Topic 3 - DNA Replication

Question 1

What does DNA replication need to be?

Answer 1

accurate and fast

Question 2

what do complementary bases suggest and why

Answer 2

that each strand of double helix could serve as a template
- due to specific pairings, each strand could be a template and create duplicate copies of both strands

Question 3

what are the three proposed models of DNA replication?

Answer 3

• conservative replication
• dispersive replication
• semiconservative replication

Question 4

what does conservative replication propose?

Answer 4

the original double helix stays intact, and a completely new, identical DNA double helix is synthesized, resulting in one “old” DNA molecule and one entirely “new” one

Question 5

what does dispersive replication propose

Answer 5

the original double helix stays intact, and a completely new, identical DNA double helix is synthesized, resulting in one “old” DNA molecule and one entirely “new” one

Question 6

what is semi conservative replication

Answer 6

method of DNA duplication where the original double helix unwinds, and each single strand serves as a template to build a new, complementary strand, resulting in two identical DNA molecules, each containing one “old” parental strand and one “new” daughter strand

Question 7

What did meselson and stahl’s experiment prove

Answer 7

that all DNA replication takes place in a semiconservative manner

Question 8

what did Meselson and Stahl use to determine that DNA rep is semiconservative

Answer 8

two isotopes of N
- 14N common form; 15N heavy form
a gradient centrifugation
- filled with a heavy salt solution
- showed one intermediate band and one light band after a second round of replication
- light band was on top (14N), heavy band was on the bottom (15N)

Question 9

Major steps in DNA replication

Answer 9

1. initation
2. unwinding
3. elongation
4. termination

Question 10

how does initiation in prokaryotic cells work

Answer 10

1. begins at the origin of replication - OriC
2. OriC gets recognized by the initiator complex (DNAa) and the DNA slightly unwinds
3. the unwinding allows helicase and other single strand binding proteins to attach to the single stranded DNA

Question 11

what happens during unwinding in prokaryotic cells

Answer 11

the double stranded DNA gets seperated into single strands by DNA helicase, single stranded binding proteins, and DNA gyrase

Question 12

what is DNA helicase function

Answer 12

• Unwinds the DNA double helix by breaking the hydrogen bonds between complementary base pairs.
• Moves along the DNA in the 5′ → 3′ direction on the template strand.
• Uses ATP to separate the two strands, creating the replication fork.

Question 13

what is the function of Single stranded binding proteins

Answer 13

• bind to the separated single stranded DNA after helicase unwinds it
• prevents secondary DNA structures
• forms tetramers
• protects ssDNA from degredation

Question 14

what is the function of DNA gyrase?

Answer 14

it is a type of topoisomerase II
- relives supercoiling and torsional strain that builds up ahead of the replication fork as helicase unwinds the DNA
- removes a twist via ATP, and reseals the strands it cut after

Question 15

what happens during elongation in prokaryotes

Answer 15

1. DNA is still being unwound and stabilized
2. Primase lays RNA primers
3. DNA pol III extends new DNA strands
4. Leading strand is continuous; lagging strand is in fragments
5. DNA pol I replaces RNA primers
6. DNA ligase seals fragments

Question 16

what is the function of primase?

Answer 16

it synthesizes short RNA primers complementary to the DNA template
- does not need a 3’ OH group to start
- primers provide the 3’ OH group to start elongation

Question 17

what is the function of DNA pol III and what does it have

Answer 17

it is a large multiprotein complex that adds nucleotides in the 5’ - 3’ direction
- has 3’ - 5’ exonuclease activity that corrects errors (removing certain nucleotides if needed)

Question 18

What are the two strands formed during elongation and what are they composed of?

Answer 18

1. leading strand: synthesized continuously towards the replication fork
   - requires only one RNA primer
2. lagging strand: synthesized discontinuously away from the fork
   - produced as okazaki fragments
   - requires multiple RNA primers on each new fragment

Question 19

what does DNA pol I do?

Answer 19

removes RNA primers using its 5’ - 3’ exonuclease activity and replaces them with DNA nucleotides

Question 20

what is the function of DNA ligase

Answer 20

• DNA ligase seals nicks in the okazaki fragments to create a continuous strand.

Question 21

what is termination of prokaryotic replication

Answer 21

DNA rep ending when two replication forks meet and the Tus protein binds to the Ter site (termination sequence)

Question 22

how is fidelity of DNA replication maintained

Answer 22

1. low error in base pair selection
2. proofreading by DNA polymerase
   - using 3’ - 5’ exonuclease activity
3. mismatch repairs occur soon after DNA replication

Question 23

what happens in theta replication

Answer 23

1. initiation at a single origin (oriC)
   - DNA unwinds and forms a replication bubble
2. Bidirectional replication
   - Two replication forks move in opposite directions around the circular chromosome.
   - Both leading and lagging strands are synthesized at each fork
3. elongation
4. termination
   - Replication ends when the two forks meet at the termination region (Ter sites).
   - The two circular daughter chromosomes are separated.

Question 24

what happens in rolling circle replication

Answer 24

1. Nicking of one DNA strand
   - A specific endonuclease makes a single-strand nick at the origin.
   - The 3′-OH end serves as a primer for DNA synthesis.
   - The 5′ end is displaced as replication proceeds.
2. Elongation
   - DNA polymerase adds nucleotides to the 3′ end, moving around the circular template.
   - The original strand is peeled off as a growing single strand.
3. Cleavage
   - releases a single stranded linear DNA and a double stranded circular DNA
   - the linear DNA may circularize and serve as a template for synthesis of a complementary strand

Question 25

What is initiation & unwinding in eukaryotic DNA replication

Answer 25

chromosomes are linear and replication begins in many origins and each origin forms a replication bubble - In eukaryotes, ORC binds replication origins and loads helicase onto double-stranded DNA; the helicase is activated in S phase to unwind DNA, while topoisomerases relieve supercoiling caused by strand separation.

