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MCDB 131 > Lecture 5 - chromosomes and DNA replication > Flashcards

Lecture 5 - chromosomes and DNA replication Flashcards

1
Q

Summary of DNA structure

A

Polymer of nucleotides connected by phosphodiester bonds. Two strands of DNA are oriented in an antiparallel manner with purines of one strand bonding to pyrimidines of the other

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2
Q

Significance of DNA grooves and fluorescence microscopy

A
  • Lots of hydrophobic interaction between stacked layers; allows DAPI and acridine orange enter grooves in DNA to fluoresce.
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3
Q

Major groove and DNA binding proteins

A

Provides sequence info for binding proteins. Binding proteins are able to read out the sequence of nucleotides based on the pattern of H donors/acceptors.

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4
Q

How do DNA proteins have sequence specificity?

A

Insertion into the major groove with the correct sequence. If there are multiple binding sites, these are usually about 10 base pairs apart to account for the natural spiral of DNA

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5
Q

Structure of the bacterial nucleoid

A

Contains topologically separated domains anchored by histone-like proteins

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6
Q

Purpose of histone-like proteins in the nucleoid

A

Anchoring proteins prevent relaxation of one region of DNA from spreading to the rest of the chromosome (maintains compaction of DNA)

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7
Q

What does supercoiling do for the overall energy state of DNA?

A

Both positive and negative supercoiling have a higher energy state than relaxed DNA.

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8
Q

Positive vs negative supercoiling

A

Positive: result from over-winding of DNA (adding an extra twist of the helix) and makes the strands harder to separate
Negative: results from under-winding of DNA (taking out one twist of the helix) and makes the strands easier to separate

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9
Q

What organisms possess positively supercoiled DNA?

A

Archaea living in acid at high temperatures; makes them more resistant to DNA degradation

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10
Q

What organisms possess negatively sueprcoiled DNA?

A

Eukaryotes, bacteria, and most archaea; makes it easier to open DNA up for reading

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11
Q

Function of topoisomerases

A

Modulate level of genomic supercoiling

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12
Q

Type I Topoisomerases

A
  • usually single subunit enzymes
  • cleave and reseal only one strand of DNA, therefore can only relieve supercoils (can’t increase supercoiling)
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13
Q

Type II Topoisomerases

A
  • multi subunit enzymes
  • cleaves both strands of DNA and uses ATP hydrolysis to change supercoiling
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14
Q

DNA Gyrase

A

Type II topoisomerase that is targeted by aminocoumarin and quinolone antibiotics

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15
Q

How does topoisomerase I work?

A

Cleaves one strand of the double helix and passes the other strand through to remove one supercoil

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16
Q

How does DNA gyrase work?

A

Cleaves both strands of the double helix and passes the remaining intact double stranded section through to add one supercoil.

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17
Q

How does novobiocin inhibit DNA gyrase activty?

A

Competitively binds GyrB (subunit of DNA gyrase) and prevents ATP from binding

18
Q

How do cipro and nalidixic acid inhibit DNA gyrase activity?

A

Prevent re-ligation of double stranded breaks

19
Q

Why did we move from nalidixic acid to cipro?

A

It was too easy for bacteria to become resistant to nalidixic acid

20
Q

Purpose of DnaA

A

Controls initiation of DNA replication. Accumulates during cell growth and triggers replication at specific sites near the origin of replication.

21
Q

How does DnaA work?

A

DnaA-ATP complexes bind to 9-bp repeats upstream of the origin –> looping in DNA to prepare for strand separation by DNA helicase (DnaB)

22
Q

What is the purpose of the clamping protein in DNA polymerase III?

A

Ensures that DNA Pol III can stay on the DNA for the entirety of replication. Otherwise, DNA Pol would fall off and have to be re-loaded onto the strand

23
Q

Purpose of RNA polymerase

A

Places RNA primer for initiation of DNA replication

24
Q

How are RNA primers removed during replication?

A
  1. RNase H recognizes the RNA-DNA mismatch and cleaves the RNA.
  2. DNA Pol I fills the gap with DNA
  3. DNA ligase repairs the nick
25
Q

How does Tus work in termination of replication

A

Binds terminator (ter) sequences and acts as a counter-helicase to stop the progress of DNA helicase

26
Q

How are linked (catenated) chromosomes separated at the end of replication?

A

Topoisomerase IV or XerC and XerD resolvases

27
Q

How are ter sequences distributed on the chromosome?

A

Along the ter macrodomain, some stop clockwise replication and some stop counterclockwise rotation. Multiple ter sequences for both directions to ensure that replication stops

28
Q

Mechanism of daughter chromosome deacatenation

A
  1. dif (deletion induced filamentation) sequence at the Ter macrodomain is recognized by XerC
  2. If two dif sequences are nearby (as in the case of linked chromosomes), XerC will bind both and form a Holliday junction
  3. XerD resolves the Holliday junction and separates the chromosomes
29
Q

Plasmids

A

Extragenomic, autonomously replicating DNA molecules.
- usually circular and sometimes much smaller than chromosomes
- require host proteins to replicate

30
Q

Copy number of plasmids in bacterial cells

A

Can range from 1 to 1000.

31
Q

Copy number vs plasmid size

A

Plasmid production exerts metabolic drag on the host cell. The bigger the plasmid, the lower the copy number

32
Q

Problems with low copy number plasmids

A
  • Need machinery to ensure that plasmids are properly partitioned into daughter cells
  • can more easily be lost by the host cell
33
Q

What advantages can plasmids give a bacteria?

A

Occupation of different niches
- Resistance to antibiotics/toxic metals
- pathogenesis
- symbiosis

34
Q

Methods of plasmid replication

A
  1. Bidirectional (“theta”) replication
  2. Rolling circle replication
35
Q

How does bidirectional (“theta”) replication work?

A

Starts at a single origin and occurs in two directions simultaneously; same as chromosomal replication

36
Q

How does rolling circle replication work?

A

Starts at a single origin and moves in one direction.

37
Q

Steps of rolling circle replication

A
  1. Rep dimer makes a nick on one strand and binds to the 5’ end
  2. 3’ end of the nick is extended by DNA pol III
  3. single strand DNA on outer strand is displaced as new DNA is made
  4. Rep releases circularized old outer strand after full replication of outer strand
  5. Old outer strand receives a complementary inner strand
38
Q

Mechanism of plasmid partitioning

A
  1. ParR binds plasmid and ParM
  2. ParM-ATP monomers bind ParR and form filaments that push one another away
  3. Plasmids are pushed apart
39
Q

What is ParM

A

Structural homolog of actin that forms filament for plasmid partitioning

40
Q

Purpose of toxin-antitoxin modules

A

Used in post-segregational killing to kill daughter cells that don’t receive the plasmid after segregation

41
Q

How does toxin-antitoxin work in post-segregational killing

A
  1. Toxin and antitoxin production are driven by the same promoter –> produce a dimer of toxin and antitoxin
  2. Toxin is held inactive when bound to antitoxin
  3. Antitoxin is degraded overtime by proteases
  4. If there is no plasmid to continue production of antitoxin molecules, the cell will die
42
Q

How does a cell die in post-segregational killing?

A

Toxin poisons DNA gyrase and prevents re-ligation of double stranded breaks

MCDB 131 (15 decks)

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