1
Q
How is DNA organized in prokaryotes?
A
Organized into a nucleoid, but is less compact, usually just a single circular chromosome and plasmids
2
Q
How is DNA stored in eukaryotes?
A
Highly compact linear chromosomes (multiple) found in the nucleus. Can be haploid or diploid
3
Q
What is the general trend as the number of chromosomes in a cell goes up?
A
Increase in complexity
4
Q
What is the genome size?
A
The total amount of DNA within one copy of the genome (haploid)
5
Q
True/False? More complex organisms have larger genomes
A
False. For example, Locusta migratoria has a genome 25x greater than Drosophila melanogaster, yet they have similar complexities
6
Q
What is gene density?
A
The average number of genes per megabase (Mb) of genomic DNA
7
Q
How does gene density change with increasing complexity? Why?
A
Lower gene density due to larger gene size and more intergenic sequences
8
Q
What causes the increase in genome size as organisms grow more complex?
A
Increasing number of introns, repetitive sequences, and longer intergenic sequences, not just gene number
9
Q
What counts as non-repetitive intergenic sequences?
A
Regulatory DNA, miRNAs
10
Q
What counts as repetitive DNA?
A
Microsatellites (simple repeats like ACACACACACACAC…) and genome-wide repeats (transposons and other mobile DNAs)
11
Q
What proportion of the human genome is made up of intergenic sequences?
A
> 60%
12
Q
What counts as non-repetitive intragenic sequences?
A
Introns, gene fragments, UTRs, and pseudogenes
13
Q
True/False? Gene fragments and pseudogenes are non-functional
A
True
14
Q
What proportion of the human genome is made up of genes and gene-related sequences?
A
<40%
15
Q
What proportion of the human genome is made up of just genes?
A
~1.5%
16
Q
What is a kinetochore?
A
A protein complex that forms on the centromeres for interacting with spindles during chromosome segregation in cell division
17
Q
What are centromeres?
A
DNA sequences that are required for the formation of the kinetochore complex. 1/chromosome
18
Q
What are telomeres?
A
TG-rich (TTAGGG) repeats that cap the ends of the chromosomes and protect them from damage and the end replication problem. 2/chromatid
19
Q
What is the origin of replication (Ori)? How many per eukaryotic chromosome? How many per prokaryotic chromosome?
A
Site where DNA replication machinery assembles and begins replication, many per EUKARYOTIC chromosome, one per PROKARYOTIC chromosome
20
Q
What may occur if chromosomes lack centromeres?
A
They will fail to attach to the spindle fibers, leading to random segregation
21
Q
What may occur if chromosomes have more than one centromere?
A
Multiple attachment points causes shearing and chromosome breakage
22
Q
What are the key functions of telomeric proteins?
A
Distinguish the chromosome ends from chromosome and other DNA breakage sites (to prevent frequent DNA recombination and degradation)
Serve as a specialized origin of replication for replicating the ends of the chromosomes
23
Q
What is the function of telomerase?
A
Can extend and maintain telomeres for chromosome integrity (end replication problem)
24
Q
When in the cell cycle do DNA structural changes occur?
A
G1, S, and M
25
What are the gap phases used for in the cell cycle?
To prepare for the following phase and to check the completion of the previous phase (checkpoint)
26
True/False? DNA replication is bidirectional
True
27
What are the key events in S phase?
Initiation of replication, replication continues as well as establishment of cohesion, replication finishes with both sister chromatids still attached together by cohesin
28
What is cohesin and its role?
A protein that forms rings to hold sister chromatids together for chromosomal integrity
29
What is bivalent attachment?
Spindle fibers from both poles attach to one chromosome (mitosis)
30
Which phase is cohesin proteolyzed in mitosis?
Anaphase
31
Which phase is cohesin synthesized in cell division?
S phase
32
Where are the microtubule organizing centers found?
The poles of the cell
33
True/False? DNA is very condensed in almost every stage of the cell cycle
False. Only at their most condensed during mitosis and much less compact during interphase due to the checkpoints, S phase, and transcription of genes needed for replication
34
What is the difference between cohesin and condensin?
