Ls3-Ch08 Dna Replication

Front 1

01: What part of the DNA do you need for replication?

Back 1

PRIMER-TEMPLATE JUNCTION with a free 3' end and 5' overhand.

Front 2

02: What is a REPLICATION ORIGIN

Back 2

where DNA is melted and primer synthesized

Front 3

03: What kind of primers do we use? what happens to them, why?

Back 3

RNA PRIMERS: are removed later. there are lots of errors in the beginning, so we will want to remove the first part.

Front 4

04: How long are primers?

Back 4

12-14 bases

Front 5

05: What is the primary enzyme of DNA replication?

Back 5

DNA POLYMERASE

Front 6

06: How does DNA Pol know it is putting in the correct base?

Back 6

new base sits in POCKET: sets up base pairing so only correct ones will fit (ADD PICTURE)

Front 7

07: How does DNA Pol prevent incorporation of RNA bases?

Back 7

DISCRIMINATOR REGION: if there is an OH on the sugar, the base will not be able to fit in correctly to pair up

Front 8

08: What happens to DNA Pol activity if incorrect base pairing happens?

Back 8

KINETIC SELECTIVITY: rate slows down dramatically

Front 9

09: how would you describe the overall shape of DNA Pol?

Back 9

HAND

Front 10

10: What is the function of DNA Pol FINGERS:

Back 10

Keep 5' strand out of the way so it does not get incorporated.

Front 11

11: What is the function of the DNA Pol PALM? What is a key mechanism?

Back 11

Primary catalysis area.

DIVALENT CATIONS:
-one draws H from old strand's 3' OH away to make it ready to bond
-other attracts Oxygen of ß and Gamma phosphates of incoming nucleotide to stabilize it.

Front 12

12: How does DNA Pol remedy incorrect base pairing?

Back 12

EXONUCLEASE: part of DNA Pol
moves incorrect bp down to a pocket to keep for later use

Front 13

13: What is a locking mechanism of DNA Pol?

Back 13

O-HELIX: locks bases down correctly so they can bond. happens at same time as divalent cations.

only locks down 1/4 of the time since base entry is random

Front 14

14: What happens to the template DNA at the active site?

Back 14

90° turn: backbone turns away to expose only the first template base for pairing

Front 15

15: What is the function of the DNA Pol THUMB?

Back 15

keeps correct position of primer+active site, strongly associates DNA Pol w/ substrate

Front 16

16: What is the term for the characteristic nature of how DNA Pol works?

Back 16

PROCESSIVITY: # of nucleotides added per time binding to DNA.

Polymerase attaches to DNA, then adds 1000's of bases before falling off. This is because binding DNA Pol to base is the slow step.

Front 17

17: How often is the wrong base put in? What is this rate reduced to?

Back 17

wrong base every 10,000 bases.

PROOFREADING via exonuclease reduces it to 1/10^10 or 1/10^12

Front 18

18: What is the term for the shape DNA makes when being replicated? What if two at once?

Back 18

REPLICATION FORK.

two of them form REPLICATION BUBBLE

Front 19

19: What are the two strands called during DNA replication?

Back 19

LEADING STRAND: synthesis follows direction of fork.

LAGGING STRAND: synthesis is in the opposite direction of the direction of the fork.

Front 20

20: What makes primers?

Back 20

PRIMASE. activated when associates w/ DNA HELICASE

Front 21

21: Where is DNA synthesis continuous or discontinuous? What is the discontinuous DNA called? How long are the discontinous bits?

Back 21

LEADING STRAND: continuous synthesis

LAGGING STRAND: discontinuous, forms OKAZAKI FRAGMENTS in between primers. 100-1000 bases long.

Front 22

22: What happens to the primers?

Back 22

RNAse H degrades all of the primer except for one base.

5' EXONUCLEASE then gets rid of sole remaining RNA base.

Now have a P-T junction, DNA Pol fills it in. LIGASE cements together.

Front 23

23: What separates the DNA strands for replication?

Back 23

DNA HELICASE: moves down one of the strands in the 5' to 3' direction, melting DNA into replication fork.

acts PROCESSIVELY: unwinds multiple pairs at a time.

Front 24

24: How do we know the direction of DNA HELICASE?

Back 24

start w/ a single stranded DNA plasmid. add a 200bp radioactive SS, which will lock on to plasmid.

Cut plasmid w/ EcoR1, on a spot on the 200bp, giving a 75bp strand at the 3' end, followed by a 125bp end.

Now add HELICASE: will kick off short end, as seen by running on gel.

Front 25

25: What happens to the DNA after it is melted?

Back 25

SINGLE STRANDED BINDING PROTEIN: stabilizes ssDNA in replication bubble.

COOPERATIVE BINDING: binding of one recruits more, rapidly coats strand.

Front 26

26: What can happen to the DNA in front of the replication fork?

Back 26

SUPERCOILS: will form.

TOPOISOMERASE will break it down, twists the helix, put back together in opposite direction so coils will balance each other.

