Topic 4 Flashcards
In prokaryotes, DNA is organized in _____, but is less _____
In prokaryotes, DNA is organized in nucleoid, but is less compact
In eukaryotes, DNA is____ within the nucleus
Highly compact
In general, as species complexity increases, the genome size…
Is this a hard and fast rule?
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.
Gene density
The average number of genes per megabase (Mb) of genomic DNA
More complex organisms have (lower/higher) gene density
- Explain why
Lower
- Due to larger gene size and more DNA between genes (intergenic sequences)
What is the increase in genome size in more complex organisms due to?
Due to increasing the number of introns, repetitive DNAs, and longer intergenic sequences, NOT just gene number
> 60% of human genome is _______, while <40% of human genome is ______
> 60% of human genome is intergenic DNA, while <40% of human genome is genes and gene related sequences
Intergenic DNA can be separated into… (2)
- Give an example for one of them
- Genome-wide 1400 Mb repeats
- e.g. transposons and other mobile DNA - Other intergenic regions (600 Mb)
Intergenic regions can be separated into… (2)
- Unique DNA regulatory regions, miRNAs
- Microstallites (90Mb)
- simple repeats, e.g. (AC)100
Repetitive DNA (1490 Mb)
Genome-wide 1400 Mb repeats and microsatellites (90Mb)
Non-repetitive intergenic sequences (510 Mb)
Unique DNA: regulatory regions, miRNAs
Gene related sequences can be divided into… (4)
- Introns
- UTRs
- Gene fragments
- Pseudogenes
Non-functional gene-related sequences
Gene fragments and pseudogenes
Kinetochore
An elaborate protein complex that forms on the centromeres for interacting with spindles during chromosome segregating in cell division
Centromeres
DNA sequences that are required for the formation of kinetochore complex.
- 1 centromere/chromosome
What gives the chromosome its “butterfly shape”?
The primary constriction site of the centromere
- Refers to the narrow, pinched region of a chromosome where the centromere is located.
Telomeres (telomeres are __-rich)
TG-rich repeats that cap the ends of a chromosome and protect from damage and loss. 2 pairs per chromosome.
Origins of replication
Multiple sites where DNA replication machinery assembles and begins replication; many origins per chromosome
If the chromosomes lack centromeres, what happens during anaphase?
Random segregation of the chromosome
- because the spindles can’t attach to move the chromatids in opposite directions
If the chromosomes have two centromeres each, what happens during anaphase?
Chromosome breakage (due to more than one centromeres)
- shearing of the genetic content
- no equal segregation of the genetic content
What are the two main key functions of telomeric proteins?
- To distinguish the chromosome ends from chromosome and other DNA breakage sites (i.e. to prevent frequent DNA recombination and degradation)
- To serve as a specialized origin of replication for replicating the ends of the chromosomes
When are the three cell cycle checkpoints?
- M phase: ensures that chromosomes will be properly segregated (e.g. ensures spindles have enough tension)
- G1/S: makes sure DNA is ready for replication
- G2/M: Makes sure DNA is ready for mitosis (replication is complete and there’s no damage)
What are the functions of the gap phases (G1 and G2) in the cell cycle?
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
True or false: DNA replication begins at the origins, and spreads in one direction
False
- DNA replication begins at the origins, and spreads in both directions
Each chromosome of a duplicated pair is a…
Chromatid
Cohesin
A protein that forms rings to hold sister chromatids together
- Important for maintaining chromosomal integrity during replication and early mitosis (before segregation)
DNA replication results in ______ changes
Topological
What happens in terms of kinetochores and spindles during chromosome segregation?
A kinetochore forms on each chromosome, and connects to spindles, which are connected to the microtubule organizing centre (MTOC or centrosomes)
What happens to cohesin during anaphase?
Cohesin is cleaved and then the sister chromatids separate to opposite poles
Interphase
G1 + S + G2
Chromosomes are (more/less) compact during interphase
Less
Describe the chromosome condensation during mitosis
During mitosis, chromosomes are in their most compact forms to facilitate their segregation
Cohesin is required for…
Holding the 2 sister chromatids together
Condensin
Required for chromosome condensation
- links the loops of DNA together, making the chromosomes compact
What happens to condensin between telophase and G1? What is this called?
Condensin is degraded to decondense the chromosomes as they go into interphase
Called the “protein turnover cycle”
During metaphase I, there is _______ attachment of the spindles to the chromosomes
Monovalent
During metaphase II, there is _______ attachment of the spindles to the chromosomes
Bivalent
What are the two forms of chromatin found in interphase?
- 10 nm fiber (beads on a string)
- 30 nm fiber
- The 10 nm fiber is further compacted into a 30 nm structure
What’s the distance between the ends of sister chromatids in the M phase?
