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Genetics > Lecture 5 > Flashcards

Lecture 5 Flashcards

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Biology 101 flashcards
1
Q

What is the problem with completely replicating linear chromosomes?

A
  1. Unfillable Gap left when RNA primer removed
  2. Dissolution of replication fork after leading strand finished (before lagging strand finished) - loss of Okazaki fragments
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2
Q

How do telomeres protect ends of linear chromosomes?

A
  • Eukaryotic chromosome ends have simple sequence repeats - telomeres
  • Telomeres from 50bp to over 20,000bp

G-rich strand extends 5’ to 3’ - terminates in ssDNA tail ~75-300nt

Form unusual structures

Telomere repeats are binding sites for proteins - mark as natural ends and distinguish DNA breaks - essential from chromosome stability

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3
Q

What maintains telomeres

A

Telomerase (a reverse transcriptase)

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4
Q

Where is telomerase active?

A

Tissue-specific stem cells and germline cells

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5
Q

What does telomerase do?

A

Synthesize one strand of telomere - G rich strand 5’ to 3’ in direction away from centromere

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6
Q

What is telomerase formed from?

A

Protein (telomerase reverse transcriptase or TERT) and RNA (template for synthesis of repeats, role in enzyme activity)

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7
Q

Explain how telomerase elongates 3’ end of telomeres

A
  • Telomerase binds ss telomere DNA
  • 3’ end of DNA base-pairs with RNA
  • RNA is template to synthesize DNA
  • Enzyme translocates and repeats synthesis to elongate G-rich 3’ end
  • c-rich strand filled in by DNA pol alpha
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8
Q

What occurs in somatic issues lacking telomerase

A

Telomere shortening over successive cell divisions

Short telomeres activate DNA damage response

Telomerase expression re-activated in many cancers

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9
Q

How long does it take Escherichia coli to divide?

A

20 mins

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10
Q

How long does it take to replicate genome in E. coli

A

50 minutes

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11
Q

What occurs in good growth conditions in E. coli replication

A

Second round of replication initiates before first finished

Re-replication is well-regulated - initiation at each oriC in a cell is synchronous - number of oriC always power of 2

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12
Q

Regulatory initiation of replication in E.coli

A

DnaA binds and multimerizes at oriC when bound to ATP - unwinds adjacent DNA

Helicase/primase/sliding clamp initiate synthesis

Sliding clamp initiates hydrolysis of DnaA-ATP to DnaA-ADP

DnaA-ADP disassociates - can’t reinitiate origin firing - prevents overstimulation of initiation

Regulatory inactivation of initiation (RIDA) blocks replication initiation - major regulatory step preventing initiation

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13
Q

Regulation of initiation by methylation status

A
  1. 11 GATC sites in oriC overlap DnaA boxes
  2. DNA adenine methylase aka Dam methylase methylates GATC sequences in E. coli genome
  3. Before replication GATC sequences fully methylated
  4. After replication DnaA boxes transiently hemi-methylated
  5. SeqA binds to hemi-methylated GATC sites
    - Prevents DnaA rebinding
    - Prevents Dam methylase methylating at GATC
  6. SeqA cause temporary block
  7. SeqA dissociates
  8. Origin fully methylated after 10 mins
  9. Fully methylated chromosome ready for DnaA binding
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14
Q

DnaA sequestration

A
  • The datA DNA region, adjacent to oriC, binds about 25% of free DnaA.
  • Post-replication, datA duplicates, sequestering double the DnaA.
  • During chromosome segregation, one datA copy frees DnaA for oriC binding, aiding initiation
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15
Q

Recycling of DnaA-ADP to DnaA-ATP

A

DnaA-ATP must be provided for next round of initiation

DnaA-ATP newly synthesized

DnaA-ADP recycled to DnaA-ATP

DnaA binds to DARS regions of DNA (DnaA reactivating sequences)

DARS serves as cofactors to stimulate exchange of ATP for ADP

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16
Q

Consequences of inaccurate cell cycle

A
  1. Loss or breakage of chromosomes
  2. Inappropriate links between daughter chromosomes
  3. Re-replication have severe consequences
17
Q

Control of eukaryotic chromosome replication

A
  • Replicated from multiple origins
  • Origin firing requires MCM loading - establishes pre-RC in G1
  • Not every pre-RC fires in every cell cycle - controlled by origin efficiency and timing
  • Origin timing relates to when origins fire early or late
  • Origin efficiency is probability an origin will fire
  • Origin firing stochastic
  • Replication of unused origins removes preRC

-MCM proteins cannot load in S

18
Q

What regulates origin licensing in mammalian cells?

A

Proteolysis - origins selected in G1 by ORC binding, cdc6 and cdt1 mediated loading of MCM

19
Q

Mechanism of yeast to prevent helicase loading outside G1

A
  • Exporting proteins from nucleus - transcription inhibition, proteolysis
  • Metazoa - Main mechanism is proteolysis of proteins required to form pre-replication complex in S-phase
  • Geminin provides additional control layer where it binds to Cdt1
20
Q
A

DNA packaged into chromatin by wrapping around nucleosomes ~146bp DNA wrapped around histone octamer

Nucleosome faithfully replicated on 2 daughter DNA molecules

Histone modifications maintained - epigenetic inheritance

21
Q

Explain how parental histones recycled

A

Nucleosomes are disrupted at replication fork by CMG helicase

Parental histones are actively transferred to daughter DNA – parental histone segregation

Provides a guide to reconstruction of daughter chromatin with same modifications as were present in parental chromatin.

Basis of epigenetic inheritance

Parental H3 and H4 are distributed equally between daughters as tetramers – half go to one daughter, half to the other

Parental H2A and H2B are disassembled and reassembled on daughters as dimers

Nucleosome disassembly and reassembly is mediated by histone chaperones

Histone chaperones interact with replication fork proteins to coordinated nucleosome disassembly and reassembly

22
Q

Synthesis of new nucleosomes

A
  • Segregation splits parental nucleosomes, giving each daughter half.

New histones made in S-phase.

H3 and H4 histones acetylated on specific lysine in N-terminal tail.

ASF1 and CAF1 aid new H3/H4 assembly into tetramer, loaded onto DNA via PCNA for half nucleosome.

NAP1 and FACT add new H2A/H2B to form complete nucleosomes.

H3/H4 deacetylated.

Parental histones guide enzyme modification of new histones (based on old nucleosome modifications).

Genetics (18 decks)

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