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Bio 209 > Lecture 3 - DNA Synthesis > Flashcards

Lecture 3 - DNA Synthesis Flashcards

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1
Q

Semiconservative mode of DNA replication and how this makes sense given what you know about the W&C model of DNA structure

A
  • Each parental strand serves as a template for new daughter strands
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2
Q

Direction of DNA synthesis? Evidence?

A

5’-3’, bidirectional

Denaturation mapping of phage

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3
Q

3 phases of DNA replication

A
  1. Initiation
  2. Elongation
  3. Termination
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4
Q

DNA polymerases: know what reactions they catalyze

A
  • DNA polymerases are proteins that catalyze DNA synthesis via covalent addition of nucleotides to pre-existing DNA
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5
Q

DNA polymerases: cofactor requirements

A
  • Mg2+ -> as nucleotide comes into catalytic site, Mg 2+ helps position dNTP properly for nucleophilic attack => phosphodiester linkage
  • dNTPs -> substrate
  • Primer DNA (primer is RNA)
  • Template DNA
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6
Q

DNA polymerases: Prokaryotic types

A

DNA Polymerase I: DNA repair, RNA primer removal
- 5’ to 3’ polymerase activity (catalyzes formation of phosphodiester bond)
- Facilitates nucleophilic attack by 3’ OH of primer strand
- Exergonic elimination of pyrophosphate coupled w/ endergonic formation of phosphodiester bond
- 5’-3’ exonuclease activity: remove RNA primer w/ RNase H, DNA polymerase I fills base gap in bacteria
- 3’-5’ exonuclease activity: removes mismatched bases

DNA Polymerase III: True “replicase” (synthesizes majority of bacterial chromosome)
- Replicates E.coli genome
- Multimeric protein (subunits, beta is important), without holoenzyme, short strands are synthesized (no beta clamp)
- Synthesizes both strands in 5’-3’ direction -> one continuous (leading), one discontinuous (lagging/Okazaki fragment)

DNA Polymerase II, IV, and V: for replication of damaged DNA (idt we need to know)

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7
Q

What is strand directed mismatch repair (SDMMR)?

A
  • Repairs errors missed by proofreading exonuclease of DNA polymerase
  • Must recognize/repair error in newly synthesized strand, NOT original base in parent strand

Strand detection:
- Bacteria: more methylated A’s => older => template | less methylated => younger => new strand
-Humans: detects nicks prior to DNA ligase sealing of Okazaki fragments

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8
Q

What are the three proofreading steps that give rise to high-fidelity DNA synthesis?

A
  • 5’ - 3’ exonuclease activity
  • 3’ - 5’ exonuclease activity
  • SDMMR
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9
Q

What is a holoenzyme?

A

The complete enzyme complex

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10
Q

Leading vs Lagging Strand? Continuous vs Discontinuous Synthesis? Okazaki fragments?

A

Both DNA strands are synthesized in the 5’‘3’ direction

One strand is continuously synthesized => leading strand

One strand is discontinuously synthesized in short stretches => lagging strand => Okazaki fragments

Okazaki fragments must be joined together to make discontinuous strand continuous

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11
Q

DNA polymerase: Eukaryotic types

A

DNA polymerase alpha
- Primer synthesis (complexed to DNA primase)
- Primer extended to ~30 nucleotides (10 RNA + 20 DNA)

DNA polymerase delta
- Lagging strand DNA synthesis
- Nucleotide and base-excision repair
- Processive synthesis of chromosomal DNA (must interact w/ PCNA and Rf-C to be active)
- 3’ to 5’ exonuclease activity

DNA polymerase epsilon
- Leading strand DNA synthesis
- Nucleotide and base-excision repair

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12
Q

What is processivity? What is responsible for processive synthesis of DNA in prokaryotes vs eukaryotes?

A

Processivity: how well polymerase can stay on track to synthesize longer stretches of DNA

Prokaryotes: Beta subunit “clamp” of DNA pol III

Eukaryotes: PCNA (sliding clamp) and Rf-C (loads PCNA onto DNA) of DNA pol delta

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13
Q

General similarities/differences b/t prokaryotic and eukaryotic DNA replication

A

All polymerases need primers for free 3’ OH, 1 for leading strand and 1 for every Okazaki fragment

Prokaryotes:
- Faster, but single origin of replication (245 bp, 3x13 bp A/T rich repeat for replication bubble, 4x9 bp repeat for protein binding site)
- No cell cycle => replication is non stop

Eukaryotes:
- Slower, but multiple origins of replication (Autonomously Replicating Sequences ARS 50 bp, core 11-bp AT rich sequence)
- More complex (replisome has more proteins)
- RNA primers and Okazaki fragments shorter
- Occurs only in S phase of cell cycle
- Multiple polymerases at replication fork
- Nucleosomes
- Telomeres

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14
Q

What is DNA primase?

