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CMMB 411- Doreen > Topic 4 > Flashcards

Topic 4 Flashcards

1
Q

In prokaryotes, DNA is organized in _____, but is less _____

A

In prokaryotes, DNA is organized in nucleoid, but is less compact

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2
Q

In eukaryotes, DNA is____ within the nucleus

A

Highly compact

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3
Q

In general, as species complexity increases, the genome size…
Is this a hard and fast rule?

A

Increases
Not a hard and fast rule become some species have similar complexities, but different genome sizes. So we can’t really say that complex organisms have a high number of genes all the time.

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4
Q

Gene density

A

The average number of genes per megabase (Mb) of genomic DNA

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5
Q

More complex organisms have (lower/higher) gene density
- Explain why

A

Lower
- Due to larger gene size and more DNA between genes (intergenic sequences)

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6
Q

What is the increase in genome size in more complex organisms due to?

A

Due to increasing the number of introns, repetitive DNAs, and longer intergenic sequences, NOT just gene number

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7
Q

> 60% of human genome is _______, while <40% of human genome is ______

A

> 60% of human genome is intergenic DNA, while <40% of human genome is genes and gene related sequences

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8
Q

Intergenic DNA can be separated into… (2)
- Give an example for one of them

A
  1. Genome-wide 1400 Mb repeats
    - e.g. transposons and other mobile DNA
  2. Other intergenic regions (600 Mb)
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9
Q

Intergenic regions can be separated into… (2)

A
  1. Unique DNA regulatory regions, miRNAs
  2. Microstallites (90Mb)
    - simple repeats, e.g. (AC)100
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10
Q

Repetitive DNA (1490 Mb)

A

Genome-wide 1400 Mb repeats and microsatellites (90Mb)

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11
Q

Non-repetitive intergenic sequences (510 Mb)

A

Unique DNA: regulatory regions, miRNAs

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12
Q

Gene related sequences can be divided into… (4)

A
  1. Introns
  2. UTRs
  3. Gene fragments
  4. Pseudogenes
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13
Q

Non-functional gene-related sequences

A

Gene fragments and pseudogenes

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14
Q

Kinetochore

A

An elaborate protein complex that forms on the centromeres for interacting with spindles during chromosome segregating in cell division

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15
Q

Centromeres

A

DNA sequences that are required for the formation of kinetochore complex.
- 1 centromere/chromosome

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16
Q

What gives the chromosome its “butterfly shape”?

A

The primary constriction site of the centromere
- Refers to the narrow, pinched region of a chromosome where the centromere is located.

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17
Q

Telomeres (telomeres are __-rich)

A

TG-rich repeats that cap the ends of a chromosome and protect from damage and loss. 2 pairs per chromosome.

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18
Q

Origins of replication

A

Multiple sites where DNA replication machinery assembles and begins replication; many origins per chromosome

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19
Q

If the chromosomes lack centromeres, what happens during anaphase?

A

Random segregation of the chromosome
- because the spindles can’t attach to move the chromatids in opposite directions

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20
Q

If the chromosomes have two centromeres each, what happens during anaphase?

A

Chromosome breakage (due to more than one centromeres)
- shearing of the genetic content
- no equal segregation of the genetic content

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21
Q

What are the two main key functions of telomeric proteins?

A
  1. To distinguish the chromosome ends from chromosome and other DNA breakage sites (i.e. to prevent frequent DNA recombination and degradation)
  2. To serve as a specialized origin of replication for replicating the ends of the chromosomes
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22
Q

When are the three cell cycle checkpoints?

A
  1. M phase: ensures that chromosomes will be properly segregated (e.g. ensures spindles have enough tension)
  2. G1/S: makes sure DNA is ready for replication
  3. G2/M: Makes sure DNA is ready for mitosis (replication is complete and there’s no damage)
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23
Q

What are the functions of the gap phases (G1 and G2) in the cell cycle?

A

Provides time for the cells to achieve two goals:
1. To prepare for the next phase of the cell cycles (M or S)
2. To check the completion of the previous phase-cell cycle checkpoint

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24
Q

True or false: DNA replication begins at the origins, and spreads in one direction

A

False
- DNA replication begins at the origins, and spreads in both directions

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25
Q

Each chromosome of a duplicated pair is a…

A

Chromatid

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26
Q

Cohesin

A

A protein that forms rings to hold sister chromatids together
- Important for maintaining chromosomal integrity during replication and early mitosis (before segregation)

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27
Q

DNA replication results in ______ changes

A

Topological

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28
Q

What happens in terms of kinetochores and spindles during chromosome segregation?

