Lectures 1.6-1.7

Question 1

How is DNA compacted to fit in the eukaryotic nucleus?

Answer 1

DNA wraps around protein cores called nucleosomes (which is composed of histone octamers). Binding to nucleosomes compacts DNA 7x. The nucleosomes are arranged into tightly spaced structured called 30nm fiber. 30 nm fiber then loop and form a rosetter around a “nuclear scaffold” which then forms coils making up a chromatid.

Question 2

properties of histones

Answer 2

• small, basic proteins
• highly conserved in eukaryotes
• H2A, H2B, H3, and H4 form histone core (nucleosome octamer)
• post translational modification of histone proteins regulates chromatin structure/function

Question 3

How do histones interact with DNA?

Answer 3

DNA wraps around histones to form chromatin

Question 4

homologous recombination (general recombination)

Answer 4

genetic exchange between homologous DNA sequences:

• basepairing occurs between sequences
• DNA is broken and rejoined
• Holliday junction intermediate
• forms heteroduplex DNA
• initiation from double stranded DNA break
• strand invasion, DNA synthesis and ligation casues formation of joint molecule (Holliday junction)
• migration and resolution of holliday junction leads to DNA exchange and results

Question 5

Which histones make up the histone core?

Answer 5

H2A, H2b, H3, H4

Question 6

Nucleosome

Answer 6

• one nucleosome every 200 bp

- N terminal tails extend out from the core and are sites of modification and regulation

Question 7

homologous recombination (general recombination)

Answer 7

genetic exchange between homologous DNA sequences:

• basepairing occurs between sequences
• DNA is broken and rejoined
• Holliday junction intermediate
• forms heteroduple DNA
• initiation from dsDNA
• strand invasion, DNA synthesis and ligation casues formation of joint molecule (Holliday junction)
• migration and resolution of holliday junction leads to DNA exchange

Question 8

Holliday junction

Answer 8

• four stranded intermediate which contains two joined DNA duplexes
• recombinase enzymes catalyze breaking ad rejoining of DNA

Question 9

What are the classes of products that can be produced when a Holliday junction is resolved?

Answer 9

• Crossover (half new chromatid/ half old chromatid) or non-crossover products (new insertion but chromatid remains the same otherwise)
• recombined DNA
• repaired break

Question 10

site-specific recombination

Answer 10

• occurs in certain cellular processes and viral infections
• occurs between specific sequences
• is carried out by recombinases or integrases that act on target sequences
• causes insertions, deletions or inversions

Question 11

transposition

Answer 11

DNA sequence that can move in the genome by recombination
two types: DNA Transposons and Retrotransposons

• DNA: mostly in bacteria, recombine into random site on the genome
• Retrotransposons: found in eukaryotes, related to retroviruses (have RNA intermediate), some produce virus particles (not found outside cell)

Question 12

transposition

Answer 12

DNA sequence that can move in the genome by recombination
two types: DNA Transposons and Retrotransposons

• DNA: mostly in bacteria, recombine into random site on the genome
• Retrotransposons: found in eukaryotes, related to retroviruses (have RNA intermediate), some produce virus particles (not found outside cell)

Question 13

recombination

Answer 13

controlled chromosome rearrangements

Question 14

roles of recombination

Answer 14

• double strand break repair
• meiosis
• programmed genetic rearrangements
• conjugation

Question 15

types of recombination

Answer 15

• site specific recombination
• homologous recombination
• “genome editing”
• DNA transposition

Question 16

CNV

Answer 16

copy number variant: deletion or duplications of regions of the genome.

• leads to loss of heterozygosity
• associated with disease

Question 17

What are the stages of replication?

Answer 17

1. Initiation of replication: process that lead synthesis and assembly of replication fork
2. Elongation: the process of DNA synthesis. replication forks move through DNA synthesizing new DNA strands.
3. Termination: when replication stops. usually when replication forks meet.

Question 18

what are the two classes of DNA polymerase ?

