Lectures 7 & 8

Question 1

What is semiconservative DNA replication?

Answer 1

when a cell copies its DNA each of the 2 new double helices is made of one original (parental) strand and one newly made strand

Question 2

DNA synthesis occurs from __ to __ and this means…

Answer 2

5’ to 3’
new nucleotides are added to the 3’ OH end of the growing DNA strand

Question 3

What DNA polymerases are used in chromosomal DNA replication?

Answer 3

alpha, epsilon, and delta

Question 4

What DNA polymerase is used in Mitochondrial DNA replication?

Answer 4

gamma

Question 5

What does DNA polymerase do?

Answer 5

catalyzes DNA synthesis, but needs a primer with a 3’ OH group to start… brings in nucleotide then makes a bond

Question 6

Semidiscontinuous Replication

Answer 6

process where one new DNA strand is synthesized continuously and the other discontinuously because DNA can only synthesize new DNA in the 5’ to 3’ direction

Question 7

What is the continously synthesized strand called?

Answer 7

Leading strand

Question 8

What is the discontinuously synthesized strand called? Special features?

Answer 8

Lagging strand
made in short fragments called Okazaki fragments before being joined together

Question 9

What is the replication bubble? Replication fork?

Answer 9

bubble: unwound, open region of DNA double helix where DNA replication takes place, formed at the origin of replication
fork: Y shaped structure that forms when a cell’s DNA double helix unwinds and separates during DNA replication

Question 10

What doe DNA primase do? What does it use as its template? Special features?

Answer 10

synthesizes RNA primers in the 5’ to 3’ direction (puts primers on 3’ end of template strand)
DNA strand can start a new polynucleotide chain by joining together 2 nucleoside triphosphates without the need for a base-paired 3’ end as a starting points… less accurate than DNA polymerase

Question 11

How are Okazaki fragments ligated? What type of bond does this create?

Answer 11

DNA ligase joins them together after their RNA primers are removed and replaced with DNA by DNA polymerase which creates a continuous phosphodiester bond between the fragments

Question 12

What does DNA ligase do?

Answer 12

makes phosphodiester bond between already existing nucleotides (to seal the nick between Okazaki fragments)

Question 13

How does proofreading work?

Answer 13

if DNA polymerase adds a wrong base, the polymerase senses a distortion in the helix and pauses, then the enzyme has a 3’ to 5’ exonuclease activity which backs up and removes the incorrect nucleotide, then DNA polymerase inserts the correct nucleotide and continues replication

Question 14

What are theta structures in a circular chromosome?

Answer 14

replication intermediates…temporary DNA structures formed while replication is in progress (“in between” DNA shapes)

Question 15

What is the autonomous replicating sequence (ARS)?

Answer 15

a specific stretch of DNA that can act as an origin of replication (found in yeast and some other eukaryotes)

Question 16

What does helicase do and how?

Answer 16

enzyme that functions as a molecular motor to unwind the double stranded DNA helix using energy from ATP hydrolysis to break hydrogen bonds between base pairs

Question 17

Do origin DNA sequences tend to be AT or GC rich?

Answer 17

AT rich because they’re easier to disrupt (they have 2 hydrogen bonds instead of 3)

Question 18

How do replication factories appear?

Answer 18

discrete subnuclear compartments or foci

Question 19

What’s weird about BrdU?

Answer 19

anywhere brdU is added, the extension of nucleotide chain will be stopped

Question 20

Why should the secondary antibody be a different species than the primary antibody?

Answer 20

If it’s the same, it will bind to the primary antibody and can’t distinguish the experimental primary antibody from all the other normal antibodies that might be present

Question 21

What would the immunofluorescence pattern look like if DNA replication did not occur in factories?

Answer 21

It would take place all over the nucleus

Question 22

What is the replisome? What are its core compounds and their functions?

Answer 22

multiprotein machine responsible for DNA replication assembling at the replication origins to coordinate the unwinding of DNA and synthesis of new DNA strands

helicase (separates DNA double helix), DNA polymerase (builds new DNA molecules), and primase (creates RNA primers to initiate synthesis)

Question 23

What phase of the cell cycle is DNA replication restricted to?