Cohesin is required for holding 2 sister chromatids together, while condensin is required for chromosome condensation
35
Condensin must be degraded for replication. Why?
To allow for DNA replication machinery access to the DNA
36
What is the difference between meiosis and mitosis in regards to the attachment of spindles?
Mitosis: only bivalent
Meiosis: monovalent in metaphase I, bivalent in metaphase 2
37
What are the two forms of chromosome condensation in interphase?
10nm and 30nm
38
How many degrees of magnitude does it take to condense DNA into a human cell nucleus? How is this achieved?
1000X-10000X; forming complexes with proteins (chromatin)
39
What are the advantages of DNA packing?
Protect DNA from damage and proper segregation during division (prevents entanglement)
40
What are the disadvantages of DNA packing?
Reduces accessibility to cellular machinery needed for cell function (genes cannot be transcribed)
41
Chromosome packing with nucleosomes results in _X compaction
6
42
How many bps are wrapped around a histone core? Linker DNA length?
147bp; 20-60bp
43
True/False? Nucleosomes are tetramers
False. Octamers. 2xH2A, 2xH2B, 2xH3, 2xH4
44
What is micrococcal nuclease (MNase)?
Sequence-nonspecific nuclease that cleaves protein-free DNA rapidly and protein-associated DNA poorly
45
What was MNase used for? How?
The discovery of the nucleosome; when **fully** digested, gel electrophoresis yielded a 147bp band, which represents the DNA associated with the nucleosome
46
What are the characteristics of histones?
Highly basic (arginine and lysine rich), positive charge so strong that SDS-Page yielded a 30kDa band despite expecting 10-14kDa band (+ve charge made it move as slow as a 30kDa protein)
47
Why is the N-term tail so long on histones?
"Pokes" out of the histone core for modifications
48
Describe the assembly of a nucleosome
Nucleosomes are octamers made up of 4 histone core subunits. H3 and H4 make up a tetramer and bind a DNA loop non-specifically. H2A and H2B form two heterodimers that sandwich H3/H4 on both sides with all histone tails extending outwards
49
How is the association of the H2A/H2B complex to DNA facilitated?
The H3/H4 tetramer binds to the middle and ends of DNA, which constrains/bends it and facilitates the association of the two dimers
50
Describe the symmetry of the nucleosome complex
Two-fold symmetry in two axes (depth-wise and length-wise)
51
What does H3/H4 binding do to the structure of DNA? What does this allow for?
DNA constraint and being extensively bent inwards; facilitates the association of H2A/H2B dimers
52
True/False? Histones interact with DNA through the major grooves
False. Minor grooves (more interactions with -ve backbone)
53
True/False? Histone contacts DNA at sequence-independent sites
True
54
What provides the energy to bend the DNA between the minor groove and histones?
Hydrogen bonds
55
List three characteristics of the histone N-term tail
Sensitive to proteases, not required for the association of DNA with the histone octamer, and are extensively modified for regulation of function
56
What do unmodified histone tails aid in?
They guide the directionality of DNA supercoiling (negatively coiled, left-handed)
57
Where can histone tails emerge from their cores?
Between and on either side of the DNA strands
58
Describe superhelical density in eukaryotes
Nucleosomal DNA is negatively supercoiled, whereas the remainders are kept relaxed by topoisomerases. Negatively supercoiled DNA favours DNA unwinding to facilitate the accessibility of the DNA during replication, transcription, and recombination (less energy req. to unwrap)
59
What are the enzymes involved in superhelical density in prokaryotes?
Gyrase introduces negative supercoils using ATP and Reverse Gyrase introduces positive supercoils using ATP
60
What is heterochromatin? Where can it be found?
Condensed structure with a higher order nucleosomal DNA assembly and dense staining; concentrated in the nuclear scaffold/matrix region on the outskirts of the nuclear envelope
61
What is euchromatin? Where can it be found?
Relatively open structure with poor staining capabilities and less organized nucleosomal DNA assembly; found in the center of the nucleus
62
What is the role of H1?