A

A kinetochore forms on each chromosome, and connects to spindles, which are connected to the microtubule organizing centre (MTOC or centrosomes)

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29
Q

What happens to cohesin during anaphase?

A

Cohesin is cleaved and then the sister chromatids separate to opposite poles

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30
Q

Interphase

A

G1 + S + G2

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31
Q

Chromosomes are (more/less) compact during interphase

A

Less

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32
Q

Describe the chromosome condensation during mitosis

A

During mitosis, chromosomes are in their most compact forms to facilitate their segregation

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33
Q

Cohesin is required for…

A

Holding the 2 sister chromatids together

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34
Q

Condensin

A

Required for chromosome condensation
- links the loops of DNA together, making the chromosomes compact

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35
Q

What happens to condensin between telophase and G1? What is this called?

A

Condensin is degraded to decondense the chromosomes as they go into interphase
Called the “protein turnover cycle”

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36
Q

During metaphase I, there is _______ attachment of the spindles to the chromosomes

A

Monovalent

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37
Q

During metaphase II, there is _______ attachment of the spindles to the chromosomes

A

Bivalent

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38
Q

What are the two forms of chromatin found in interphase?

A
  1. 10 nm fiber (beads on a string)
  2. 30 nm fiber
  • The 10 nm fiber is further compacted into a 30 nm structure
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39
Q

What’s the distance between the ends of sister chromatids in the M phase?

A

1400 nm

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40
Q

What are two advantages of DNA packaging?

A
  1. Histones protect DNA from damaging
  2. Helps with proper segregation during cell division (prevents DNA entanglement)
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41
Q

What’s a disadvantage of DNA packaging?

A

Reduces accessibility to cellular machinery needed for cell function
(e.g. DNA replication, transcription, repair and recombination)
- Unwrapping is required for gene expression and replication

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42
Q

Building blocks of chromosomes

A

Nucleosomes

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43
Q

Each nucleosome is composed of what? (in general)

A

8 histone proteins and ~2 rounds of DNA wrapped around each histone core

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44
Q

Nucleosomes alone result in what fold compaction?

A

6X

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45
Q

Describe how nucleosomes were discovered using gel electrophoresis

A

Micrococcal nuclease (MNase) is a sequence nonspecific nuclease that cleaves protein-free DNA rapidly and protein-associated DNA poorly.
- When DNA was extensively digested with the MNase, a 147 bp strand appeared on the gel, as linker DNA was fully degraded by the MNase and only the wrapped DNA was present.
- When the DNA was lightly digested, some longer fragments (such as 200 bp or 400 bp) still include both nucleosome-associated DNA and linker DNA.

46
Q

Histones are highly (acidic/basic)
- Name two amino acids found in histones

A

Histones are highly basic (arginine and lysine rich)

47
Q

Where do histones appear on SDS-PAGE?

A

Should be at 10-20 kDa but appear at 30 kDa instead because they’re positively charged so they’re repelled by the positive charge at the end of the gel

48
Q

Describe the structure of histone proteins

A
  • All have a long N-terminus which is important for stabilization of DNA packaging
  • Composed of α helical regions
49
Q

Describe how histones are assembled

A
  1. 2 H3 and 2 H4 form a heterodimer
  2. H2A and H2B form a heterodimer
  3. H3 and H4 tetramer can bind a DNA loop
  4. H2A/H2B heterodimer adds on to the tetramer on both sides (sandwich the DNA and H3/H4 complex)
  5. Amino tails extend outward
50
Q

True or false: Histones as asymmetrical

A

False
- The histone octamer has an approximate two-fold symmetry, meaning the structure can be divided into two mirror-image halves along two axes.

51
Q

How does the H3-H4 tetramer facilitate the association of the H2A-H2B dimer to the DNA?

A

The H3-H4 tetramer binds to the middle and ends of DNA -> DNA contraint and being extensively bent
- Facilitate the association of H2A-H2B dimer to the DNA

52
Q

Where do histones primarily contact the DNA and how?

