Mmbio 240 Midterm #2 Winter

Front 1

Describe supercoiling in DNA, including why it happens. Tell when you would find negative supercoils and when you would find positive supercoils

Back 1

Each cell has about 3 m of DNA in it, so it needs to be condensed (enter supercoil)
supercoils: coil wraps around itself in order to relieve structural strain
(+Supercoils) wound too tightly (pulling two strands of DNA apart causing it to wind tightly-caused by helicase), results in a left-handed superhelix
(-Supercoils) wound loosely , found only in circular, end fixed or long DNA strands. (caused by topoisomerase) DNA is neg supercoiled. Results in a right-handed superhelix

Front 2

Predict what would happen to supercoiling given the actions of helicases and topoisomerase

Back 2

Helicase-Unwind DNA helix by separating strands. Induce Positive supercoils.
Topoisomerase-Relax and underwind DNA, leading to negative supercoils.

Front 3

Histones

Back 3

Highly alkaline proteins that package and order the DNA into structural units called nucleosomes.They are the chief protein components of chromatin, acting as spools around which DNA winds, and play a role in gene regulation. Without histones, the unwound DNA in chromosomes would be very long . For example, each human cell has about 1.8 meters of DNA, but wound on the histones it has about 0.09 mm of chromatin.
Histones form Octamers.

Front 4

Describe histone-DNA interactions:

Back 4

8-core Histone is wrapped up by DNA. Wraps 1.66 times per core.Held together by hydrogen bonds. Postively charged (because made of lysine and argine) Histone attracts negatived DNA to allow packing.

Front 5

List and describe levels of chromatin structure from nucleosomes to visible chromatids

Back 5

1. DNA: double helix
2. Nucleosomes: 8 Core histone wrapped up by DNA.
3. 30nm fiber (filament): nucleosomes condense into a compact filament 30nm wide
4. extended form of chromosomes: 30 nm fibers are wound up so they can fit into a chromosome. The loops are connected to the scaffold in a radial fashion and each turn forms a rosette of six loops held together by SMC proteins.
5. condensed section of chromosomes: 30 rosettes forms a coil
6. mitotic chromosome: a chromosome consists of several coils

Front 6

Role of promoters in positioning nucleosomes

Back 6

Promoter: A region of DNA that facilitates the transcription of an RNA.
Promoters are bound by multiple proteins that push histones out of the way(histones are regularly spaced after promoter sites)

Front 7

Describe histone variants and their roles

Back 7

H3.3 and H2AZ used in highly expressed genes. maintains transcriptionally active open state.
CENPA (H3 variant) found in centromeres. Maintains kinectochore attachment
H2AX associated with recombination and repair (attracts DNA repair enzymes)
macro H2A shuts down extra X chromosome (suppresses melanoma expression)

Front 8

Identify characteristics of DNA replication.

Back 8

Semi-conservative- Copies each include one original and one new strand. Bi-directional
Steps:
1. Open DNA (with helicase)
2. Synthesize RNA primer
3. Start DNA synthesis
4. Proceed.
5. Terminate
Bi-directional
Semidiscontinuous

Front 9

Explain the concept of a replication fork, origin of replication, termination site.

Back 9

Replication Fork- The location where the helicase breaks the hydrogen bonds holding the double strand together. This forms 2 single strands that branch out. These are the templates for the leading and lagging strand.Diagram of DNA Synth
Origin of Replication- Initial site of replication synthesis
Termination Site-final site of DNA synthesis

Front 10

Describe the directionality of DNA replication

Back 10

Synthesized 5’>>>>3’, Bidirectional

Front 11

Explain leading strand and lagging strand synthesis.

Back 11

Leading Strand- This strand is continuously replicated via DNA Polymerase. The Polymerization is towards the replication fork. It is read 3’>>>5’ and synthesized 5’>>>3’
Lagging Strand- Replicated discontinuously b/c this strand runs 5’>>>>3’. As one fragment(Okazaki fragments) is synthesized, a loop is spooled out(like a trombone) and waits to be filled in.

