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

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

1
Q

DNA structure

(1, 2, 3 / B form)

A

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)

  1. 4 bp/turn
  2. 34nm helical rise

2nm standard width

proteins access DNA at the major groove

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2
Q

Replication protein A

A
  • Euk equivalent of single-stranded binding proteins (which are in proks)
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3
Q

Deamination of 5-methylcytosine

A

Forms thymine

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4
Q

Types of DNA Mutations

(3 main types + subtypes)

A
  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
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5
Q

Telomeres

A
  • 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
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6
Q

Addition of telomeres + 3’ overhang

A
  • 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)
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7
Q

Nick translation

A
  • 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
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8
Q

DNA methylation in eukaryotes

A
  • 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
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9
Q

Excinuclease

A

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

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10
Q

DNA polymerase gamma

A
  • Eukaryotic DNA polymerase
  • Found in the mitochondria, involved in mitochondrial DNA replication
  • Prok equiv: one of the functions of DNA pol III
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11
Q

Nucleotide excision repair (NER)

A
  • 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
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12
Q

Directionality in DNA Synthesis

(+ Leading strand/Lagging strand)

A
  • 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
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13
Q

DNA polymerase alpha

A
  • Eukaryotic DNA polymerase
  • In the nucleus
  • Makes RNA primers
  • Prokaryotic equiv: dnaG
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14
Q

PCNA

A
  • proliferating cell nuclear antigen
  • In eukaryotes
    • Increases the processivity of DNA pol delta + epsilon
  • Prokaryotic equivalent: ß subunit in DNA pol III
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15
Q

Base Excision Repair

A

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
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16
Q

dna B/dna C complex

A
  • Prok helicase
  • Euk equiv: mini chromosome maintenance proteins (MCM)
17
Q

OriC

A
  • Origin of replication in prokaryotes
    • one in the circular bacterial chromosome
  • Euk equivalent: ARS (autonomous replicating sequences)
    • multiple sites in euks
18
Q

Deamination of cytosine

A

Forms uracil

19
Q

Methods of DNA Repair

A
  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)
  1. Excision repair
  • nucleotide excision repair
    • repairs DNA lesions that cause large structural changes (eg, thymine dimers)
  • base excision repair
    • remove incorrect bases in DNA
  1. Mismatch repair
    * Proofreading
20
Q

dna A complex

A
  • 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
21
Q

RNase H and Fen 1 (flap endonuclease 1)

A
  • Removal of RNA primers in Euk
  • Prok equiv: DNA pol I
22
Q

DNA polymerase delta

A
  • Eukaryotic DNA polymerase
  • Found in nucleus
  • Involved in lagging strand replication
  • Requires PCNA
  • Prok equiv: one of the functions of DNA pol III
23
Q

dna G

A
  • In prok, makes RNA primer
  • Euk equiv: DNA pol alpha
24
Q

DNA polymerase epsilon

A
  • Eukaryotic DNA polymerase
  • Found in nucleus
  • Involved in leading strand replication
  • Requires PCNA
  • Prok equiv: one of the functions of DNA pol III
25
Q

Mismatch Repair

(with the Mut HLS system)

A
  • 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.
26
Q

Repication factor C (RFC)

A
  • Euk equiv of prok y-complex of DNA pol III
27
Q

DNA polymerase ß

A
  • Eukaryotic DNA polymerase
  • DNA repair
28
Q

Methylation of prokaryotic DNA

A
  • Protects bacterial DNA from cleavage by restriction endonucleases

Only modifications in prokaryotes:

  • N6-methyladenine
  • N4-methylcytosine
29
Q

Prokaryotic DNA polymerases

(5 known)

A

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

30
Q

Stabilizing / Destabilizing

factors in secondary DNA structure

(+Tm)

A

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

31
Q

Type I topoisomerases

A

DNA is often negatively supercoiled

Single strand breaks to relax helix

32
Q

Type II topoisomerases

A

obtains energy from ATP hydrolysis

double strand breaks

changes supercoiling by 2 turns of the double helix

33
Q

Histones

A
  • 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
34
Q
A

Guanine

35
Q
A

Adenine

36
Q
A

Cytosine

37
Q
A

thymine

38
Q
A

uracil

MFoM (6 decks)

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