Interacts with linker DNA between nucleosomes for further condensation, which protects it from MNase digestion
63
How many extra bases does H1 protect from MNase?
20-60bp
64
A chromatin fiber lacks H1. What is its diameter?
10nm
65
A chromatin fiber has H1 in its makeup. What is its diameter?
30nm
66
Describe a solenoid structure. What determines this structure?
Nucleosomes organize themselves into a 6-pointed star with a hole in the middle and linker DNA + H1 between each nucleosome; short linker region
67
Describe a zigzag structure. What determines this structure?
Nucleosomes organize themselves into a 6-pointed star with linker DNA + H1 crossing into the middle; long linker region
68
How do histone tails stabilize 30nm fiber?
Interact with DNA of adjacent nucleosomes and facilitate compaction due to charge neutralization
69
How many orders of magnitude does the 30nm fiber compact the DNA?
40X
70
How is DNA compacted further than 30nm?
30nm fibers are organized into loops around a chromosome scaffold. Each loop contains 40-90kbp
71
What is the role of Topo II as a nuclear scaffold protein?
Holds DNA at the base of the 30nm fiber loops and ensures they are topologically isolated from one another to prevent entanglement
72
What is the role of the SMC proteins? What is its full name?
Condenses and holds sister chromatids after chromosome duplication and provides an underlying foundation for interactions between the nuclear scaffold and chromosomal DNA; Structural Maintenance of Chromosome protein
73
What are the nuclear scaffold proteins?
Topo II and SMCs (cohesin and condensin)
74
Describe the model of condensin role in the minimization of DNA entanglements
1. Cohesin generates and stabilizes DNA loops to organize interphase chromatin into topological domains
2. Topo II can introduce random DNA strand passages by forming knots, intra-molecular, or inter-molecular DNA interlinks, thereby introducing chromatin compaction
3. Condensin might use its DNA loop-extrusion activity to constrict DNA entanglements
75
What is H2A.X?
H2A variant that is phosphorylated and found at a site of a double-strand break. Can be recognized by DNA repair enzymes
76
What is CENP-A?
Replaces H3 in centromeric nucleosomes to serve as a binding site for kinetochore proteins. Creates genomic stability
77
Why is mammalian sperm an exception to nucleosomes?
Uses protamine instead of histones which are arginine rich. Packs DNA tighter to fit into small sperm nucleus. Also helps in epigenetic reset of DNA
78
What are the factors that regulate chromatin accessibility?
Dynamic nature of histone-DNA interactions, nucleosome-remodeling complexes and DNA binding proteins bend DNA to restrict nucleosomes binding at certain positions, modification of histone tails
79
What is the difference between the nucleosome-remodeling complex and histone modifier complex?
NRC modifies the histone core while HMC modifies the histone tails
80
How are histone-DNA interactions dynamic?
Nucleosomal DNA can be unwrapped where the most accessible protein DNA-binding sites are found close to the entry and exit points wrapped around the nucleosome
81
True/False? NRCs use ATP for their tasks
True
82
What ways can NRCs modulate histone-DNA interactions?
Sliding, ejection, and dimer exchange
83
Explain sliding by NRCs
Nucleosomes are physically moved over to expose a segment of DNA
84
Explain ejection by NRCs
Entire nucleosomes are ejected from the DNA that they hold
85
Explain dimer exchange by NRCs
Replaces H2A with H2A.X, which binds looser
86
Explain the importance of nucleosome positioning
Restricting nucleosome location allows the DNA-binding site in the linker region to remain accessible for regulatory proteins
87
In what ways can nucleosome positioning be achieved?
DNA-binding protein dependent nucleosome positioning, nucleosomes prefer to bind bent DNA
88
Explain DNA-binding protein dependent nucleosome positioning
1. If two proteins are bound at both ends of a 150bp sequence, they prevent nucleosomes from binding as they require 147 bp to bind
2. A bound protein may recruit a nucleosome
89
Explain preferential histone binding to certain regions over others
AT-rich sequences along the minor groove have a greater negative electrostatic charge than GC (remember histones are positive). These pairs also have less H-bonds so the DNA is more flexible to bend in these regions
90
In what ways can histone tails be modified? What do each of them do?