A

Histones contact the DNA mainly through hydrogen bonding near the minor grooves

53
Q

The histone and DNA contacts are mostly sequence (dependent/independent)

A

Independent

54
Q

What provides the energy to bend the DNA in histone/DNA interactions?

A

Hydrogen bonds

55
Q

True or false: The N-terminal tails of histones are sensitive to protease treatment

A

True
- they are not tightly packed structures in comparison to the histone-fold regions

56
Q

True or false: The N-terminal tails are required for the association of DNA with the histone octamer

A

False

57
Q

The histone ____ are sites of extensive modifications, which regulate nucleosome functions

A

Tails

58
Q

Where do histone tails emerge relative to the DNA helices?

A

Histone tails emerge in between and on either side of the DNA helices

59
Q

How do histone tails aid in nucleosome formation if they are not required for the association of the DNA with the histone octamer?

A

The histone tails direct the DNA to wrap around the histone octamer in a left-handed manner, which introduced negative supercoils to the DNA

60
Q

In eukaryotes, nucleosomal DNA is _____ supercoiled, whereas the remainders are kept…

A

In eukaryotes, nucleosomal DNA is negatively supercoiled, whereas the remainders are kept relaxed by topoisomerases

61
Q

What does negatively supercoiled DNA favour?

A

Negatively supercoiled DNA favours DNA unwinding to facilitate the accessibility of the DNA during DNA replication, transcription and recombination

62
Q

Gyrase and reverse gyrase in prokaryotes

A

Gyrase: A special topoisomerase that introduces negative superhelicity in expense of ATP
Reverse gyrase: keeps the genome positively supercoiled in expense of ATP

63
Q

What types of prokaryotes is reverse gyrase found in?

A

Thermophils

64
Q

Describe heterochromatin regions of DNA

A
  • Condense structure
  • Dense staining
  • higher order nucleosomal DNA assembly
65
Q

Describe euchromatin regions of DNA

A
  • Relatively open structure
  • Staining poorly
  • Less organized nucleosomal DNA assembly
66
Q

Where is heterochromatin found in the nucleus?

A

Found in the nuclear scaffold/matrix which is located closer to the nuclear envelope

67
Q

Histone H1 function

A

Interacts with the linker DNA between nucleosomes, which leads to further condensation of DNA
- also protects extra 20-60 bp of DNA from MNase digestion

68
Q

Chromatin structure in absence/presence of histone H1

A

Absence: 10 nm fiber only, beads on a string pattern
Presence: 30 nm structure

69
Q

What are the two types of chromatin packing?

A
  1. Solenoid
  2. Zigzag
70
Q

How are nucleosomes organized in a solenoid?

A

Linker DNA contained between each nucleosome shaped like a star with a hollow center

71
Q

How are nucleosomes organized in a zigzag?

A

Some linker DNA passes through the center of a star structure (cross section of zigzag) with no hollow center

72
Q

the __ nm fiber makes DNA less accessible to DNA-dependent enzymes (e.g. DNA polymerase, RNA polymerase)

A

30 nm

73
Q

How are histone N-terminal tails required for the formation of the 30 nm fiber?

A

The positively charged N-terminal tails stabilize the 30 nm fiber by interacting with DNA of adjacent nucleosomes
- Tails facilitate compaction due to charge neutralization of the phosphate backbone

74
Q

The nucleosomes arranged in a 30 nm fiber result in only ~40 X of DNA compaction (i.e. not enough compaction). What results in even greater compaction?

A

Each 30 nm fiber forms a DNA loop, and the loops are all held together by a chromosome scaffold

75
Q

How many times does DNA wrap around nucleosomes?