Front 12

Identify key proteins necessary for DNA replication

Back 12

Helicase
Single Stranded Binding Proteins
Topoisomerase
Primase
DNA polymerase
Ligase
Gyrase
Polymerase III holoenzyme
Polymerase I

Front 13

Role of Helicase in DNA replication

Back 13

unwinds the DNA double helix and forms a replication fork on both ends of the origin of replication - ultimately forming a replication bubble.

Front 14

Role of Single Stranded Binding Proteins in DNA replication

Back 14

after the double helix has been unwound, the SSB proteins hold the single strands in place so they don’t wind back as double helices

Front 15

Role of topoisomerase in DNA replication

Back 15

as replication bubbles are being formed, topoisomerase releases the tensions by breaking and reforming the tightened fragments of the dna double helix.

Front 16

Role of primase in DNA replication

Back 16

adds an RNA primer on the unwound fragment of the DNA.
DNA polymerase: begins replication by using the parental strand as a template, and synthesis occurs 5’ to 3’ direction.

Front 17

Role of ligase in DNA replication

Back 17

After DNA poly replicates DNA strands, ligase connects the lose ends of the newly synthesized strand to the neighboring DNA strand. In short, it ties the lose ends.

Front 18

Role of gyrase in DNA replication

Back 18

Type II topoisomerase, relieves strain while DNA is unwound by helicase, induces negative supercoils

Front 19

Role of polymerase III holoenzyme in DNA replication

Back 19

does the synthesis at replication forks
ATPase binds polymerase III and DNAB(Helicase)

Front 20

Role of polymerase I in DNA replication

Back 20

Okazaki fragment processing, removes RNA

Front 21

List the roles of prokaryotic DNA polymerases

Back 21

Pol 1: synthesizes okazaki fragments on the lagging strand and functions in DNA repair. Pol 1 also has exonucleaseses for both directions 5’ to 3’ and 3’ to 5’.
3’ to 5’ exonuclease: functions as proofreading by removing an incorrect dNMP.
5’ to 3’ exonuclease: functions as nick translation. It functions the same direction as the polymerase and because of it, it can degrade DNA or RNA strand and simultaneously add dNTP’s behind it.

Pol3: Functions as the main polymerase in chromosome replications (leading strands). It also only has the 3’ to 5’ exonuclease (proofreading).

Front 22

Components necessary to begin DNA replication:

Back 22

Helicase
Single stranded binding proteins (SSB)
Topoisomerase
Primases
DNA polymerase
Ligase.

Front 23

Chemical and mechanical events DNA polymerase uses to synthesize DNA

Back 23

DNA polymerase holds the DNA strand similar to a hand. Only the base that fits the niche in the polymerase will be added to the strand. The 3’ prime hydroxyl group attack the tri-phosphate on the last added nucleotide resulting in the phosphodiester bond that attaches the new nucleotide that DNA polymerase holds.(Polymerase holds the next nucleotide in just the right position to encourage the reaction)

Front 24

Describe proofreading and how it contributes to fidelity of replication

Back 24

If the wrong nucleotide is added, 3’ to 5’ exonuclease will remove the nucleotide and the polymerase will replace it with the correct nucleotide. Because these mistakes happen thousands of times a day, without proofreading, mutations will occur in abundance possibly leading to cancer.

Front 25

Define DNA damage and contrast it with mutation and carcinogenesis

Back 25

DNA Damage: a chemical change in a DNA base or a backbone. (physical damages, sometimes causes mutation-but not always, can be repaired)

Mutation: A chemical change in a DNA base sequence.(Base sequence change, cannot be repaired)

Carcinogenesis: the creation of cancer cells. (from multiple mutations that lead to cancer) -its a process

Front 26

Describe proofreading and how it contributes to fidelity of replication

Back 26

If the wrong nucleotide is added, 3’ to 5’ exonuclease will remove the nucleotide and the polymerase will replace it with the correct nucleotide. Because these mistakes happen thousands of times a day, without proofreading, mutations will occur in abundance possibly leading to cancer.

