DNA Replication

Question 1

DNA replication

Answer 1

how DNA produces a copy of itself

Question 2

DNA polymerase

Answer 2

catalyzes DNA replication (addition of dNTPs to a DNA strand in a 5’ to 3’ direction)

Question 3

polymerization

Answer 3

the 3’ hydroxyl end of the alpha phosphate nucleophilically attacks the incoming dNTP
a phosphodiester bond is formed, and pyrophosphate is released

Question 4

what was the first polymerase purified? who did this?

Answer 4

E. Coli DNA polymerase I; Arthur Kornberg

Question 5

DNA polymerase I

Answer 5

a 928 amino acid polypeptide encoding by the PolA gene

Question 6

the enzymatic activities of DNA polymerase I

Answer 6

5’ to 3’ polymerase activity
3’ to 5’ exonuclease activity
5’ to 3’ exonuclease activity

Question 7

Klenow fragment

Answer 7

carries the 5’ to 3’ polymerase activity and the 3’ to 5’ exonuclease activity (which is beneath the polymerase active site)
is shaped like a right hand
has 3 domains (finger, thumb, and palm)
the thumb and finger domains wrap around the DNA
the active site is located in the palm domain

Question 8

2-metal mechanism of DNA polymerase

Answer 8

the active site of DNA polymerase has 2 metal ions (normally Mg) that are coordinated by 2 Asp side chains in the palm domain

Question 9

metal ion A

Answer 9

bridges the alpha phosphate of dNTP and the primer’s 3’ hydroxyl group, which activates the 3’ hydroxyl group for an inline attack on the alpha phosphate group

Question 10

metal ion B

Answer 10

is coordinated by all 3 phosphate groups of the bound dNTP

orients the bound triphosphate group and electrostatically shield their negative charges

Question 11

factors that affect the specificity of DNA replication

Answer 11

correct base pairing between the incoming dNTP and the DNA template
the shape of the incoming base

Question 12

proofreading

Answer 12

removing incorrectly inserted nucleotides from the 3’ end of DNA, correcting mistakes during DNA replication due to its inherent 3’ to 5’ exonuclease activity
increases the fidelity of DNA replication approximately 1000-fold
~1 in 20 correct bases are excised by E. Coli polymerase I

Question 13

why DNA polymerase I doesn’t replicate DNA in E. Coli

Answer 13

the enzyme is too slow
the enzyme is not processive enough
cells containing the PolA1 mutation are viable to mutagenic agents

Question 14

at least how many DNA polymerases does E. Coli have?

Answer 14

5
Pol 1 is involved in DNA replication (contributes to important 5’ to 3’ exonuclease activity)
Pol 3 is the primary enzyme of DNA replication

Question 15

DNA helicase

Answer 15

the separation of the 2 strands of DNA is necessary for DNA replication
separates DNA double strands
helicases uses the energy from ATP hydrolysis to power strand separation

Question 16

possible mechanism of DNA helicase

Answer 16

ATP binding and hydrolysis causes a conformational change that pulls the lagging strand through the central hole, acting as a wedge to force separation of the 2 DNA strands

Question 17

topoisomerase

Answer 17

enzyme that induces or eliminates supercoils by breaking one or both DNA strands

Question 18

topoisomerase I

Answer 18

relaxes supercoils, its reaction mechanism breaks 1 DNA strand
doesn’t require ATP

Question 19

topoisomerase II

Answer 19

can relax or add supercoils, its reaction mechanism breaks both DNA strands
adding supercoils does require ATP

Question 20

single strand binding protein (SSB)

Answer 20

prevents single stranded DNA (ssDNA) from reannealing, forming intramolecular secondary structures, and protects ssDNA from nucleases

Question 21

primase

Answer 21

makes primers that are necessary for DNA replication to begin
the primer is ~10 nucleotides long