A

about twice
~147 bp

76
Q

Role of topo II as a nuclear scaffold protein

A

Holds DNA at the base of loops and ensures loops are topologically isolated from each other

77
Q

Structural maintenance of chromosome (SMC) function

A

Condenses and holds sister chromatids after chromosome duplication
- Provides an underlying foundation for interactions between nuclear scaffold and chromosomal DNA
- Holds heterochromatin in nuclear scaffold

78
Q

Describe the model of condensin’s role in the minimization of DNA entanglements

A
  • Cohesin generates and stibilizes DNA loops to organize interphase chromatin into topological domains.
  • Topo II can introduce random DNA strand passages by forming knots, intra- or inter-molecular DNA links. Thereby, introducing chromatin compaction.
  • Condensin might use its DNA loop-extrusion activity to constrict DNA entanglements.
79
Q

H2A.X

A
  • H2A variant that is phosphorylated
  • Found at the site of a double-strand break
  • Can be recognized by DNA repair enzymes
  • Strand breakage can happen often, even during meiosis (recombination) so doesn’t need to be a disastrous break.
80
Q

CENP-A

A

Replaces histone H3 in centromeric nucleosomes to serve as a binding site for kinetochore proteins

81
Q

The sequences that tend to face the histone are ____-rich, because…

A

AT-rich, because these sequences tend to bend toward the minor groove and favours histone core facing
- less hydrogen bonds in AT base pairs, which allow DNA to be more flexible

82
Q

What replaces histones during spermatogenesis?

A

Protamines

83
Q

What does protamine allow for in sperm? (3)

A
  1. Protamine allows tighter DNA packaging for sperm’s streamline shape
  2. Protects sperm’s DNA from damage
  3. Helps epigenetic resetting
84
Q

True or false: Many DNA-binding proteins prefer to interact with histone-bound DNA

A

False
- Many DNA-binding proteins prefer to interact with histone-free DNA
- Histone octamer sterically hinders DNA accessibility

85
Q

What 4 factors regulate chromatin accessibility?

A
  1. Dynamic nature of histone octamer-DNA interactions
  2. Nucleosome remodelling complexes and DNA-binding proteins bend DNA and restrict nucleosome at certain positions
  3. Modifications of histone N-terminal tails by histone modifier (modification) complex
  4. A combination of these factors
86
Q

The unwrapping of nucleosomal DNA is important for what?

A

DNA accessibility

87
Q

Which binding sites on nucleosomal DNA are the most accessible?

A

The entry and exit points

88
Q

Nucleosome-remodelling complexes (NRC) aka chromatin-remodelling complexes

A

Modulate histone-DNA interaction using the energy of ATP hydrolysis

89
Q

Through what 3 ways can the nucleosome-remodelling complex change DNA accessibility?

A
  1. Sliding DNA
  2. Ejecting nucleosomes
  3. Dimer exchange (e.g. when H2A needs to be replaced with H2A.X during meiosis)
90
Q

What does restricting nucleosome location allow for?

A

Allows the DNA-binding site in the linker region to remain accessible for regulatory proteins

91
Q

What two ways can nucleosome positioning be achieved by?

A
  1. DNA-binding protein-dependent nucleosome positioning (e.g. DNA-binding proteins either preventing nucleosomes from forming or facilitating nucleosome assembly)
  2. Nucleosomes prefer to bind bent DNA
92
Q

Explain how MNase and gel electrophoresis helped analyze nucleosome positioning

A
  • micrococcal nuclease has a strong preference to cleave DNA between nucleosomes, rather than DNA tightly associated with nucleosomes.
  • Restriction enzyme digestions create a defined end point for all cleaved fragments. This ensures that the fragments have uniform starting points for analysis.
  • Southern blot
  • Positioned nucleosomes: If nucleosomes are regularly positioned, the enzyme cuts will occur at specific intervals between nucleosomes. This will produce discrete, regularly spaced bands on the gel.
  • Random nucleosomes: If the nucleosomes are randomly positioned, the cutting pattern will be irregular, leading to a broad smear on the gel with no clear pattern, as shown in the left diagram.
  • The gaps of 160–200 bp detected by the probe reveal the positions of the nucleosomes.
93
Q

Histone code

A

Modifications at specific site(s) affecting protein association, which regulates specific nucleosome function and gene expression

94
Q

Which residues are phosphorylated?

A

Ser, Thr, Tyr

95
Q

Acetylation

A
  • COCH3 group added
  • Reduces positive charge of histones which decreases the affinity for the negatively charged DNA backbone to bind the histone
  • increases gene expression
96
Q

Modifications of histone tails creates binding site(s) for domain specific DNA binding proteins, which further recruit modifying enzymes to act on chromatin. Give three examples of these proteins.