Front 27

Define DNA damage and contrast it with mutation and carcinogenesis

Back 27

DNA Damage: a chemical change in a DNA base or a backbone. (physical damages, sometimes causes mutation-but not always, can be repaired)

Mutation: A chemical change in a DNA base sequence.(Base sequence change)

Carcinogenesis: the creation of cancer cells. (from multiple mutations that lead to cancer) -its a process

Front 28

List types of mutations. Rank them in order of effect on protein products.

Back 28

Point mutation: this is a change in a single base pair, switching out one nucleotide and putting in another.

There are 3 types:
Silent: Produces a different codon for the same amino acid/ No Change in sequence
Missense: Changes from one amino acid to another/ there is a change in the sequence
Nonsense: Changes amino acid to stop codon-(Can cause the most change of the three)

Front 29

transition mutation:

Back 29

change of purine-pyrimidine for the other other purine-pyrimidine (C=G to T=A, or vice versa)

Front 30

transversion mutation:

Back 30

replacement of a purine-pyrimidine is another purine-pyrimidine (e.g. original base pair C=G to G=C or A=T

Front 31

Insertion/deletion mutations

Back 31

Insertion mutation: addition of one or more nucleotides into the original strand( from the spot it changes the entire sequence of amino acids)

Deletion mutation: one or more nucleotides gets deleted off original strand(from that spot it changes entire sequence of amino acids)

Front 32

Types of DNA damage:

Back 32

Water Damage
Oxidative
Alkylation
Damage from chemotherapeutic agents
Strand breaks
UV-induced damage

Front 33

Suicide enzyme (methyltransferase)

Back 33

- not really an enzyme because it does change in reaction
- 06-Methylguanine binds with thymine
- suicide enzyme takes the methyl group from 06-MG which restores it to normal working order
- enzyme can’t do anything else and degrades

Front 34

Direct repair:

Back 34

1) enzyme recognizes specific type of DNA damage
2) enzyme fixes DNA damage

Front 35

Key steps to base excision repair:

Back 35

- DNA glycosylase finds the damaged base and cuts the nucleotide base out by hydrolyzing the bond leaving an apurinic or apyrimidinic site.
- Instead of just putting a new base on, the backbone is nicked by AP endonuclease
- One or a few new bases are inserted
- Ligase seals the nick

Front 36

Uses of base excision repair:

Back 36

- Used to fix small damage to single bases (ex. Point Mutations)
- Oxidative damage, small alkylations, apurinic sites, uracil
- This type is more broad and can fix many kinds of damage

Front 37

Key enzymes in nucleotide excision repair:

Back 37

BACTERIAL
-UvrA: recognizes lesion
-UvrB: Unwinds DNA
-UvrC: Excinuclease (cuts)
-UvrD: Helicase(removes damaged strand)
-Pol1: Fills in gap
-Ligase: Seals in gap

EUKARYOTIC
- XPC or RNA polymerase II: recognizes lesion
- XPB and XPD: unwinds DNA
- XPF and XPG: cut DNA
- Pol o/RFC/PCNA: fills in gap
-Ligase: seals in gap

Front 38

Uses of nucleotide excision repair

Back 38

- Bulky or multibase damage

- Repairs damage from UV radiation
Cuts out DNA damage along with a stretch of the damaged strand
Resynthesizes damaged strand
Seals DNA

Front 39

Mismatch repair

Back 39

When DNA polymerase adds the wrong nucleotide

Front 40

Predict consequences of failure in DNA repair:

Back 40

Can lead to damage or modification of important genes such as tumor suppressors, oncogenes and those in charge of genome maintenance. Multiple mutations can lead to cancer

Front 41

Explain translesion synthesis (bypass repair) and why it exists:

Back 41

Translesion synthesis is a method of DNA repair that is error prone. When a DNA polymerase runs into a lesion (DNA abnormality) it stalls the DNA polymerase. Unless this is repaired, then the replication is no good and will degrade.

Front 42

Explain the importance of recombination:

Back 42

- Mixes up chromosomes
- Prevents loss of good DNA due to detrimental or lethal ones
- allows pairing of homologous chromosomes during meiosis

Front 43

How is recombination initiated?

