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Outline for Replication section of BIO 319 Fall 2007 Chapter 11 Problems: C8, C16, C24, C28, C30 I. General Features of Replication A. Semi-Conservative B. Starts at Origin C. Bidirectional D. Semi-Discontinuous II. Identifying Proteins and Enzymes of Replication

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slide1

Outline for Replicationsection of BIO 319 Fall 2007

Chapter 11

Problems: C8, C16, C24, C28, C30

I. General Features of Replication

A. Semi-Conservative

B. Starts at Origin

C. Bidirectional

D. Semi-Discontinuous

II. Identifying Proteins and Enzymes of Replication

III. Detailed Examination of the Mechanism of Replication

A. Initiation

B. Priming

C. Elongation

D. Proofreading and Termination

slide2

5’

3’

Identical base sequences

5’

3’

3’

5’

5’

3’

Watson/Crick proposed mechanism of DNA replication

Figure 11.1

proposed models of dna replication
Proposed Models of DNA Replication
  • In the late 1950s, three different mechanisms were proposed for the replication of DNA
    • Conservative model
      • Both parental strands stay together after DNA replication
    • Semiconservative model
      • The double-stranded DNA contains one parental and one daughter strand following replication
    • Dispersive model
      • Parental and daughter DNA are interspersed in both strands following replication
slide5
Matthew Meselson and Franklin Stahl experiment in 1958
    • Grow E. coli in the presence of 15N (a heavy isotope of Nitrogen) for many generations
      • Cells get heavy-labeled DNA
    • Switch to medium containing only 14N (a light isotope of Nitrogen)
    • Collect sample of cells after various times
    • Analyze the density of the DNA by centrifugation using a CsCl gradient
slide6

DNA

14N

15N

CsCl Density Gradient Centrifugation

slide7

Generation

15N

0

Shift cells to 14N

1

2

3

slide8

Interpreting the Data

After one generation, DNA is “half-heavy”

After ~ two generations, DNA is of two types: “light” and “half-heavy”

This is consistent with only the semi-conservative model

bacterial replication
BACTERIAL REPLICATION
  • DNA synthesis begins at a site termed the origin of replication
      • Each bacterial chromosome has only one
  • Synthesis of DNA proceeds bidirectionally around the bacterial chromosome
    • eventually meeting at the opposite side of the bacterial chromosome
      • Where replication ends
slide11

Autoradiography: Radioactivity darkens film

Radioactive bacterial colonies on an agar petri dish

slide13

Directionality of the DNA strands at a replication fork

Fork movement

Lagging strand

Leading strand

slide14

Experimental demonstration of semi-discontinuous Replication

3H labeled Okazaki fragments seen in sucrose density gradients

Wild-type cells DNA Ligase deficient cells

Sucrose gradient

Top=

Smaller

Bottom=

Bigger

slide15

1. Genetic Approach: Obtain Mutants that are defective in Replication

  • Such mutations are Lethal!

b. Conditional lethal

c.Temperature sensitive (ts) lethal

2. Method:

a. Mutagenize cells

b. Plate the cells on agar plates and grow at 30 oC

c. Replica plate and grow at 37oC

II. Identifying Proteins and Enzymes involved in Replication

A. Combine Genetics and Biochemistry

slide16

3. Identifying which ts lethal mutants have defects in Replication

a. pick ts colonies from 30oC plate and grow them in liquid medium at 30oC.

b. shift them to 37oC

c. add Bromodeoxyyridine (BrdU) and continue growth for a short time at 37oC

d. remove the BrdU and irradiate the cells with UV light

1). if BrdU is incorporated into the DNA the UV light will kill the cells

e. return the cells to 30 oC

f. the cells that revive and continue to grow did not incorporate BrdU

because they have a defect in Replication!!!

g. Those ts cells that had other defects continued to replicate their DNA

and incorporate BrdU, and hence the UV light killed them.

dna replication in vitro
DNA Replication In Vitro
  • The in vitro study of DNA replication was pioneered by Arthur Kornberg
    • Nobel Prize in 1959

Kornberg mixed the following

    • An extract of proteins from E. coli
    • Template DNA
    • Radiolabeled nucleotides
  • Incubated to allow the synthesis of new DNA strands
    • Addition of acid will precipitate these DNA strands
    • Centrifugation will separate them from the radioactive nucleotides
slide18

ts mutant

ts mutant

WT

ts mutant WT

DNA

DNA

DNA

DNA

37C

37C

30C

37C

In vitro Complementation of mutants in DNA Replication

initiation of replication
Initiation of Replication
  • The origin of replication in E. coli is termed oriC
    • origin of Chromosomal replication
  • Important DNA sequences in oriC
    • AT-rich region
    • DnaA boxes
slide21

Figure11.6 Initiation of Replication at oriC

  • DNA replication is initiated by the binding of DnaA proteins to the DnaA box sequences
  • causes the region to wrap around the DnaA proteins and separates the AT-rich region
slide22

SSB

SSB

SSB

SSB

  • Uses energy from ATP to unwind the duplex DNA

Figure 11.6 continued

slide23

DNA helicase separates the two DNA strands by breaking the hydrogen bonds between them

  • This generates positive supercoiling ahead of each replication fork
    • DNA gyrase travels ahead of the helicase and alleviates these supercoils
  • Single-strand binding proteins bind to the separated DNA strands to keep them apart
  • Then short (10 to 12 nucleotides) RNA primersare synthesized by DNA primase
    • These short RNA strands start, or prime, DNA synthesis
fig 11 9a te art
Fig. 11.9a(TE Art)

DNA Polymerase Cannot Initiate new Strands

5’

Unable to

covalently link

the 2 individual

nucleotides together

3’

5’

5’

3’

5’

Able to

covalently link

together

3’

3’

5’

Primer

5’

3’

slide25

Figure 11.10

Innermost phosphate

11-30

slide27

Figure 11.8 Schematic representation of DNA Polymerase III

Structure resembles a human right hand

Template DNA thread through the palm;

Thumb and fingers wrapped around the DNA

slide28

Direction of synthesis

on leading strand

3’

5’

3’

5’

3’

Direction of synthesis

on lagging strand

5’

Figure 11.7 Two dimensional view of a replication fork

slide29

Figure 11.13 “Three Dimensional” view of Replication Fork

Direction of synthesis

of leading strand

Direction of synthesis

Of lagging strand

Direction of fork movement

slide30

Proofreading by the 3’  5’ exonuclease activity of DNA polymerases during DNA replication.

slide31

Knicks are something else all together!

Nicks are single strand breaks in double stranded DNA

slide33

DNA polymerases can only synthesize DNA only in the 5’ to 3’ direction and cannot initiate DNA synthesis

  • These two features pose a problem at the 3’ end of linear chromosomes

Figure 11.24 Problem at ends of eukaryotic linear Chromosomes

slide34

If this problem is not solved

    • The linear chromosome becomes progressively shorter with each round of DNA replication
  • The cell solves this problem by adding DNA sequences to the ends of chromosome: telomeres
    • Small repeated sequences (100-1000’s)
  • Catalyzed by the enzyme telomerase
  • Telomerase contains proteinand RNA
    • The RNA functions as the template
    • complementary to the DNA sequence found in the telomeric repeat
      • This allows the telomerase to bind to the 3’ overhang
slide35

The complementary

strand is made by primase, DNA polymerase and ligase

Step 1 = Binding

The binding-polymerization-translocation cycle can occurs many times

Step 2 = Polymerization

This greatly lengthens one of the strands

Step 3 = Translocation

Figure 11.25

RNA primer

11-80