A
  1. bromo-domain containing proteins interact with acetylated histone tails.
  2. Chromo-domain-TUDOR-domains and PHD-finger containing proteins interact with methylated histone tail
  3. SANT-domain containing proteins interact with unmodified histone tail
97
Q

Combinations of the activities of histone-modifying enzymes influence what?

A

Combinations of the activities of histone-modifying enzymes modulate chromatin structures and influence gene expression

98
Q

What 6 things are dynamic histone-DNA interactions achieved by?

A
  1. Specific DNA sequence
  2. Competition between histones and specific DNA-binding proteins
  3. Interaction of nucleosome with nucleosome remodelling proteins
  4. Modifications of histone N-terminal tails by histone modifying enzymes
  5. Combinatorial modification of histone N-terminal tails
  6. Combinatorial effects of interactions with nucleosome-remodelling complexes to change the accessibility of the DNA
99
Q

In terms of nucleosome assembly, what does replication of DNA during the S-phase require?

A

Partial disassembly of the nucleosome

100
Q

Newly replicated DNA is rapidly packaged into nucleosomes. How did researchers determine if all the old histones are lost and only new histones are assembled into nucleosomes?

A

Used different radioisotopes to differentially label old and new histones.

101
Q

Describe histone disassmebly/reassembly during replication (4 steps)

A
  1. As the replication fork passes, histones disassemble into subunits
  2. Parental H3-H4 tetramers are randomly transferred to the new strand, but not released into the free pool of histones
  3. Newly synthesized H3-H4 tetramers form nucleosomes on the strand that does not have the parental tetramer
  4. In contrast, parental H2A-H2B dimers are released into the soluble pool and compete for H3-H4 association with newly synthesized H2A-H2B
    SO A MIX OF BOTH OLD AND NEW HISTONES ARE FOUND ON THE DAUGHTER CHROMOSOMES
102
Q

Describe how nucleosome modifications are inherited during DNA replication

A

The old, modified H3-H4 tetramer recruits the histone modifying enzymes to add similar modifications to adjacent nucleosomes of the daughter chromosome to maintain states of modification after DNA replication

103
Q

Why is it important that nucleosome modifications are inherited during DNA replication?

A

The modifications strongly influence gene expression, so this mechanism is important for maintaining cell identity from one generation to the next.

104
Q

Histone chaperones

A

Negatively charged proteins that form complexes with histones and escort them to the site of nucleosome assembly
- Negatively charged so they can interact with histones.

105
Q

Describe how histone chaperones facilitate nucleosome assembly and disassembly during replication (2 steps)

A
  1. DNA undergoes replication, histone chaperones assemble free H3-H4 tetramers (CAF-I) and H2A-H2B dimers (NAP-I) to the site of newly replicated DNA
  2. histone chaperons are recruited to the newly replicated DNA and interacts with the ring-shaped DNA sliding clamp protein, PCNA, and releases it as replication fork moves
106
Q

How is genome-wide DNA methylation at the single-base resolution level studied?

A

Using Whole Genome Bisulfite Sequencing (WGBS)

107
Q

Describe Whole Genome Bisulfite Sequencing (WGBS)

A

Unmethylated cytosines are converted into uracil through deamination, while methylated cytosines remain unchaged with bisulfite treatment (bisulfite removes the amine groups_
- The methylation of the whole genome is identified at single-base resolution by comparing with the reference genome
- Used to determine which cytosines are part of CpG islands (methylated)

108
Q

What technology enables comprehensive mapping of chromatin interactions across the entire genome, providing a detailed view of the genome’s 3D architecture?

A

High-throughput Chromosome Conformation Capture (Hi-C)

109
Q

What does Hi-C measure?

A

The frequency at which 2 DNA fragments physically associate in 3D, linking chromosomal structure to the genomic sequence

110
Q

What are the 5 steps of Hi-C?

A
  1. DNA fragmented with restriction enzyme
  2. Biotinylated residue is incorporated
  3. Blunted ligation under dilute conditions that favour ligation events between cross-linked DNA fragments.
  4. Library is sheared, and the junctions are pulled down with streptavidin beads.
  5. The purified junctions can subsequently be analyed using a high-throughput sequencer, resulting in a catalogue of interacting fragments
111
Q

Structural maintenance of chromosome (SMC) proteins

A
  1. Condensin
  2. Cohesin

CMMB 411- Doreen (9 decks)

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