DNA and Chromatin Structure

Question 1

Pyrimidines

Answer 1

Question 2

Purines

Answer 2

Question 3

Describe the structure of a nucleotide

Answer 3

Question 4

Difference between deoxyribose and ribose

Answer 4

Deoxyribose has a “H” at the 2’ carbon Ribose has a “OH” at the 2’ carbon

Question 5

What is a nucleotide comprised of

Answer 5

Sugar, nitrogenous base, phosphate group

Question 6

In which direction is DNA written

Answer 6

5’ to 3’ direction

Question 7

How are the two strands in DNA configured

Answer 7

Antiparallel

Question 8

What are the major and minor grooves of DNA and why are they important

Answer 8

Major grooves (12 angstroms wide) Minor grooves (6 angstroms wide) Both can hydrogen bond with foreign molecules - DNA binding proteins will typically target major grooves (because of larger surface area) and affect DNA or RNA synthesis

Question 9

How tall is each turn of B DNA

Answer 9

10.5 residues, 36 angstroms = 2 major + 2 minor turns

Question 10

What forces help to hold together DNA

Answer 10

Resonance of aromatic rings from nitrogenous bases on top of one another (orbital overlap) Hydrogen base pairing between nitrogenous bases

Question 11

Explain why Adenine pairs with Thymine and why Guanine pairs with Cytosine, which pairing is stronger

Answer 11

These pairings maximize the number of hydrogen bonds between nitrogenous bases A:T form 2 hydrogen bonds, G:C form 3 hydrogen bonds G:C are thus stronger

Question 12

What are a-form and z-form DNA

Answer 12

B-form DNA is the most stable and commonly witnessed biological state of DNA

a-form and z-form DNA are mutagenic variants and are less stable

a-form is more compact - 28A per turn (vs 36A of B DNA)

Question 13

How does base pairing affect DNA hybridization

Answer 13

DNA strands will spontaneously hybridize in the most complementary form possible (most A:T and G:C overlap) so as to optimize thermodynamic stability DNA form keeps relatively hydrophobic nitrogenous bases on the inside

Question 14

How can DNA denaturation be observed

Answer 14

Nitrogenous bases absorb UV light at 260nm (this only occurs when they are not hydrogen bound to other bases) Relative UV absorbance will increase as temperature is increased (thus increasingly denaturing the DNA)

Question 15

What is the Tm (melting temperature), what does it depend on

Answer 15

The temperature at which DNA is 50% denatured G:C content of the DNA (more G:C = higher Tm) and ionic strength of solution (cations neutralize the repulsive force of the negatively charged phosphate backbone = more stabile DNA = higher Tm)

Question 16

What are the different types of supercoiling, what categories of DNA can supercoil

Answer 16

There is positive and negative supercoiling - circular DNA and long strands of linear DNA can both supercoil - must be of B-form DNA

Question 17

How is supercoiling relieved, why must this be done

Answer 17

Supercoiling can make DNA inaccessible to transcription, topoisomerases cleave the DNA, relieve tension, and repair the DNA Type 1 topoisomerases snip single strands of DNA and do not require energy Type 2 topoisomerases snip double strands of DNA but require energy to do so

Question 18

How are topoisomerases clinically relevant

Answer 18

Certain antibacterials and cancer treatments target topoisomerase function to either (1) prevent target cell relief of supercoiling OR (2) prevent the repair of DNA after unwinding

Question 19

What comprises a nucleosome

Answer 19

An octamer of histone proteins wrapped in 140 bp of DNA with 60 bp of linking DNA on either side

Question 20

What are the different families of histones and their functions

Answer 20

H1 - linker histone H2A, H2B, H3, H4 - histones forming the nucleosome core. 2 of each

Question 21

What is the function of histones

Answer 21

Providing scaffolding for chromosomes and inhibiting DNA transcription

Question 22

How are histone modifications characterized

Answer 22

Rapid short-term changes that regulate gene expression

Question 23

What histone modifications inhibit transcription, how is this accomplished

Answer 23

Hyper phosphorylation of histones condenses the chromatin Methylation of H3 (carried out by histone methyltransferase (HMT)) Deacetylation by histone deacetylase (HDAC) Encourages the condensation of chromatin

Question 24

What histone modification activates transcription, what carries out this process

Answer 24

Acetylation of lysine residues on H3 and H4 histones carried out by Histone acetyl transferase (HAT) - inhibits ability of histones to interact with DNA

Question 25

Where does histone modification occur

Answer 25

On the exposed amino terminal tails of the core histones

Question 26

What is an epigenetic modification

Answer 26

A modification that produces a heritable change in gene function WITHOUT actually changing the DNA sequences

Question 27

What is DNA methylation an example of, what does it do

Answer 27

DNA methylation is a form of epigenetic modification Attaches a methyl group to the 5’ of cytosine in CGCG sequences (CpG’s) Blocks transcription factors, resulting in repression of the gene

Question 28

How does DNA methylation work with histone modification to condense chromatin

Answer 28

Methyl CpG can be targeted by methyl-CpG binding protein (MBP) MBP then recruits histone deacetylase (HDAC) to remove acetyl groups MBP also recruits histone methyltransferase (HMT) to methylate histones - methylated histones are recognized and bound by the HP-1 protein which silence gene expression

Question 29

What is the clinical significance of epigenetic modifications

Answer 29

epigenetic modifications that suppress gene expression can suppress tumor suppressor genes thus leading to cancer - medications will either protect the DNA from processes such as methylation or inhibit enzymes that would promote gene repression

Question 30

Describe a transcriptionally active segment of chromatin

Answer 30

Known as euchromatin Linking strands are free of H1 histone, the first two nucleosomes are missing to allow for polymerase binding Nuclease (DNA cutters) can access the chromatin Core histones H3 and H4 are acetylated on lysine residues (by HAT)

Question 31

How are histone acetyl transferases (HAT) activated to acetylate histones 3 and 4?

Answer 31

Expression of various combinations of growth factors (LIF and BMP) causes transcription factors A and B to be imported into the nucleus A and B attach to the DNA - HAT anchors to A and B and proceeds to acetylate the histones on lysine residues - inhibits histone’s ability to interact with DNA

Question 32

What is a 30nm fiber? How is it formed

Answer 32

Fiber formed by 6 nucleosomes coiling together H1 binds to the linking strands on euchromatin which promotes the condensation of euchromatin into hetero-chromatin

Question 33

What happens during the formation of a chromatin loop

Answer 33

Fifty sequential chromatin fibers form loops that are anchored to the nuclear matrix (the attachment points are highly active for transcription because they contain proteins such as RNA polymerase and transcription factors)

Question 34

What is a miniband

Answer 34

A ring of 18 chromatin loops

Question 35

What are the orders of chromatin structure? number of base pairs in each?

Answer 35

nucleosome - beads on a string - 200 bp total - 140 bp wrapped around the histone octamer - 60 bp in between each ‘bead’ 30nm fiber - 1200 bp - H1 attaches to ‘string’, causes 6 nucleosomes to coil together chromatin loop - 50x 30nm fibers form a loop - 60,000 bp - each loop can potentially contain several genes miniband - 18x chromatin loops - 1 million base pairs chromosomes - 75 minibands - 75 million base pairs - formation is mediated by hyper-phosphorylation