Answer 23

S phase

Question 24

What is replication licensing?

Answer 24

the process in the G1 phase of the cell cycle where DNA replication machinery is loaded onto potential replication origins on the chromosome making them ready for replication in the S phase

Question 25

What is the replication complex?

Answer 25

a group of proteins and molecules that come together to duplicate a cell's genetic material either in DNA or RNA genome

Question 25

How is replication licensing regulated?

Answer 25

the activity levels of cyclin-dependent kinases (CDKs)

Question 26

What is a starting point for replication? What is needed?

Answer 26

licensed origins can be starting points for the replication process but only if they have successfully loaded the Mcm2-7 protein complex (the licensing factor)

Question 27

What does Pre-RC do?

Answer 27

licenses DNA origins of replication in G1 ensuring that DNA is copied only once per cell cycle by assebling ORC, Cdc6, and MCM proteins

Question 28

What does the Post-RC do?

Answer 28

activated form of the pre-RC that forms in S phase, once kinase activity triggers the initiation of DNA replication

Question 29

What is Topoisomerase 1 and 2 and what do they do?

Answer 29

Topoisomerase 1 cuts single stranded DNA and topoisomerase 2 can cut double stranded DNA... after cutting it twists and relaxes converting positive supercoils to relaxed

Question 30

How does positive supercoiling occur and what does it do?

Answer 30

occurs when the double helix is over-would, meaning it is twisted in the same right handed direction as the helix itself making it more compact and tightly coiled

Question 31

What is negative supercoiling?

Answer 31

left handed twist that under-winds or "loosens" the DNA double helix, reducing its helical turns and making it easier to separate into 2 strands

Question 32

What do DNA polymerase alpha and primase do in leading/lagging strand synthesis?

Answer 32

add RNA primer and initiators

Question 33

What is synthesized first, 25-35 nt initiator DNA or 7-10 nt RNA primer?

Answer 33

RNA primer first, then DNA polymerase/primase adds the 25-35 initiator DNA

Question 34

What is PCNA and what does it do?

Answer 34

PCNA is a sliding clamp with many protein partners that keeps DNA polymerase from falling off and plays a role in trading places

Question 35

What is Replication factor C (RFC) and how does it play a role in the PCNA clamp?

Answer 35

it is the clamp loader that forms a spiral that matches the minor grooves of DNA the clamp loader required ATP and RFC is loaded onto the PCNA

Question 36

How does proofreading work?

Answer 36

DNA polymerases detect and remove incorrect nucleotides during DNA replication...if an incorrect base is added, the enzyme shifts to the exonuclease site, removes the error, then returns the strand to the polymerase site to continue synthesis

Question 37

What does nucleotide selectivity largely depend on? Why?

Answer 37

the geometry of Watson-Crick base pairs because they provide the ideal geometry and chemical cues for DNA polymerase to catalyze high-fidelity DNA replication

Question 38

What is the function of RNase H?

Answer 38

enzyme that directly degrades the RNA portion of RNA:DNA hybrids, leaving the DNA strand in tact... it removes most of the RNA primer but leaves a single ribonucleotide still attached to the DNA which is removed by FEN-1

Question 39

What is the function of FEN-1?

Answer 39

specialized enzyme that cleaves 5' single stranded DNA "flaps" to remove RNA primers (specifically in lagging strand)... has endo and exonuclease activity

Question 40

What's the difference between exonuclease and endonuclease?

Answer 40

Exo: only phosphodiester cleavage in the terminal nucleotide Endo: can cleave phosphodiester in the middle of nucleotides

Question 41

What is the function of DNA ligase 1?

Answer 41

catalyzes the formation of phosphodiester linkages between Okazaki fragments, encircles DNA by interacting with the minor groove, and stabilized distorted structures with A-form helix upstream of gap

Question 42

What are the basic steps of DNA replication?

Answer 42

1. at the origin, helicase unwinds DNA and single stranded binding proteins keep it separated 2. topoisomerase keeps the DNA from supercoiling 3. primase marks strands and DNA polymerase builds new strands (only in 5' to 3' direction) 4. ligase glues Okazaki fragments together