 exo. activity and synthesizes new
  complementary DNA simultaneously.
• DNA ligase ligates the adjacent fragments

Question 24

Joining of Okazaki adjacent fragments in eukaryotes

Answer 24

Two-step process:
• DNA polymerase δ and helicase displace the primer, creating a 5’ flap. Simultaneously the DNA polymerase fills the gap
• Flap endonuclease I (FEN1) cleaves the flap, removing the primer.
• DNA ligase ligates the adjacent fragments

Question 25

Replication of the lagging strand should be slower:
How does it keep the same synthesis rate in both strands?

Answer 25

It would be slower because of the many Okazaki fragments.
The replisome (a complex of two DNA polymerases + associated proteins) keeps both DNA pol. linked and assures a parallel synthesis of the leading and lagging strands

Question 26

Replisome

Answer 26

The replisome is a multiprotein structure that assembles at the replication fork to undertake synthesis of DNA. It contains:
- 2x DNA polymerase ( for lagging and leading strand -> DNA pol. in lagging strand must repetitively associate /dissociate from DNA.)
- 2x dimerizing subunit T that link DNA pol. together
- 2x sliding clamps form β-rings that encircles the DNA and assure contact between DNA and DNApolymerase
- Clamp loader (group of 5 proteins) places the clamp on DNA, keeping all the structure together

Question 27

Termination of replication in E. coli

Answer 27

• DNA replication is bidirectional and starts at OriC
• Chromosome is circulair -> Replication forks meet and halt in a region halfway from the origin,
• The replication fork trap.
• At both sites of the termination region there are five Ter sites (23 bp) that are recognized by Tus (Terminator utilization substance) proteins.
• Tus proteins bind to DNA in a specific orientation and stops the replication fork

Question 28

Linear chromosomes could become shorter after
replication because one of these reasons

Answer 28

1)Final Okazaki fragment cannot be primed (primase doesn’t have space to add a primer)
2) The primer of the last Okazaki fragment is at the very last 3’ extreme, it cannot be removed and synthesized again by DNA polymerase.

Question 29

Telomeres

Answer 29

Short, tandemly repeated sequences that “seals” the ends of chromosomes. A G-rich tail structure in the 3’ end Between 100 to 1000 repeats (2 to 15 kb long)

Question 30

Telomere functions

Answer 30

1. Cell distinguish telomeres from other free ends caused by damage
2. Telomeres can be extended to avoid short of chromosomes
3. Telomeres form a T-loop that stabilizes the end of the chromosome by invasion of G-rich 3’-end in an upstream region of the telomere

Question 31

Shelterin

Answer 31

complex of six proteins that:
- Catalyze the T-loop formation
- Protect telomeres from DNA repair mechanisms
- Regulate telomeres length control

Question 32

Telomerase

Answer 32

a Reverse transcriptase enzyme (RNA dependent-DNA polymerase) that synthesizes DNA from an RNA template.
Consists of protein + RNA (RNA template is the reverse complement of the G-rich strand). It uses the 3’ end of the G-rich strand as a primer to elongate the DNA.Then, a new Okazaki fragment can be primed
Telomerase is ONLY active in dividing cells