Answer 18

classical DNA polymerase and Translesion DNA polymerase

Question 19

Classical DNA Polymerase

Answer 19

includes replicative and repair polymerases:

• have 3’ to 5’ exonuclease activity (proofreading)
• replicative is highly processive (synthesize long stretches of DNA without dissociating from template) and have a high rate of synthesis
• repair polymerases have low to medium processivity and have lot to high rates of synthesis

Question 20

translesion DNA polymerase

Answer 20

involved in DNA repair and DNA synthesis of damaged DNA

Question 21

What are the functions of DNA polymerase accessory factors?

Answer 21

to help achieve high processivity by being topologically linked to DNA

• sliding clamp
• Clamp loader

Question 22

What are the seven types of protein involved in the replication fork?

Answer 22

1. DNA Polymerase
2. Primase
3. DNA helicases
4. ssDNA binding proteins (SSBs)
5. DNA nucleases
6. DNA ligases
7. Topoisomerases

Question 23

sliding clamp

Answer 23

toroid shaped sliding clamp interacts with core polymerase and holds it on the DNA

Question 24

Clamp loader

Answer 24

sliding clamps are loaded with double stranded DNA by clamp loading complexes

Question 25

DNA Polymerase

Answer 25

enzymes that synthesize DNA - synthesize 5' to 3' direction - require an existing DNA strand to copy (ie.. template) - require a primer; add to a 3' OH

Question 26

Primase

Answer 26

- synthesize sort RNA oligonucleotide primes - can initiate synthesis on SSDNA de novo (no 3'=OH needed) - most primases start synthesis at random sites - usually part of a protein complex at replication fork

Question 27

DNA helicase

Answer 27

- utilize energy of ATP hydrolysis to disrupt the double helix (each strand then becomes a helix) - a type of motor protein. Move along ssDNA in one direction disrupting hydrogen bonds. (either move 5'-3' or 3'-5') - necessary for movement of a replication fork; usually interact with primase. - nomenclature: direction of helicase movement is defined on the strand the helicase binds.

Question 28

ssDNA binding proteins

Answer 28

- bind ssDNA - prevent formation of secondary structure in ssDNA - usually interact with other replication proteins to promote efficient replication

Question 29

DNA Nucleases

Answer 29

- degrade (cleaves the nucleotides off) RNA or DNA from its end (exonucleases) or internally (endonucleases) - a 5' to 3' exonuclease required in replication to remove RNA primers to give continuous DNA strand

Question 30

DNA Ligase

Answer 30

- form phosphodiester nods that join strands of DNA | - require high energy cofactor (ATP or NAD)

Question 31

Topoisomerases

Answer 31

- parental strands start replication as a single helix but end replication as one strand of each of two separate helices - required to release the links between the parental DNA strands

Question 32

What is the problem in replicating linear chromosomes?

Answer 32

The DNA at the very end of the chromosome cannot be fully copied in each round of replication, resulting in a slow, gradual shortening of the chromosome. When the replication fork reaches the end of the chromosome, however, there is (in many species, including humans) a short stretch of DNA that does not get covered by an Okazaki fragment—essentially, there's no way to get the fragment started because the primer would fall beyond the chromosome end

Question 33

steps of enzymatic activity of telomerase

Answer 33

The enzyme binds to a special RNA molecule that contains a sequence complementary to the telomeric repeat. It extends (adds nucleotides to) the overhanging strand of the telomere DNA using this complementary RNA as a template. When the overhang is long enough, a matching strand can be made by the normal DNA replication machinery (that is, using an RNA primer and DNA polymerase), producing double-stranded DNA.

Question 34

What is the role of telomerase in the cell?

Answer 34

enzyme that extends the telomeres of chromosomes. Telomerase is an RNA-dependent DNA polymerase, meaning an enzyme that can make DNA using RNA as a template.

Question 35

How is replication initiated?

Answer 35

- special complexes form at origin - origin is activated causing local unwinding of the DNA helix and loading of DNA helicase. - primase binds and synthesizes initial RNA primers - DNA polymerase extends primers - leading and lagging strand synthesis established - primary specificity in DNA replication is initiated