lecture 3 & 4 - dna replication

Question 1

is dna replication semi-conservative or conservative

Answer 1

semi-conservative

Question 2

what was discovered in 1953

Answer 2

Watson and Crick discovered that DNA is symmetrical (important idea for DNA rep)

Question 3

explain the meselson-stahl experiment

Answer 3

proves that DNA rep is semi-conservative

E. coli (fast replicator) is grown in 15N which is rather heavy, then it was switched to 14N which is lighter. All the replications were the same weight in the first generation but in the second generation they started to be different weights which proves semi-conservative

Question 4

where does replication begin in prokaryotes

Answer 4

in the middle of DNA in an A-T area

Question 5

which direction along the DNA does replication move

Answer 5

bidirectional

Question 6

who identified dna polymerase

Answer 6

arthur kornberg

Question 7

what does DNA Pol III do in prokaryotes

Answer 7

catalyzes addition of nucleotide to growing DNA chain

Question 8

what does DNA poly III require in prokaryotes

Answer 8

primer and template

Question 9

which way does dna pol iii move in prokaryotes

Answer 9

5’ to 3’ with respect to growing chain (antiparallel to template)

Question 10

what do the other dna polymerases do in prokaryotic replication

Answer 10

help with repair reactions

Question 11

how do the leading and lagging strands differ in DNA rep

Answer 11

leading is synthesized continuously while lagging is made with short Okazaki fragments that are joined together afterwards

Question 12

what is primase

Answer 12

a RNA polymerase in E coli that provides a priming end

Question 13

what happens to the RNA left my primase once replication is complete in prokaryotic replication

Answer 13

the RNA primer is removed 5’ -> 3’ by exonuclease activity of DNA Pol I and then the gaps are filled in by DNA Pol I

Question 14

do okazaki fragments require primers

Answer 14

yes

Question 15

what does DNA ligase do in DNA replication

Answer 15

connects the 3’ end of one okazaki frag to 5’ end of the next

Question 16

what makes up the DNA Pol III holoenzyme

Answer 16

900 kD dimeric structure

each monomeric unit has catalytic core, dimerisation subunit and a beta clamp. there is a single clamp loader complex

each core is associated with one strand of DNA. core has the polymerase and the 3’-5’ exonuclease

the clamp loader places the sliding clamp (beta complex) onto the DNA

Question 17

what is a 3’-5’ exonuclease

Answer 17

proof reads to make sure the correct base was added

Question 18

what is a single stranded binding protein

Answer 18

helps protext the single stranded dna to prevent from degradation and secondary structure formation

Question 19

what does topoisomerase do in prokaryotes

Answer 19

prevents DNA from getting tangled (relieves pressure in supercoiled DNA)

Question 20

what does helicase do in DNA replication

Answer 20

disrupt the h bonds that hold the 2 dna strands together

Question 21

what makes up the replisome in dna replication

Answer 21

helicase + primosome + polymerase

Question 22

how can the 2 DNA polymerases complexed be attached and move in opposite direction in DNA rep

Answer 22

the lagging strand is looped back

Question 23

where does dna replication termination occur in prokaryotes

Answer 23

opposite oriC by contrahelicase

Question 24

how many origins of replication are there in euk dna rep

Answer 24

multiple

Question 25

when does dna rep happen in euk

Answer 25

during s phase of mitosis

Question 26

explain the pre-replication process in euk dna rep

Answer 26

origin recognition complex (ORC) recognizes origin and then recruits other proteins which in turn recruit DNA pol

Question 27

what proteins make up the pre-replication complex in euk dna rep

Answer 27

ORC complex, Cdc6, Cdt1, MCM

Question 28

what is Cdc6 protein function in euk dna rep

Answer 28

ATP binding

Question 29

what is MCM in euk dna rep

Answer 29

minichromosome maintance

Question 30

what units are in primase in euk dna rep

Answer 30

heterodimer, catalytic and regulatory subunits

Question 31

what do DNA polymerase a/primase complex do in euk dna rep

Answer 31

synthesize short RNA primer and extend it by about 15 deoxynucleotides (primase does RNA and DNA pol a takes over for the DNA)

Question 32

how do different DNA polymerase units deal with strands in euk dna rep

Answer 32

leading strand taken by DNA Pol ε and lagging strand by DNA Pol δ (can switch but it is rare)

Question 33

what does PCNA do in euk dna rep

Answer 33

it the sliding clamp that is attached to DNA pol complexes

Question 34

what removes RNA primers in euk dna rep

Answer 34

RNAseH and FEN1 (endonuclease)

Question 35

what can defects in ORC cause in humans

Answer 35

meirer-gorlin syndrome, dwarfism, microcephaly

Question 36

what is the issue that occurs at the end of the chromosome in euk dna rep and how is this dealt with

Answer 36

there is an issue filling in the primer gaps at the end because the chromosome is linear which means that the DNA will shorten and cause problems. this is dealt with by adding telomeres to the 3’ end of chrosomes by telomerase

Question 37

what does a telomere consist of

Answer 37

a single repeating unit with protruding single stranded end

Question 38

what is the telomere repeat for humans

Answer 38

TTAGG

Question 39

what makes up telomerase

Answer 39

2 subunites (one is reverse transcriptase) and a stretch of RNA that is used as template