Front 27

27: What are the overall enzymes acting at the replication fork?

Back 27

PRIMASE
DNA HELICASE
SSBP
TOPOISOMERASES

Front 28

28: What are the different DNA Pol's in EUKARYOTES:

Back 28

Pol Alpha: lays down 10bp

Pol Delta or Epsilon lays down lots (depends on lagging or leading)

Delta=Lagging
Epsilon=Leading

Front 29

29: What are the different DNA Pols in Prokaryotes?

Back 29

Polymerase I: removes primer

Pol III: x-some replication

Front 30

30: What is a major thing that eukaryotes have for DNA replication that prokaryotes do not have?

Back 30

SLIDING CLAMPS: helps DNA Pol reassociate w/ DNA when it falls off. increases processivity.

Front 31

31: How are clamps put onto DNA?

Back 31

CLAMP LOADER: attaches to a sliding clamp. ATP comes and binds ALLOSTERICALLY, changing loader conformation to open clamp.

When interacts w/ DNA, ATP gets hydrolyzes, loader closes clamp around DNA.

Front 32

32: What is the overall complex at the replication fork?

Back 32

REPLISOME: DNA POL III HOLOENZYME: clamp loader w/ clamp, bound to TWO DNA Pol III cores by flexible linkers.

also binds to HELICASE (between linkers) when it gets close, increasing helicase activity so it moves ahead.

Front 33

33: What is the overall model for the replisome?

Back 33

TROMBONE MODEL: PRIMASE associates w/ HELICASE, makes new primer on lagging strand.

SSBP bounds to DNA.

lagging POL synthesizes OKAZAKI fragments 5' to 3', then releases from clamp.

LOADER then puts clamp at new P-T junction.

DNA POL associates w/ clamp.

Front 34

34: Why are both strands replicated at once?

Back 34

REDUCES amount of ss DNA, therefore reducing mutations and breaks.

Front 35

35: What is a REPLICON?

Back 35

all DNA replicated from a certain origin

Front 36

36: What initiates replication?

Back 36

REPLICATOR (CIS): start point of DNA. short sequence that signals start of replication.

INITIATOR: proteins that initiate replication. recognize and bind to replicator.

Front 37

37: What kinds of regions do initiators bind to?

Back 37

A-T rich regions, will unwind easily (due to helicase)

Front 38

38: How does the E. Coli initiation happen?

Back 38

DnaA binds to 9-MERS, then 13-MERS sequences, pulling one strand apart.

then helicase and loaders jump one.

primase associates w/ helicase.

Front 39

39: How do you identify a replication origin?

Back 39

Clone it w/ plasmid w/ resistance. add replicator proteins.

DNA in process of replication will form various "Y" shaped fragments.

CUT dna w/ restriction enzyme.

run a 2D GEL: first separate by shape+size, then just by size.

then add probe to tell you shape+stage of replication

Front 40

40: What do the probes show when identifying origin of replication?

Back 40

Y ARC: origin not on fragment.

BUBBLE ARC: origin on probe

BUBBLE TO Y ARC: origin not over probe, but on your fragment.

(ADD DIAGRAM)

Front 41

41: What is the FACTORY HYPOTHESIS?

Back 41

either replication bubbles remain together as one big bubble, or move apart with machinery staying stagnant.

now favor idea that machinery stays in one place.

Front 42

42: How is DNA replication regulated in E. Coli?

Back 42

Newly synthesized DNA will be HEMI-METHYLATED: old half still has methyl groups on it.

SEQ A Protein quickly binds to unmethylated strand, preventing DAM METHYLASE from interacting it.

DnaA then can't find 9-MERS to initiate replication.

SEQ A falls off after 20 min, DAM METHYLASE methylates DNA, then replication can happen.

insures the DNA copied only ONCE per S phase of cell cycle.

Front 43

43: How happens to eukayryotic DNA before it can begin to replicate?

Back 43

ORC: origin replication complex. binds to replicator.

other proteins bind, then 2 helicases, to form PRE-REPLICATION COMPLEX: in G Phase.

dormant until triggered in S phase

Front 44

44: What happens to the PRE-RC in S phase?

Back 44

protein holding helicases is destroyed, letting them loose.

CYCLIN-DEPENDENT KINASES: activate PRE-RC, but can also act on new PRE-RC formation

Front 45

45: How do CDK's affect replication?

Back 45

LOW CDK activity:
-stimulates new PreRC formation
-inhibits PreRC activation

HIGH CDK activity:
-inhibits PreRC formation
-stimultes PreRC activation

Front 46

46: What happens to the ends of DNA during replication?

Back 46

END REPLICATION PROBLEM: last okazaki fragment will be slightly too short due to loss of RNA primer on lagging end. therefore DNA keeps getting shorter throughout life.

TELOMERASE: extends ends in cancer, sperm, egg cells. carries own template to extend one end, then polymerase fills in other strand.