lecture 7 - DNA replication

Question 1

what is the relationship between daughter and parental strand?

Answer 1

parental strand is template for daughter, are complementary to one another

Question 2

which end are new polymers added onto?

Answer 2

3’ end

Question 3

how does the addition of a polymer on DNA replication work (bond-wise)?

Answer 3

phosphate group in the incoming dNTP (nucleotide) reacts with 3’ hydroxyl group (OH) in the original chain

Question 4

what is the semiconservative mechanism of DNA replication?

Answer 4

DNA is made from one old strand and one new strand

Question 5

how did they figure out the semiconservative mechanism of DNA replication?

Answer 5

used isotopes that contain versions of heavier atoms and feed to cells, and incorporate it into DNA. used high speed centrifugation to separate heavy from light (Meselson and Stahl)

Question 6

what is step one of DNA replication?

Answer 6

the opening/splitting of dsDNA creating a replication fork

Question 7

what is DNA helicase?

Answer 7

protein that starts the unwinding of DNA (moves on the DNA strand)

Question 8

what is DNA polymerization?

Answer 8

the process of building a new DNA strand by adding new nucleotides

Question 9

what is required for DNA polymerization?

Answer 9

• DNA polymerase
• dNTPs
• primer
• proceeds 5’ to 3’

Question 10

what is DNA polymerase?

Answer 10

protein that reads DNA and takes dNTPs and sticks it onto chains

Question 11

what is an issue with the growing replication fork?

Answer 11

as you unwind more of the replication fork, there will be more twists and torsions, which will prevent polymerase from continuing

Question 12

what is topoisomerase?

Answer 12

protein that relieves supercoils/twists in the replication fork

Question 13

in DNA replication what is the primer?

Answer 13

primer is RNA because DNA cannot generate themselves

Question 14

due to the primer being RNA in DNA replication, which end will RNA and DNA be at?

Answer 14

RNA is at the 5’ end (beginning) and DNA is at 3’ end

Question 15

what is primer?

Answer 15

the strand that starts DNA synthesis (RNA)

Question 16

what is primase?

Answer 16

enzyme that synthesizes RNA primer used for DNA replication

Question 17

how does primase start DNA replication?

Answer 17

primase is a special RNA polymerase that forms a short RNA molecule complementary to a single-stranded region of the unwound dsDNA (DNA polymerase extends primer to make new daughter dsDNA)

Question 18

what is the problem with leading-lagging strand synthesis being antiparallel?

Answer 18

the lagging strand must be formed in the opposite direction of the movement of the fork, which means as the fork progresses, gap increases so primers must be generated more often

Question 19

what are okazaki fragments?

Answer 19

short discontinuous fragments on the lagging strand consisting of DNA and RNA

Question 20

what is the composition of each Okazaki fragment?

Answer 20

RNA at the beginning of each strand (primer) and DNA at the end (polymerase)

Question 21

how are the discontinuities of the lagging strand (Okazaki fragments) fixed?

Answer 21

enzymes recognize RNA and replace it with DNA and the two DNA molecules are joined with DNA ligase

Question 22

what is the CMG helicase composed of?

Answer 22

3 complexes that make up helicase:
- Cdc45
- GINS
- MCM

Question 23

what does the CMG helicase bind to?

Answer 23

leading strand (start moving on DNA to separate strands)

Question 24

what is the structure of CMG helicase and what’s the purpose?

Answer 24

donut shape with a pore in the center where DNA goes to get separated

Question 25

what does replication protein A (RPA) do?

Answer 25

it binds single-stranded DNA to keep it in optimal conformation (extended form) for DNA polymerase

Question 26

difference between leading strand and lagging strand synthesis in budding yeast?

Answer 26

- leading strand is immediately synthesized - lagging strand must have a synthesized primer so polymerase is recruited

Question 27

where is DNA sensitive to breaks?

Answer 27

sections of the lagging strand that are in single strand DNA form

Question 28

at does single-stranded DNA tend to do that's a problem (purpose of RPA)?

Answer 28

it tends to start base pairing and start forming secondary structures (RPA binds to keep in extended form)

Question 29

in eukaryotes how many different polymerases are used in our replisome? what are they?

Answer 29

3 - DNA pol epsilon - primase/pol alpha complex - pol delta/PCNA complex

Question 30

what does DNA pol epsilon do?

Answer 30

carry out leading strand DNA synthesis

Question 31

what is processivity?

Answer 31

how long the enzyme can do its job for before detaching from substrate (PCNA lengthens time)

Question 32

what is PCNA and what does it do?

Answer 32

donut-shaped, homotrimeric protein that surrounds DNA - prevents the pol epsilon or delta from disassociating from the template which makes it work longer

Question 33

what is a homotrimeric protein?

Answer 33

a trimer produced by the same polypeptide 3 times (ex. PCNA)

Question 34

how does PCNA work?

Answer 34

it grabs DNA and slides on and interacts with a polymerase that keeps holding onto DNA

Question 35

what is the primase/pol alpha complex?

Answer 35

primase forms the RNA component of the primer and DNA polymerase alpha (pol alpha) extends the primer with DNA

Question 36

how does pol alpha serve as primase?

Answer 36

it synthesizes primers of RNA that DNA polymerases can use

Question 37

in eukaryotes, what are primers made of?

Answer 37

partly RNA partly DNA

Question 38

how does the pol delta/PCNA complex work?

Answer 38

it replaces pol alpha/primase complex and completes the synthesis of an Okazaki fragment

Question 39

what does pol delta mainly do?

Answer 39

it does most synthesis on lagging strand

Question 40

what is RFC? what does it do?

Answer 40

the PCNA loader; it opens the PCNA ring and loads it at a primer on DNA

Question 41

what happens after RFC loads PCNA onto the primer?

Answer 41

pol delta (lagging) or epsilon (leading) binds to PCNA to start high-processivity DNA synthesis

Question 42

what is ribonuclease H?

Answer 42

an enzyme that digests and degrades RNA

Question 43

what is FEN-1?

Answer 43

enzyme that displaces RNA

Question 44

what do ribonuclease H and FEN-1 do?

Answer 44

they are enzymes that help remove the primer (RNA) produced by pol alpha on the Okazaki fragments

Question 45

what does pol delta do in the removal of primer process?

Answer 45

replaces the RNA with DNA (glued together by ligase)

Question 46

what are the specific regions where unwinding of DNA occurs called?

Answer 46

origins of replication

Question 47

what do origins of replication tend to be rich in?

Answer 47

AT-rich (2 H-bond makes easier to separate than C-G)

Question 48

what is the origin recognition complex (ORC)?

Answer 48

six subunit protein that marks replication origins and load helicase to open dsDNA

Question 49

what are CDKs?

Answer 49

cyclin-dependent kinases

Question 50

in which phase of replication is helicase loaded in?

Answer 50

G1 (inactive phase)

Question 51

in which phase of replication is helicase activated in? how?

Answer 51

S-phase (through phosphorylation)

Question 52

in which phases is there high CDK levels, no helicase loading, and helicase activation?

Answer 52

S-phase, G2, and M (mitosis)

Question 53

in which phases is there low CDK levels, helicase loading, and no helicase activation?

Answer 53

G1 phase

Question 54

at the origin of replication how many helicases are there?

Answer 54

2 (work bidirectionally, one for each fork)

Question 55

unwinding of the CMG helicase is driven by what?

Answer 55

ATP hydrolysis (requires energy)