DNA Synthesis

Question 2

Repeated trinucleotide inherited diseases

(3)

Answer 2

1. Fragile X syndrome (CGG): mental retardation
2. Huntington’s disease (CAG): chorea-involuntary movement, progressive dementia, death
3. Myotonic dystrophy (CTG): muscle weakness, myotonia-slow relaxation of muscles after contraction

Question 2

Problems in Replicating and Maintaining DNA

(3)

Answer 2

1. Immense size
2. Inevitable errors and mutations introduced in replication apparatus and environment
3. Integration of recombination and repair pathways with replication (goal is to maintain fidelity)

Question 3

DNA replication general characteristics

Answer 3

1. 3’OH of last nucleotide makes a nucleophilic attack on α phosphate of infomring dNTP → PPi released
2. Semi-conservative; strand stays hydrogen bonded to template, allows for correction

Question 4

Leading and Lagging strands

Answer 4

1. Highly processive (semi-discontinuous)
2. RNA primer is required for DNA polymerase
3. lagging: Okazaki fragments can be1-2kb

• DNA pol I
• DNA ligase requires ATP/ NAD → AMP/NMN

Question 5

Replisome

DNA replication at replication fork

(7)

Answer 5

1. DNA pol III holoenzyme

• active site for polymerization
• 3’ - 5’ exonuclease proofreading
• leading strand synthesis; highly processive
• lagging strand synthesis; suited for multiple associates and disassociations
• leading and lagging subunits anchored

2. Helicase: DNAB also directs primse to site
3. Primase: catalyzes synthesis of RNA primers
4. SS binding protein or helix-destabilizing proteins prevents SS to fold on itself
5. Topoisomerase/gyrase: relieves positive supercoils ahead and negative supercoils behind
6. DNA pol I: low processivity, removes RNA primer in Okazaki fragments
7. DNA ligase: seals nicks

Question 6

DNA polymerase actions

Answer 6

1. Polymerize in 5’ → 3’ direction
2. Requires a template & a primer (made by primase)
3. Processivity: ability to polymerize without dissociating from template (allows for continuous addition)
4. Forms an active site for specific dNTP
5. Nucleotide discrimination: prefers low Km and high Vmax pairings
   * non-standard bp have regions that protrude outside of active site; doesn’t fit well into act
6. 3’ → 5’ exonuclease active site = proofreading

Question 7

Types of DNA polymerase

Answer 7

Pol I: removed RNA primers

Pol III: active in replicaiton fork

Eukaryotic replication proteins

Pol α: forms tight complex with primase

Pol ß: base excision repair

Pol δ: may be the analog of DNA

Pol ε: similar to Pol δ

Pol γ: replicates mitochondrial DNA

Question 8

Initiation of DNA replication: prokaryotes

Answer 8

1. Initiator protein DnaA binds to OriC-E.Col**i **(AT rich region and consensus seq); opens the duplex and recruites helicase and primase to origin *requires ATP
2. A recruits DnaB helicase
   * DnaC is molecular matchmaker promoting interaction btn DnaA and DnaB
3. DNA pol III holoenzyme

Question 9

Initiation of DNA replication: eukaryotes

Regulation of initiation

Licensing (4)

Answer 9

1. ORC (origin recognition complexes) are always bound
2. cdc6/18 and cdt1 bind in early G1
3. Recruit MCM (mini chromosome maintenance) → helicase activity
4. During S phase: dissociation

• cdt1 binds to geminin until M phase
• cdc6/18 gets degraded, expressed after M

*Licensing allows replication to be activated once and only once in cell cycle & is not activated until after M phase

In G_o → cell loses capacity to license origins and prevents from becoming a tumor cell

Question 10

Aberrations in initiation of DNA synthesis (2)

Answer 10

1. Overinitiation at origin → gene amplification → drug resistant tumor cells
2. Oncogens: mutant genes of signal transducing proteins → persistent signal for cell division