Back 43

- DNA damage or specific enzymes (during meiosis) cause a double stranded break
- Spo11 cuts the DNA (5’ ends are resected) and attaches to each strand through a tyrosine amino acid.

Front 44

Compare the Double-strand break repair process with the synthesis-dependent strand annealing process:

Back 44

- Double strand break repair:
DNA can cut at X or Y region giving patch (X+X) and splice (X+Y) recombination, both strand pairs end up with new information on them.

- Synthesis dependent strand annealing
The D-loop kicks out the invading strand and only the invading strand ends up with new information on it.

Front 45

Non-homologous end joining vs. homologous recombination

Back 45

Non-homologous end joining mechanism:
- not very accurate, DNA is “smushed” together. A matching place is found and the rest is cut off.
- Executed by Ku proteins

Homologous recombination:
- more accurate
- specific DNA is cut and exchanged at the holiday junction
- Spo11 cuts the DNA

Front 46

Compare splice recombinants with patch recombinants

Back 46

Splice recombinants: X + Y cuts
Patch recombinants: X + X cuts

Front 47

Gene conversion:

Back 47

Heteroduplex DNA that is mispaired and then repaired resulting in new info on the strand.

Front 48

Describe yeast mating type selection.

Back 48

-Two types, a and alpha
-two haploid forms come together can mate
- Have to mate with the opposite type
-sequence is at MAT, double stranded break at mat can be recombined with either HMLalpha of HMRa causing gene conversion.
-Yeast can not mate with the same type, but they have the ability to change which type they are.

Front 49

Common types of transposons

Back 49

DNA
-Replicative (copy and paste)
-Non-Replicative (cut and paste)

RNA-MEDIATED
-”retro” transposons
-gene sequence that transcribes RNA out of the sequence, DNA made and inserted later
-Copy and paste type

FUNCTIONAL OR DEFECTIVE
-functional: has the gene to build transposase and is free to replicate and plant itself
-defective: can transpose, but doesn’t have the gene for transposase. Only replicates when it borrows that enzyme from another transposon

Front 50

What are transposase and integrase and what do they do?

Back 50

TRANSPOSASE
Provides the reaction for the transposon to remove itself to it’s current position and place itself in a new position

INTEGRASE
Enzyme that helps the insertion of a retroviris or retrotransposon into its target site

Front 51

Explain how transposons lacking transposase can still function:

Back 51

They borrow the transposase from other transposons

Front 52

Name the scientist who discovered transposons, and in general how she did so:

Back 52

Discovered by Barbara McClintock in corn because of color variation in corn. The color variation is due to these transposons (DNA that can move in and out of the genome).

Front 53

LTR

Back 53

Long terminal repeats
-makes an RNA template and has 2 complete strands of cDNA made together
-integrase puts this freshly made strand of cDNA into the target DNA
-primer for reverse transcriptase is tRNA
-very similar to retroviruses(only difference is retrovirus have extra gene-env)

Front 54

Non-LTR:

Back 54

-doesn’t have long terminal repeats
-makes an RNA template
-enzyme brings the RNA template to the target strand and cuts the strand in one place to allow entry of that one piece of RNA
-that RNA then uses the cut strand as a primer for reverse transcriptase and copies itself straight into the genome
-other strand that has been left alone is now cut
-that freshly made strand is then copied (but anti-sense) to fit into the other side

Front 55

Retroviruses

Back 55

-practically the same as LTR BUT it has one extra gene called env
-env gives the virus the viral envelope
-enables the virus to jump from cell to cell and not just within a genome
-An example of a retro viruses is HIV

Front 56

Explain the role of primers in retrotransposition. Describe what is used for a primer in each type

Back 56

As with the usual use of primers, primers in retrotransposition are used to start the transcription process in DNA (or RNA in the case of LTM Retrotransposons) that has been cut.

Long Terminal Repeat (LTM) Retrotransposons
-Use tRNA as the primer to make the cDNA strand needed.

Non-LTM Retrotransposons
-Uses end of cut strand of DNA as the primer (see picture)