Question 26

what is the origin recognition complex (ORC)

Answer 26

it is the complex loads helicase onto dsDNA - ORC binds to replication origins on double-stranded DNA. - ORC helps load the helicase (MCM complex) onto the DNA. - At this point, the helicase is not active yet

Question 27

when is helicase activated during euk DNA rep?

Answer 27

it is activated during the S phase of the cell cycle - kinases actives the helicase and it starts seperating the DNA strand

Question 28

what causes the relief of supercoiling in euk DNA rep?

Answer 28

topoisomerase similar to DNA gyrase - specifically topoisomerase I and II

Question 29

what is elongation in euk DNA rep?

Answer 29

During eukaryotic elongation, DNA polymerase α synthesizes RNA–DNA primers, DNA polymerase ε extends the leading strand, DNA polymerase δ synthesizes Okazaki fragments on the lagging strand, RNA primers are removed, and DNA ligase joins fragments together.

Question 30

what is the function of DNA polymerase α

Answer 30

it starts elongation - it has primase activity so it: - synthesizes a short RNA primer - then adds a short string of DNA nucleotides - this creates a RNA-DNA primer with a free 3' OH

Question 31

what is the function of DNA polymerase ε

Answer 31

after priming, epsilon takes over and synthesizes DNA continuously in the 5' - 3' direction (makes the leading strand) - only one primer is needed for each leading strand

Question 32

what is the function of DNA pol δ

Answer 32

this synthesizes the lagging strand discountinously. - Each okazaki fragment starts with an RNA-DNA primer made by DNA pol α - DNA pol sigma then extends each fragment and fills in the DNA between primers

Question 33

what stitches the okazaki fragments together in euk DNA rep?

Answer 33

- DNA ligase

Question 34

what happens during euk DNA rep termination?

Answer 34

occurs due to the two forks running into each other and converge

Question 35

what are the four challenges associated with Eukaryotic DNA replication?

Answer 35

1. eukaryotic genomes are much larger - slower replication - multiple origins of replication 2. euk DNA wrapped around Histones - Histones + DNA = nucleosomes 3. Euk DNA replication needs to be coordinated with the cell cycle 4. eukaryotic genomes are linear and not circular - free ends lead to chromsome degredation

Question 36

what is a replicon?

Answer 36

unit of replication consisting of DNA independently replicated starting from one origin of replication.

Question 37

why are eukaryotic chromosomes associated with histone proteins?

Answer 37

to package the enormous size of euk chromosomes into a cell nucleus, its DNA must be highly organized and condensed and histone proteins help package the DNA

Question 38

what is a nucleosomes?

Answer 38

the fundamental unit of chromatin

Question 39

how does replication proceed with nucleosomes present?

Answer 39

nucleosomes rapidly disassemble and reassemble 1. rep fork disrupts original nucleosomes on the DNA 2. Redistribution of preexisting histones on the new DNA 3. The addition of newly synthesized histones to complete the formation of new nucleosomes

Question 40

what is the G1/S checkpoint?

Answer 40

after this checkpoint, the cell is committed to dividing (S phase)

Question 41

what is the G2/M checkpoint?

Answer 41

after this checkpoint the cell can divide

Question 42

How do eukaryotic cells make sure DNA is replicated only once per cell cycle?

Answer 42

The licensing system during G1 phase 1. ORC binds first 2. ORC + 2 licensing factor recruits MCM2-7 - Together, they load the MCM2–7 complex onto DNA. - MCM2–7 is the helicase, but it is inactive at this point. 3. In S phase, MCM2–7 + cofactors form active helicase 4. After, S phase, MCM cannot bind and initiate replication again until after cell cycle (mitosis) is completed

Question 43

what is the end-replication problem and how is it solved?

Answer 43

The end replication problem occurs because DNA polymerase cannot replace the final RNA primer on the lagging strand of linear chromosomes, leading to progressive shortening unless telomerase acts.

Question 44

what are the events in which telomeres protect chromosome ends

Answer 44

1. The RNA component of telomerase binds to a complementary sequence on the 3' G overhang of DNA 2. nucleotides that are complementary to the RNA component of telomerase are added to the 3' end of the DNA 3. telomerase moves along the newly synthesized DNA strand towards the 3' end 4. Additional nucleotides are added to the 3' end of the DNA 5. telomerase is removed from the DNA strand entirely 6. synthesis occurs on the 5' end of the complementary strand of DNA to fill the gap created by telomerase

Question 45

what is telomerase?

Answer 45

ribonucleoprotein. Enzyme that elongates the ends of eukaryotic chromosomes - reverse transcriptase (RNA -> DNA)

Question 46

what are the 3 protection mechanisms associated with telomeres

Answer 46

1. special proteins bind G rich overhangs to protect ends 2. Shelterin (multiprotein complex) binds to telomeres and prevents DNA repair mechanisms from recognizing telomere ends as a double stranded break 3. Some cells form a T-loop functions to protect ends

Question 47

what is Werner syndrome?

Answer 47

an autosomal recessive disease - caused by a mutation in WRN gene (type of helicase), which is necessary for telomere replication