LEC29: DNA Replication

Question 1

where and how (broadly) does DNA replicaton begin?

Answer 1

ORI, origins of replication, must be bound by ORC, origina recognition complex

throughout the genome, located on average 100,000 bp apart

Question 2

what signals it’s time to replicate DNA?

Answer 2

all of the ORI of the genome fire at the same time, simultaneously

(S phase)

Question 3

during which phase of cell cycle does replication occur?

Answer 3

S phase

Question 4

how does replication from an ORI occur?

Answer 4

bi-directional

double stranded DNA opens into a replication fork as **DNA polymerase **synthesizes new DNA bidirectionally from that fork, in both directions simultaneously

Question 5

what opens DNA for replication?

Answer 5

helicase opens dbl stranded DNA helix for replication

Question 6

what keeps double stranded DNA unwound for replication?

Answer 6

**SSB, single stranded binding protein **keeps helix unwound once helicase opens it up

**topoisomerase unwinds the double helix, **creates tension

Question 7

what is the nature of DNA replication?

Answer 7

it is processive: once initiated, DNA polymerase synthesizes chain w/o releasing the template strand for hundrands of thousands of nucleotides

Question 8

what does this show?

explain process of PIC formation for DNA replication

Answer 8

DNA replication occurs when ORC binds the ORI

helicase opens up the DNA; single stranded binding protein (SSB)ensures strands stay separate;topoisomerase creates tension by unwinding the double helix

Question 9

what do DNA polymerases require in order to initiate DNA replication?

Answer 9

they need a primer w/ an existing 3’OH to which they can add the next nucleoside

DNA Primase does this

Question 10

what is the direction of DNA replication?

how is this dealt w/ for double stranded DNA replication?

Answer 10

always 5’ to 3’ synthesis of new strand

**bi-directional DNA replication **away from an origin thus must be discontinuous, i.e. 1 template strand DNA polymerase synthesizes new DNA continuously (leading strand), other strand in short fragments (lagging strand)

Question 11

which DNA polymerases do elongation on which strands?

Answer 11

leading strand: alpha, epsilon

lagging strand: delta

Question 12

what is DNA primase?

Answer 12

a **DNA-dependent RNA polymerase **that is a subunit of DNA Pol-alpha

non-specific, non-processive

action: starts at different ORIs; lays down a short RNA primer, which DNA polymerase then extends

happens once on leading strand, multiple times on lagging strand

Question 13

what happens to RNA primers involved w/ DNA replication?

Answer 13

they are eventually replaced w/ DNA

DNA is then ligated together to form a continuous double-helix from 1 end of the chromosome to the other

Question 14

describe process of leading strand synthesis

Answer 14

DNA primase of DNA pol-alpha starts at different ORIs

DNA pol-alpha extends the primer a bit along the strand

DNA pol-epsilon continues synthesis from DNA-pol alpha’s initiation

Question 15

describe time sequence of leading/lagging strand synthesis

Answer 15

leading/lagging strand synthesis occur simultaneously **as replication forks go bidirectionally away from the ORI **

Question 16

how many times does DNA primase act on each strand?

Answer 16

once on leading strand, many times on lagging strand

Question 17

what happens to RNA primers used during replication on lagging strand?

Answer 17

they are removed from Okazaki fragments

1) “old” RNA primers displaced by DNA polymerase

this creates a “flap” structure where RNA is not annealed to DNA template strand

2) FEN1, flap endonuclease, endonucleolytically cleaves off this RNA primer

creates “nick” between 5’ end of “old” Okazaki DNA and 3’ end of “new” Okazaki DNA

3) DNA ligase seals gap

Question 18

what does DNA replication for mitochondria?

Answer 18

DNA polymerase gamma

mitochondrial DNA has its own genome, so has its own DNA polymerase

Question 19

how can viral DNA replication be blocked?

Answer 19

selective DNA polymerase inhibitors stop viral replication while allowing cellular replication

can do something like **AZT, azidothymine, HIV drug: **blocks replication b/c does not have a 3’ OH from which polymerase would act

these drugs are nucleoside analogues

Question 20

how do nucleoside analogue drugs work?

how do they not inhibit endogenous DNA replication?