Question 22

primase activity in bacteria

Answer 22

helicase and primase form a primosome

RNA primers are removed by the 5’ to 3’ exonuclease activity of DNA Pol I

Question 23

replication fork

Answer 23

the site of active DNA replication
moves in 1 direction
the leading strand is continuously synthesized in the 5’ to 3’ direction
the lagging strand is discontinuously made in the 5’ to 3’ direction as Okazaki fragments
both strands are synthesized simultaneously by DNA Pol III

Question 24

DNA ligase

Answer 24

catalyzes the joining of Okazaki fragments

Question 25

DNA ligase mechanism

Answer 25

the AMP moiety of NAD+ is used to adenylylate the 5'-phosphorous terminus of the nick. NMD is made as a byproduct (in eukaryotes, ATP and pyrophosphate is used) DNA ligase catalyzes the attack of the 3' hydroxyl end on the 5' phosphoryl group, sealing the nick

Question 26

lagging strand DNA synthesis

Answer 26

RNA primers are synthesized by primase using parental DNA as the template DNA Pol III elongates RNA primers with new DNA DNA Pol I removes 5'-RNA at the end of the neighboring fragments and replaces it with DNA DNA ligase joins adjacent fragments

Question 27

parts of E. Coli DNA Pol III

Answer 27

core (alpha subunit, epsilon subunit, theta subunit) beta sliding clamp clamp loader (Y complex)

Question 28

alpha subunit

Answer 28

responsible for polymerization

Question 29

epsilon subunit

Answer 29

responsible for proofreading

Question 30

theta subunit

Answer 30

stabilizes the epsilon subunit

Question 31

beta sliding clamp

Answer 31

donut-shaped structure formed from 2 beta subunits binds DNA Pol III and prevents it from the template DNA strand (is responsible for its high processivity) it encircles the DNA helix and slides along the DNA as replication proceeds

Question 32

clamp loader (Y complex)

Answer 32

loads the beta sliding clamp onto the DNA | made up of 5 subunits of 4 different types

Question 33

how the clamp loader loads the beta sliding clamp onto DNA

Answer 33

binding of clamp loader >> strain on clamp >> opens ring at 1 subunit interface >> primed DNA stand is slipped through the break into the ring >> the clamp loader hydrolyzes ATP >> the sliding clamp is released and closes around the DNA

Question 34

trombone model

Answer 34

the lagging strand is looped >> an Okazaki fragment is made >> the loop is released >> a new loop is formed

Question 35

mechanism of trombone model

Answer 35

helicase unwinds the duplex DNA >> the lagging strand is looped so that it passes through the other core polymerase of Pol III for Okazaki fragment synthesis >> when Okazaki fragment synthesis is nearly done, primase will generate a new DNA primer >> a new sliding clamp is loaded onto the new template primer by the clamp loader >> after the Okazaki fragment synthesis is complete, the lagging strand core polymerase is transferred to the new clamp >> Initiation of the creation of a new Okazaki fragment

Question 36

where does DNA replication of E. Coli begin?

Answer 36

at the origin of replication (oriC locus)

Question 37

challenges of DNA replication in eukaryotes

Answer 37

a lot more DNA and multiple chromosomes the chromosomes are linear DNA molecules, which presents unique problems in replication chromatins and histones

Question 38

unique features in eukaryotic DNA replication

Answer 38

multiple origins of replication are required | licensing factors limit the assembly of pre-replication complex at each replicon to only once every cell cycle

Question 39

the end problem in replicating linear DNA

Answer 39

due to the nature of DNA replication, the extreme 5' end of the strands gets shorter with each replication cycle

Question 40

telomere

Answer 40

the end of a chromosome contains 100s of tandem repeats of a 6 nucleotide sequence (AGGGTT) the 2 telomeric DNA strands are of unequal lengths (the leading strand has a 3' overhang)

Question 41

telomerase

Answer 41

a special type of DNA polymerase used by eukaryotic cells to extend the leading strand of the telomere the key to telomerase activity is RNA template which anneals to the leading strand in rapidly dividing cells telomeres must be maintained by the telomere to prevent shortening of chromosome ends, which would lead to cell death high telomerase activity is a characteristic of cancer cells