1400 nm
What are two advantages of DNA packaging?
- Histones protect DNA from damaging
- Helps with proper segregation during cell division (prevents DNA entanglement)
What’s a disadvantage of DNA packaging?
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
Building blocks of chromosomes
Nucleosomes
Each nucleosome is composed of what? (in general)
8 histone proteins and ~2 rounds of DNA wrapped around each histone core
Nucleosomes alone result in what fold compaction?
6X
Describe how nucleosomes were discovered using gel electrophoresis
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.
Histones are highly (acidic/basic)
- Name two amino acids found in histones
Histones are highly basic (arginine and lysine rich)
Where do histones appear on SDS-PAGE?
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
Describe the structure of histone proteins
- All have a long N-terminus which is important for stabilization of DNA packaging
- Composed of α helical regions
Describe how histones are assembled
- 2 H3 and 2 H4 form a heterodimer
- H2A and H2B form a heterodimer
- H3 and H4 tetramer can bind a DNA loop
- H2A/H2B heterodimer adds on to the tetramer on both sides (sandwich the DNA and H3/H4 complex)
- Amino tails extend outward
True or false: Histones as asymmetrical
False
- The histone octamer has an approximate two-fold symmetry, meaning the structure can be divided into two mirror-image halves along two axes.
How does the H3-H4 tetramer facilitate the association of the H2A-H2B dimer to the DNA?
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
Where do histones primarily contact the DNA and how?
Histones contact the DNA mainly through hydrogen bonding near the minor grooves
The histone and DNA contacts are mostly sequence (dependent/independent)
Independent
What provides the energy to bend the DNA in histone/DNA interactions?
Hydrogen bonds
True or false: The N-terminal tails of histones are sensitive to protease treatment
True
- they are not tightly packed structures in comparison to the histone-fold regions
True or false: The N-terminal tails are required for the association of DNA with the histone octamer
False
The histone ____ are sites of extensive modifications, which regulate nucleosome functions
Tails
Where do histone tails emerge relative to the DNA helices?
Histone tails emerge in between and on either side of the DNA helices
How do histone tails aid in nucleosome formation if they are not required for the association of the DNA with the histone octamer?
The histone tails direct the DNA to wrap around the histone octamer in a left-handed manner, which introduced negative supercoils to the DNA
In eukaryotes, nucleosomal DNA is _____ supercoiled, whereas the remainders are kept…
In eukaryotes, nucleosomal DNA is negatively supercoiled, whereas the remainders are kept relaxed by topoisomerases
What does negatively supercoiled DNA favour?
Negatively supercoiled DNA favours DNA unwinding to facilitate the accessibility of the DNA during DNA replication, transcription and recombination
Gyrase and reverse gyrase in prokaryotes
Gyrase: A special topoisomerase that introduces negative superhelicity in expense of ATP
Reverse gyrase: keeps the genome positively supercoiled in expense of ATP
What types of prokaryotes is reverse gyrase found in?
Thermophils
Describe heterochromatin regions of DNA
- Condense structure
- Dense staining
- higher order nucleosomal DNA assembly
Describe euchromatin regions of DNA
- Relatively open structure
- Staining poorly
- Less organized nucleosomal DNA assembly
Where is heterochromatin found in the nucleus?
Found in the nuclear scaffold/matrix which is located closer to the nuclear envelope
Histone H1 function
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
Chromatin structure in absence/presence of histone H1
Absence: 10 nm fiber only, beads on a string pattern
Presence: 30 nm structure
What are the two types of chromatin packing?
- Solenoid
- Zigzag
How are nucleosomes organized in a solenoid?
Linker DNA contained between each nucleosome shaped like a star with a hollow center
How are nucleosomes organized in a zigzag?
Some linker DNA passes through the center of a star structure (cross section of zigzag) with no hollow center
the __ nm fiber makes DNA less accessible to DNA-dependent enzymes (e.g. DNA polymerase, RNA polymerase)
30 nm
How are histone N-terminal tails required for the formation of the 30 nm fiber?
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
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?
Each 30 nm fiber forms a DNA loop, and the loops are all held together by a chromosome scaffold
How many times does DNA wrap around nucleosomes?
about twice
~147 bp
Role of topo II as a nuclear scaffold protein
Holds DNA at the base of loops and ensures loops are topologically isolated from each other
Structural maintenance of chromosome (SMC) function
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
Describe the model of condensin’s role in the minimization of DNA entanglements
- 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.
H2A.X
- 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.
CENP-A
Replaces histone H3 in centromeric nucleosomes to serve as a binding site for kinetochore proteins
The sequences that tend to face the histone are ____-rich, because…
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
What replaces histones during spermatogenesis?