Answer 20

nucleoside analogues replace the 3’OH that would be used for replication w/ a N group (azide group, azidothymine, AZT) or by taking it away (dideoxythymine)

Km of AZT for HIV reverse transcriptase is low, and Km of DNA polymerase epsilon for the drug is high

thus if dose correctly, are at [drug] where HIV will use it as substrate rather than DNA polymerase, so replication does not occur

Question 21

what are telomeres? structure?

Answer 21

**end of chromosomes **

**telomeric DNA = repeating TTAGGG sequence **

this DNA folds back into a “T-loop” structure which distinguishes it from broken DNA end and prevents joing to other chromosomes

Question 22

how is end of a chromosome distinguishable from a break in the middle of DNA, aka how does it not get attacked?

Answer 22

1) t-loop structure at of telomere is repeating TTAGGG

extended G-rich strand folds back, anneals to C-rich strand, creating a D-loop that overall results in T-loop

2) t-loop prevents end-to-end joining of chromosomes
3) shelterin complex, telomere-specific proteins, binds along telomere repeat sequences and to end of telomere to protect it

Question 23

what is the “end replication problem”?

Answer 23

each successive round of lagging strand synthesis means at end, lagging strand will be slightly shorter than leading

without mechanism to repair this, telomeres always would be short and would be unable to form T-loop

continuous successive rounds of replication on lagging strand cause shortening of the chromosome

**telomerase **solves this problem by extending lagging strand

Question 24

what is telomerase?

how does telomerase work?

Answer 24

ribonucleoprotein complex that carries own RNA template & can extend DNA on lagging strand to make telomere full length:

1) binds to 3’ flanking end of telomere that’s complementary to telomerase RNA
2) bases added using RNA as template

telomerase then relocates

3) bases added again, using RNA as template
4) DNA polymerase complements the lagging strand that telomerase RNA activity just added

does not occur in normal, undifferentiated somatic cells

Question 25

what is the hayflick limit? why does it occur?

Answer 25

normal, differentiated somatic cells do not have telomerase activity

therefore can only undergo limited number of cell divisions before become senescent

this number is “hayflick limit”

Question 26

how might cancer be caused in differentiated somatic cell re: lagging strand synthesis?

Answer 26

if telomerase is reactivated, allows cells to continue to divide unchecked

Question 27

why don’t stem cells have hayflick limit?

Answer 27

because have telomerase activity, which extends lagging strand template to maintain telomere length

stem/germ cells need to be able to divide constantly, so need this active telomerase

Question 28

what are the components of telomerase?

Answer 28

ribonucleoprotein, hTR, human telomerase RNA

hTERT, human telomerase reverse transcriptase

it’s an RNA dependent DNA enzyme aka reverse transcriptase that carries its own template complementary to the laggin strand template and onto which DNA can be extended

Question 29

what protein activities assist DNA polymerase in the initiation of DNA replication?

Answer 29

1) Binding of the Origin Recognition Complex (ORC) to the ORI recruits additional proteins which then recruit replication machinery
2) Primase, of DNA Pol-alpha, lays down an RNA primer that DNA pol alpha extends leading/lagging strands’ synthesis off of

Question 30

are RNA polymerases primer dependent

Answer 30

no! can start “de novo” unlike DNA polymerases

Question 31

why does initiation of DNA replication from multiple origins of replication have to be strictly controlled?

Answer 31

Because synthesis must proceed in a 5’ to 3’ direction and this is happening off of both sides of the double stranded DNA – bi-directionally and on 2 single strands of DNA – so it must occur in a very controlled way

Question 32

why would an inhibitor of viral DNA polymerase not also inhibit cellular DNA polymerase, since both catalyze the same DNA synthesis rxn?

Answer 32

DNA polymerases need a free 3’OH in order to replicate. Inhibitors of this integrate a molecule analogous to a nucleotide that do not have a 3’OH so they cannot be replicated.

These only target viral DNA polymerase and not cellular because the drug has a Km for HIV that is very low, aka binds w/ high affinity; whereas the drug’s Km for cellular DNA polymerase epsilon is very high, aka does not bind easily/well, so only the viral DNA polymerase is going to be used as a substrate