DNA Structure, Function, Replication + Modification (Lectures 6, 10, 14)

Question 1

DNA structure

(1, 2, 3 / B form)

Answer 1

Primary structure: base sequence

Secondary structure: B form DNA, stabilized by H bonds, van der waals, hydrophobic interactions

Tertiary structure: supercoiling

B-form DNA: (right handed helix)

10. 4 bp/turn
11. 34nm helical rise

2nm standard width

proteins access DNA at the major groove

Question 2

Replication protein A

Answer 2

• Euk equivalent of single-stranded binding proteins (which are in proks)

Question 3

Deamination of 5-methylcytosine

Answer 3

Forms thymine

Question 4

Types of DNA Mutations

(3 main types + subtypes)

Answer 4

1. Point mutations–> single base pair change
   
   • Transition mutation: most common, usually repaired
     
     • purine –> purine (G <–> A)
       
       • 8-oxoguanine mispaired with adenine
     • pyrimidine –> pyrimidine (C <–> T)
       
       • 5 methylcytosine deamination –> thymine
   • Transversion mutation: rare, only repaired spontaneously
     
     • purine –> pyrimidine (A <–> T; C <—> G)
2. Deletion/Insertion mutations ​

• Often caused by intercalating agents that insert btw bp (ie, ethidium bromide)
• Can disrupt reading frame, cause pre-mature termination of transcription

3. Inter/intra strand breaks / cross-links

• Caused by UV/ionization radiation, chemicals like bleomycine, mitomycin C, etc.
• Photodimerization can result in intra-strand dimerization of adjacent thymines

Question 5

Telomeres

Answer 5

• Highly repetitive seq at 3’ end of linear chromosomes
• Added by telomerase:
  
  • Protein component: reverse transcriptase
    
    • hTERT: human telomerase reverse transcriptase
  • RNA component: template + primer for DNA pol
    
    • hTR: human telomerase RNA

Question 6

Addition of telomeres + 3’ overhang

Answer 6

• 3’ overhang left by removal of terminal primer
• Telomerase uses RNA component to add repeats
  
  • Daughter strand is synthesized (complementary base pairing) by DNA pol alpha
  • Process is repeated, forming the long telomere
• When telomerase moves away single stranded 3’ overhang is left (forms D-loop-t-loop)
  
  • Loops + base-pairs with strand on the other side
    
    • remaining single strands are stabilized by telomere-binding proteins (TRF-1 and TRF-2)

Question 7

Nick translation

Answer 7

• Only in prokaryotes?

1. DNA pol I 5’ –> 3’ exonuclease hydrolyses the RNA primers
2. Simultaneously the 3’ end of the Okazaki fragment (DNA) is extended by incorporation of dNTPs
3. DNA ligase seals the nick

Question 8

DNA methylation in eukaryotes

Answer 8

• only 5-methylcytosine
  
  • 3-5% of DNA cytosine content (in CPG islands)
• usually in residues 5’ to G
• when C in one strand is methylated, the C in the complementary strand is also methylated
• heritable
• done by maintenance methylase after replication
• one method of controlling gene expression
  
  • methylated promoters are not expressed

Question 9

Excinuclease

Answer 9

An endonuclease that cuts both sides of the site of damage in DNA

Question 10

DNA polymerase gamma

Answer 10

• Eukaryotic DNA polymerase
• Found in the mitochondria, involved in mitochondrial DNA replication
• Prok equiv: one of the functions of DNA pol III

Question 11

Nucleotide excision repair (NER)

Answer 11

• Repairs DNA lesions that cause large structural changes
  
  • eg, intra-strand thymine dimers caused by UV irradiation
• In prokaryotes:
  
  1. DNA is scanned for damage such as pyrimidine dimers by UvrAB.
  2. Repair proteins stall at damage
  3. Bend DNA
  4. UvrA displaced, UvrC joins complex
  5. Endonuclease cuts 3’ then 5’ to the damage and a helicase removes damaged piece
  6. DNA pol I replaces the excised DNA
  7. Nick in backbone sealed by DNA ligase

Question 12

Directionality in DNA Synthesis

(+ Leading strand/Lagging strand)

Answer 12

• DNA template read 3’ —> 5’
• DNA synthesized 5’ —->3’
• Lagging strand: synthesized away from the replication fork opening (in fragments)
• Leading strand: synthesized toward replication fork opening

Question 13

DNA polymerase alpha

Answer 13

• Eukaryotic DNA polymerase
• In the nucleus
• Makes RNA primers
• Prokaryotic equiv: dnaG

Question 14

PCNA

Answer 14

• proliferating cell nuclear antigen
• In eukaryotes
  
  • Increases the processivity of DNA pol delta + epsilon
• Prokaryotic equivalent: ß subunit in DNA pol III

Question 15

Base Excision Repair

Answer 15

DNA-N-glycosylases remove incorrect bases in DNA

• Prokaryotes have one for each base

1. Recognition of damage
2. Base removed from backbone (creates apyrimidinic / apurinic site by removing base and keeping backbone), cuts glycosidic bond
3. Endonuclease cuts backbone 5’ to the damage
4. DNA pol I removes the deoxyribose phosphate residue in the 5’—> 3’ direction, and replaces it and several other nucleotides through nick translation
5. Nick in backbone sealed by DNA ligase

