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DNA REPLICATION. BIT 220 MCCC Chapter 11. Replication. Meselson and Stahl. CsCl Equilibrium Density Gradient Centrifugation. DNA will position itself in the salt solution where the DNA density equals the salt density If use a heavy isotope in formation of nucleotides,

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Dna replication l.jpg

DNA REPLICATION

BIT 220

MCCC

Chapter 11


Replication l.jpg
Replication

Meselson

and

Stahl


Cscl equilibrium density gradient centrifugation l.jpg
CsCl Equilibrium Density Gradient Centrifugation

DNA will position itself in the salt solution where the DNA density equals the salt density

If use a heavy isotope in formation of nucleotides,

density will increase

Protocol:

Grow E coli in presence of 15N.

Replace medium with 14N

As multiple rounds of replication ...

you will see bands of DNA of different densities.

Figure 11.3 and Technical sidelight


Requirements of replication l.jpg
Requirements of Replication

1. Primer

5’-phosphate of new nucleotide must be added to

a free 3’-OH group

DNA can not synthesize de novo

2. Template

DNA strand which is used to make complementary strand

3. Synthesis is ONLY in 5’ to 3’ direction


Terms l.jpg
Terms

  • Replication Fork: Y-shaped structure

  • Bidirectional

  • Replication Bubble

  • Theta Structures in circular DNA

  • Figure 11.5

  • Replicon


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Enzymes Involved

DNA Polymerase

Helicase

Topoisomerase (Gyrase)

Primase

Ligase

Telomerase

Figure 11.26


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Origin of Replication

  • unique site in bacteria and viruses for beginning of replication

  • multiple sites in eukaryotes

  • OriC - E. coli

    • 245 base pairs

  • Three tandem repeats of 13mer

  • AT rich

  • allows strand separation

  • Figure 11.6


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Separating DNA strands

  • 1. HELICASE

    • Break attachment between strands opening DNA

    • Unwindind DNA

    • Replication Fork (Two = bubble)

      • Y shaped region where 2 new DNA strands elongate

  • 2. Single Stranded DNA Binding Protein (SSBs)

    • hold strands apart

    • prevents DNA strand from folding on itself


  • Dna polymerase can only work in 5 3 direction l.jpg
    DNA polymerase can only work in 5’-3/ direction

    1. