Replication

Question 1

Purpose of DNA Packaging

Answer 1

Protects Against Damage

Reduces the amount of space taken up

Question 2

Eukaryotic DNA Packaging

Answer 2

Chromatin (DNA+ Protein): 2 forms

• Euchromatin- Loose; site of transcription/Gene expression
• Heterochromatin: Condensed; no transcription

Negative Charged on Phosphates Neutralized by:

1) Polyamines
- Putrescine
- Spermidine
- Spermine
2) Cations (Na+, K+, Mg2+)
3) Proteins
- Non histone proteins (Topoisomerase etc)
- Histones

Question 3

Histones

Answer 3

1) High Concentration of Lys & Arg
-helps neutralize Phosphate
2) 5 total
H1
H2a
H2b
H3
H4
3) Octameric Complex
-all except H1
-contains 2 copies of each

Question 4

DNA histone Complex

Answer 4

1) Winding of DNA around histone core
- Forms Nucleosome core
- H2a, H2b, H3, H4 (octameric core)
- Superhelicity

2) Add more DNA + H1= Chromatosome

Question 5

New Model of DNA Packaging

Answer 5

Levels of Organization

1) Formation of Nucleosomes
- DNA wound around histone core
2) Folding of Chromatin to form Loops
- Loops= TADs
3) Formation of Compartments; 2 types
- A= Euchromatin (active + Nuclear Speckles)
- B=Heterochromatin (inactive)
4) Formation of Chromosome Territories

Question 6

How are TADs maintained?

Answer 6

1) 2 proteins: CTCF + Cohesin
- CTCF binds to CTCF. binding site on DNA
- Cohesin attaches to forms loop

Question 7

Nuclear Speckles

Answer 7

1) Associated with Euchromatin which produces precursor mRNA
2) Contains Splicing factors which splices mRNA
3) AKA Interchromatin Granule Clusters

Question 8

Remodeling Chromatin

Answer 8

1) Domains can switch Compartments
2) Used to Express certain proteins during cell cycle
3) Polycomb
- contains DNA that codes for PcG
4) Polycomb Group Proteins
- cause TADs to switch from Hetero and Euchromatin

Question 9

Packaging DNA in Sperm Heads

Answer 9

1) No Histones
- replaced by Protamines (high Arg + Cys)
2) . Neutralize Phosphate
- Polyamines
- Cations (Na+,K+, Mg2+)
3) Steps
- Protamines bind to Major groove inducing Donut Structure
- Donut compressed to form Donut Loop
4) Crosslinking b/w cysteine and protamines by disulfide bonds causes more condensed structure

Question 10

Replication

Answer 10

1) S phase of Cell cycle
2) Semiconservative process
- both eukaryotes and prokaryotes
3) DNA polymerase
- synthesizes 5’ to 3’
4) Bidirectional from origin of replication

Question 11

DNA polymerase requires

Answer 11

1) Substrates: dATP, dTTP, dCTP, dGTP
2) Mg2+ (Cofactor)
3) Template (DNA)
4) RNA Primer w/free 3’ OH

Question 12

DNA polymerase Types

• function
• Location
• Role

Answer 12

Alpha

• 1) Polymerase:
• Synthesizes Leading and Lagging strand
• low Processivity
• DNA Repair
• replaced by Delta or Epsilon
  2) Primase
• Synthesizes RNA primers
  3) Nucleus

Beta

1) Polymerase
- DNA Repair
2) Nucleus

Gamma

1) Polymerase
- Mitochondrial DNA Replication
2) 3’–>5’ exonuclease activity
3) Mitochondria

Delta

1) Polymerase
- Lagging Strand Synthesis
- DNA Repair
2) 3’->5’ exonuclease
3) Nucleus

Epsilon

1) Polymerase
- leading strand
- DNA repair
2) 3’->5’ exonuclease
3) 5’->3’ exonuclease
4) Nucleus

Question 13

3’->5’ exonuclease Activity

Answer 13

1) Proofreading Activity

- removes misincorporated DNA

Question 14

Processivity

Answer 14

1) Ability continue synthesizing without releasing

Question 15

RFA

Answer 15

1) Single Stranded DNA Binding Protein

2) Blocks reannealing(keeps apart) and degradation of ssDNA

Question 16

PCNA

Answer 16

1) Proliferating Cell Nuclear ANtigen
2) 3 Subunits=CLAMP
3) Stimulates Poly Delta & Epsilon
- increases Processivity

Question 17

RNase H1

Answer 17

1) Removes RNA Primers (with FEN1/RTH1)

2) Degrades RNA BUT H=HYBRid so only degrades RNA base paired to DNA

Question 18

FEN1/RTH1

Answer 18

Removes RNA primers (w/RNase H1)

Question 19

Leading Strand

Answer 19

1) synthesized Continuously by DNA Poly Epsilon

2) Switches from Epsilon to Delta during Replication Termination

Question 20

Lagging Strand

Answer 20

1) Synthesized discontinuously by DNA poly Delta

2) produces Okazaki fragments

Question 21

Origin of replication

Answer 21

1) Where Replication Begins in either direction

2) Multiple per chromosome

Question 22

DNA Ligase

Answer 22

1) Requires ATP

2) Ligates DNA fragments together

Question 23

Problem at the end of replication:

• What occurs without solution
• What is the solution

Answer 23

1) Incomplete synthesis of lagging strand at end of chromosome creates 3’ overhang In parental strand
2) Without solution the chromosome will get shorter after each round of replication

3) Solution=Telomerase
-Telomerase Binds to 3’ overhang and uses RNA as a template to produce more DNA
-Telomere shifts 6 nucleotides to synthesize another repeat
* STep 1 and 2 occur multiple times
Why? Elongates parental strand of 3’ Overhang

Question 24

Telomerase

Answer 24

1) Composed of RNA + proteins

2) RTase activity

Question 25

Why is the telomerase activity for the 3’ Overhang beneficial?
-steps

Answer 25

1) Elongation of parental strand gives DNA Poly A more room to bind to synthesize RNA Primer

Steps:

1) DNA Poly A has low processivity so displaced by RFC
2) Replaced by PCNA (Clamp) on parental strand
3) Stimulates DNA Poly E/D to fill in missing DNA
4) DNA Ligatin and removal of RNA primer

Question 26

How does the 3’ Overhang protect itself?

Answer 26

1) Invades dsDNA and Base pairs to form D and T loops