lecture 4: dna rep1 - basic concepts & classic experiments

semi-conservative dna rep

each new molecule contains one original and one new strand

conservative dna rep

original two strands stay together, completely new molecule

dispersive dna rep

each new strand contains a mixture of original and newly made dna segments

meselson-stahl experiment

1. grow bacteria in medium containing heavy isotope of nitrogen (15N)

2. both strands become 15N labelled (heavy-heavy dna)

3. transfer dnas to a medium containing normal nitrogen isotope (14N)

4. newly synthesised dna will contain light isotope 14N

5. centrifuge dna through CsCl to seperate dna based on density

density-gradient centrifugation

1. dna spun at high speed in CsCl solution

2. molecules settle at positions matching their density

conservative replication experiment results

2nd gen: one heavy(HH) one light (LL) bands. HH remain in later gens

semi-conservative replication experiment results

every dna molecule should have one heavy and one light strand → intermediate density band

2nd gen: HL and LL molecules

random dispersive replication experiment results

all dna molecules should have mixed heavy and light segments → intermediate bands

2nd gen: one mixed band → later gens: move towards the light positions

chromosomal replication start point

replication origins where dna unwound to form a replication bubble

how does replication forks move

bidirectionally

method: bidirectional replication by fibre autography

1. replication start at an origin

2. supply low radioactive thymidine

3. supply higher level later

4. view labelled dna by autoradiography

5. results: low-high-low pattern

direction of synthesis at the fork

two strands run in opposite directions

semi-discontinuous replication

two strands cannot be made continuously in the direction the fork moves

discovery of Okazaki fragments

1. pulse label cells with 3H-thymidine

2. prepare dna and centrifuge to determine the size of the labelled dna

3. possible to isolate small fragments with pulse labelling

4. labelled dnas incorporated into larger pieces

how was it discovered that ligase is needed for Okazaki fragments to join

bacteriophage T4 mutant with defective dna ligase

which strand is made as Okazaki fragments

lagging strand

how does dna polymerase start

primase makes a short rna primer

evidence for priming with RNA

1. label 5’ end of an Okazaki fragment with radioactive isotope

2. digest the DNAase to remove DNA portion

3. determine size of remaining labelled RNA fragment by electrophoresis

describe the trambone-loop model

1. lagging strand forms a loop → allowing polymerase to work alongside the leading-strand polymerase

2. loop grows as new Okazaki fragments are made

3. loop released when fragment is complete

4. new primer starts next fragment and new loop grows

helicase

unwinds parental dna strands

single stranded binding proteins (SSB)

avoid separated strands from pairing again

primase

makes rna primers for new Okazaki fragments

dna polymerase 3

makes new dna

sliding clamp

helps polymerase stay attached to dna

dna polymerase 1

works at junctions between fragments

replace rna primers with dna

dna ligase

seals the remaining joints

replicative dna polymerase

can remove incorrectly added base and replace it