A
  • An RNA polymerase that requires only a DNA template (no primer/free 3’ OH) that creates a primer and RNA/DNA heteroduplex
  • The RNA primer provides a free 3’ OH that can nucleophilic attack and form a phosphodiester linkage
  • RNA primers are excised and replaced by DNA polymerase I 5’-3’ exonuclease activity in prokaryotes, ribonuclease H1 and ribonuclease FEN-1 in eukaryotes
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15
Q

DNA primase and Okazaki Fragments - elaborate

A
  • 1 primer per Okazaki fragment
  • On lagging strand, extension stops when encounters next RNA primer
  • Adjacent Okazaki fragment 3’ OH is used as primer for DNA polymerase I
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16
Q

What is DNA ligase?

A
  • An enzyme that catalyzes the covalent closure of nicks in DNA
  • Covalently links adjacent Okazaki fragments
  • Does not work to fill gaps i.e. where bases are missing => filled by DNA polymerase I in prokaryotes, DNA polymerase delta in eukaryotes, then DNA ligase seals backbone nick
17
Q

What is DNA helicase?

A
  • 6 subunit enzyme that unwinds DNA using ATP, producing two single strands of DNA
18
Q

What prevents single stranded DNA from H-bonding to itself and staying extended after unwinding by DNA helicase? Proks vs Euks

A

In prokaryotes: single-stranded DNA binding protein (SSB)

In eukaryotes: replication protein A (Rp-A)

19
Q

What is DNA topoisomerase? Type 1 vs Type 2?

A
  • Enzyme that catalyzes transient breaks in DNA to prevent overwinding, positive supercoils

Topoisomerase I: Single stranded break
- Removes supercoils one at a time
- Transient break allows opposite sides of break to spin independently around intact phosphodiester bond
- Reseal break

Topoisomerase II: Introduces negative supercoils
- 2 (-) supercoil added
- Double stranded break
- Ex: DNA gyrase, responsible for negative supercoiling found in E. coli genome
- In eukaryotes -> found where 2 DNA helices cross one another -> breaks both strands of double helix, allows other double helix to pass through gate, reseals double stranded break -> separates two interlocked DNA circles

20
Q

What is DNA gyrase?

A
  • A topoisomerase II in e. coli
  • W/o it, bacterial genome would have a lot of positive supercoil, overwound DNA => block bacterial replication
  • Antibiotics work on this to block bacterial genome replication
21
Q

Components of prokaryotic and eukaryotic DNA replication that serve as accessory proteins to major enzymes discussed

A
  • SSBs in prokaryotes, RP-A in eukaryotes => keep unwound single strand DNA extended
  • Sliding clamp: beta subunit of DNA pol III of prokaryotes, PCNA in DNA pol delta of eukaryotes
  • Clamp loader: gamma subunit of DNA pol III of prokaryotes, Rf-C in DNA pol delta of eukaryotes
22
Q

What experiment was used to determine dispersive mechanism of nucleosome replication?

A

Method: density transfer experiments (use light and heavy isotopes of certain atoms to determine age of histone complex)

Result: Nucleosome on progeny DNA contains old and new histone complexes => nucleosome duplication occurs through dispersive mechanism

23
Q

Telomeres: General types of sequences associated , where found on the chromosome, issues related to replication of telomere lagging strand, importance of telomerase, relationships b/w telomere length and aging vs. cancers

A

Sequence: 500-3000 copies of TTAGGG repeat in humans, 12-16 bp single stranded 3’ overhang

Location: Ends of eukaryotic chromosomes

Issues related to replication of telomere lagging strand: Cannot produce terminal Okazaki fragment b/c no primer to provide free 3’ OH (refer to slide 69 lecture 3)

Telomerase:
- Binds to G rich telomere overhang using internal RNA template
- Adds single telomere repeats to parent strand
- After several additions => RNA primer made, DNA polymerase synthesizes new strand

Telomere length and aging vs cancers:
- Older cells have shorter telomere lengths
- Cancer -> tumor cells display increased telomerase activity and longer telomeres vs normal cells
- Progeria -> rare human disease characterized by premature aging i.e. extremely short telomeres

Bio 209 (10 decks)

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