Question 16

dna B/dna C complex

Answer 16

• Prok helicase
• Euk equiv: mini chromosome maintenance proteins (MCM)

Question 17

OriC

Answer 17

• Origin of replication in prokaryotes
  
  • one in the circular bacterial chromosome
• Euk equivalent: ARS (autonomous replicating sequences)
  
  • multiple sites in euks

Question 18

Deamination of cytosine

Answer 18

Forms uracil

Question 19

Methods of DNA Repair

Answer 19

1. Direct repair of damaged base

• O6 methylguanine DNA methyl transferase –> could be incorrectly paired with thymine during replication
• Transfers methyl group from O6-methylguanine to itself (self alkylates and restores guanine)

2. Excision repair

• nucleotide excision repair
  
  • repairs DNA lesions that cause large structural changes (eg, thymine dimers)
• base excision repair
  
  • remove incorrect bases in DNA

3. Mismatch repair
   * Proofreading

Question 20

dna A complex

Answer 20

• binds to the origin of replication in proks
  
  • initiator protein
• Euk equiv: (ORC) origin of replication complex
  
  • requires MCM (DNA helicase) + activation by cyclins + CDKs to open replication forks and initiate replication

Question 21

RNase H and Fen 1 (flap endonuclease 1)

Answer 21

• Removal of RNA primers in Euk
• Prok equiv: DNA pol I

Question 22

DNA polymerase delta

Answer 22

• Eukaryotic DNA polymerase
• Found in nucleus
• Involved in lagging strand replication
• Requires PCNA
• Prok equiv: one of the functions of DNA pol III

Question 23

dna G

Answer 23

• In prok, makes RNA primer
• Euk equiv: DNA pol alpha

Question 24

DNA polymerase epsilon

Answer 24

• Eukaryotic DNA polymerase
• Found in nucleus
• Involved in leading strand replication
• Requires PCNA
• Prok equiv: one of the functions of DNA pol III

Question 25

Mismatch Repair (with the Mut HLS system)

Answer 25

* Recognizes and repairs mis-matche base pairs (recognizes new strand is unmethylated) * MutS: motor protein that scans DNA looking for damage; stalls at damage and pulls DNA from both directions * MutH and Mut L complex with MutS. It pulls DNA trough it, searching for nearest GATC sequence (A is normally methylated in EColi in this squence ) * MutH (endonuclease) cuts 5' to damage site,, then DNA is removed by a helicase, the DNA unwinds past the damage site, and a 3' exonuclease removes the nucleotides. DNA pol III and DNA ligase finish the repair.

Question 26

Repication factor C (RFC)

Answer 26

* Euk equiv of prok y-complex of DNA pol III

Question 27

DNA polymerase ß

Answer 27

* Eukaryotic DNA polymerase * DNA repair

Question 28

Methylation of prokaryotic DNA

Answer 28

* Protects bacterial DNA from cleavage by restriction endonucleases Only modifications in prokaryotes: * N6-methyladenine * N4-methylcytosine

Question 29

Prokaryotic DNA polymerases (5 known)

Answer 29

**DNA pol I:** RNA primer excision (5' exonuclease) and DNA repair (polymerase and 3' exonuclease) **DNA pol II:** SOS DNA repair * activated as a cellular stress responsive to extensive DNA damage **DNA pol III:** Chain replication/elongation (and repair) during synthesis; highly processive * Epsilon subunit: 3' --\> 5' exonuclease (proof-reading; removes mismatched bases) * Alpha subunit: polymerase * ß subunit: sliding clamp, has low affinity for DNA * y complex: loads sliding clamp onto DNA **DNA pol IV & V:** SOS DNA repair

Question 30

Stabilizing / Destabilizing factors in secondary DNA structure (+Tm)

Answer 30

Stabilizing factors * Hydrogen bonds between base pairs * AT; 2 bonds * CG; 3 bonds * Van der waals interactions * GC rich DNA is more stable than AT rich, because adjacent GC have stronger van der waals interactions * Ions in cells Tm: temperature at which hald the DNA has denatured, depends on the % of AT and GC base pairs

Question 31

Type I topoisomerases

Answer 31

DNA is often negatively supercoiled Single strand breaks to relax helix

Question 32

Type II topoisomerases

Answer 32

obtains energy from ATP hydrolysis double strand breaks changes supercoiling by 2 turns of the double helix

Question 33

Histones

Answer 33

* add negative supercoils, reduce length, directly involved in the regulation of access to DNA * there are five histones, they are (-) charged * **nucleosomes :** first level of folding in eukaryotes, dsDNA is around an octamer histone core

Question 34

Answer 34

Guanine

Question 35

Answer 35

Adenine

Question 36

Answer 36

Cytosine

Question 37

Answer 37

thymine

Question 38

Answer 38

uracil