Acetylation, phosphorylation, methylation, and ubiquitination; acetylation and phosphorylation loosen interactions, methylation tighten interactions, and ubiquitination marks the tail for degradation
91
What residues are modified for phosphorylation?
Ser, Thr, Tyr
92
What residue is modified for acetylation, methylation, and ubiquitination?
Lys
93
What does acetylation do?
Reduces positive charge of histone core and decreases the affinity for the negatively charged DNA backbone (increases expression)
94
What does methylation do?
Increases affinity between DNA and histone, decreases expression
95
What does a bromo-domain bind to? What is its effect?
Acetylated histone tail; increased expression
96
What does a chromo-domain bind to? What is its effect?
Methylated histone tail; decreased expression
97
What does a TUDOR-domain bind to?
Methylated histone tail
98
What does a PHD-finger domain interact with?
Methylated histone tail
99
What does a SANT-domain interact with?
Unmodified histone tails
100
What is the histone code?
Combinations of different enzymes and modification statuses of histones modulates chromatin structures and gene expression
101
True/False? Histone modifications and interactions with NRCs are mutually exclusive
False. Both have combinatorial effects on gene expression
102
Are all the old histones lost and only new histones assembled into nucleosomes following DNA replication?
No, there is a random mix of both old and new nucleosomes on the parent and daughter strands
103
Describe the process of nucleosome distribution following replication
As the replication fork passes, histone 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.
Parental H2A-H2B dimers are released into the soluble pool and compete for H3-H4 association with newly synthesized dimers
104
Are nucleosome modifications inherited during DNA replication? If so, how?
Yes; the old, modified H3-H4 tetramer recruits the modifying enzymes to add similar modifications to the adjacent nucleosomes of the daughter chromosome to maintain states of modification
105
Why is it important for nucleosome modifications to be inherited following replication?
Maintains cell identity
106
What charge do histone chaperones have? What is their function?
Negative; form complexes with histones and escort them to the site of nucleosome assembly
107
Explain the steps histone chaperones take to facilitate nucleosome assembly
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 chaperones are recruited to the newly replicated DNA by interactions with ring-shaped DNA sliding clamp protein PCNA
108
What does WGBS stand for?
Whole genome bisulfite sequencing
109
What is WGBS?
Combines bisulfite conversion with next-gen sequencing. Detects genome-wide DNA methylation at the single-base resolution level. Unmethylated cytosines are converted into uracil while methylated cytosines remain unchanged. The methylation of the whole genome is identified at single-base resolution by comparing with the reference genome after PCR and sequencing
110
Why is it important that only unmethylated cytosines are converted using WGBS?
Otherwise, they would turn into thymines which would be undetectable
111
What does Hi-C stand for?
High-throughput chromosome conformation Capture
112
What is Hi-C?
A high-throughput method that enables comprehensive mapping of chromatin interactions across the entire genome, providing a detailed view of the genome's architecture. The frequency at which 2 DNA fragments physically associate in 3D, linking chromosomal structure to the genomic sequence
ChatGPT: Hi-C is a technique used to study the 3D architecture of the genome within the nucleus of a cell. It provides insights into how chromosomes are organized, how different regions of the genome interact, and how this spatial organization influences gene expression and other cellular processes. Overall, Hi-C is valuable in studying how chromatin structure influences gene expression, development, differentiation, and diseases like cancer.
CMMB 411
flashcards
Decks in class (12)
# Cards
-
Topic 1 (Genetic switch)56
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Topic 2 (DNA-protein interactions)37
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Topic 3 (DNA Structure and Topology)86
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Topic 4 (Epigenetics)112
-
Topic 5 (DNA Replication)95
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Topic 6 (DNA Damage and Repair)181
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Topic 7 (Homologous Recombination)57
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Topic 8 (Transcription)155
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Topic 9 (RNA Structure)63
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Topic 10 (Post-transcriptional Regulation)84
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Topic 11 (Translation)57
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Topic 12 (Regulatory RNA)41
