LEC29: DNA Replication Flashcards Preview

MCG > LEC29: DNA Replication > Flashcards

Flashcards in LEC29: DNA Replication Deck (32):
1

where and how (broadly) does DNA replicaton begin?

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

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

 

2

what signals it's time to replicate DNA?

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

(S phase) 

3

during which phase of cell cycle does replication occur?

S phase

4

how does replication from an ORI occur?

bi-directional 

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

5

what opens DNA for replication?

helicase opens dbl stranded DNA helix for replication

6

what keeps double stranded DNA unwound for replication?

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

topoisomerase unwinds the double helix, creates tension 

 

7

what is the nature of DNA replication?

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

8

what does this show?

explain process of PIC formation for DNA replication

Q image thumb

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

 

9

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

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

DNA Primase does this

10

what is the direction of DNA replication? 

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

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)

11

which DNA polymerases do elongation on which strands?

leading strand: alpha, epsilon 

lagging strand: delta

12

what is DNA primase?

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

13

what happens to RNA primers involved w/ DNA replication?

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

 

14

describe process of leading strand synthesis

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

 

15

describe time sequence of leading/lagging strand synthesis

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

16

how many times does DNA primase act on each strand?

once on leading strand, many times on lagging strand

17

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

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

18

what does DNA replication for mitochondria?

DNA polymerase gamma 

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

19

how can viral DNA replication be blocked?

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 

20

how do nucleoside analogue drugs work? 

how do they not inhibit endogenous DNA replication?

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 

21

what are telomeres? structure?

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

22

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

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

23

what is the "end replication problem"?

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 

24

what is telomerase? 

how does telomerase work?

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 

25

what is the hayflick limit? why does it occur? 

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" 

26

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

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

27

why don't stem cells have hayflick limit?

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 

28

what are the components of telomerase?

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 

 

29

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

 

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 

30

are RNA polymerases primer dependent

no! can start "de novo" unlike DNA polymerases

31

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

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 

32

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

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 

Decks in MCG Class (77):