Protamines
What does protamine allow for in sperm? (3)
- Protamine allows tighter DNA packaging for sperm’s streamline shape
- Protects sperm’s DNA from damage
- Helps epigenetic resetting
True or false: Many DNA-binding proteins prefer to interact with histone-bound DNA
False
- Many DNA-binding proteins prefer to interact with histone-free DNA
- Histone octamer sterically hinders DNA accessibility
What 4 factors regulate chromatin accessibility?
- Dynamic nature of histone octamer-DNA interactions
- Nucleosome remodelling complexes and DNA-binding proteins bend DNA and restrict nucleosome at certain positions
- Modifications of histone N-terminal tails by histone modifier (modification) complex
- A combination of these factors
The unwrapping of nucleosomal DNA is important for what?
DNA accessibility
Which binding sites on nucleosomal DNA are the most accessible?
The entry and exit points
Nucleosome-remodelling complexes (NRC) aka chromatin-remodelling complexes
Modulate histone-DNA interaction using the energy of ATP hydrolysis
Through what 3 ways can the nucleosome-remodelling complex change DNA accessibility?
- Sliding DNA
- Ejecting nucleosomes
- Dimer exchange (e.g. when H2A needs to be replaced with H2A.X during meiosis)
What does restricting nucleosome location allow for?
Allows the DNA-binding site in the linker region to remain accessible for regulatory proteins
What two ways can nucleosome positioning be achieved by?
- DNA-binding protein-dependent nucleosome positioning (e.g. DNA-binding proteins either preventing nucleosomes from forming or facilitating nucleosome assembly)
- Nucleosomes prefer to bind bent DNA
Explain how MNase and gel electrophoresis helped analyze nucleosome positioning
- 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.
Histone code
Modifications at specific site(s) affecting protein association, which regulates specific nucleosome function and gene expression
Which residues are phosphorylated?
Ser, Thr, Tyr
Acetylation
- 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
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.
- bromo-domain containing proteins interact with acetylated histone tails.
- Chromo-domain-TUDOR-domains and PHD-finger containing proteins interact with methylated histone tail
- SANT-domain containing proteins interact with unmodified histone tail
Combinations of the activities of histone-modifying enzymes influence what?
Combinations of the activities of histone-modifying enzymes modulate chromatin structures and influence gene expression
What 6 things are dynamic histone-DNA interactions achieved by?
- Specific DNA sequence
- Competition between histones and specific DNA-binding proteins
- Interaction of nucleosome with nucleosome remodelling proteins
- Modifications of histone N-terminal tails by histone modifying enzymes
- Combinatorial modification of histone N-terminal tails
- Combinatorial effects of interactions with nucleosome-remodelling complexes to change the accessibility of the DNA
In terms of nucleosome assembly, what does replication of DNA during the S-phase require?
Partial disassembly of the nucleosome
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?
Used different radioisotopes to differentially label old and new histones.
Describe histone disassmebly/reassembly during replication (4 steps)
- As the replication fork passes, histones disassemble into subunits
- Parental H3-H4 tetramers are randomly transferred to the new strand, but not released into the free pool of histones
- Newly synthesized H3-H4 tetramers form nucleosomes on the strand that does not have the parental tetramer
- 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
Describe how nucleosome modifications are inherited during DNA replication
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
Why is it important that nucleosome modifications are inherited during DNA replication?
The modifications strongly influence gene expression, so this mechanism is important for maintaining cell identity from one generation to the next.
Histone chaperones
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.
Describe how histone chaperones facilitate nucleosome assembly and disassembly during replication (2 steps)
- 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
- 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
How is genome-wide DNA methylation at the single-base resolution level studied?
Using Whole Genome Bisulfite Sequencing (WGBS)
Describe Whole Genome Bisulfite Sequencing (WGBS)
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)
What technology enables comprehensive mapping of chromatin interactions across the entire genome, providing a detailed view of the genome’s 3D architecture?
High-throughput Chromosome Conformation Capture (Hi-C)
What does Hi-C measure?
The frequency at which 2 DNA fragments physically associate in 3D, linking chromosomal structure to the genomic sequence
What are the 5 steps of Hi-C?
- DNA fragmented with restriction enzyme
- Biotinylated residue is incorporated
- Blunted ligation under dilute conditions that favour ligation events between cross-linked DNA fragments.
- Library is sheared, and the junctions are pulled down with streptavidin beads.
- The purified junctions can subsequently be analyed using a high-throughput sequencer, resulting in a catalogue of interacting fragments
Structural maintenance of chromosome (SMC) proteins
- Condensin
- Cohesin
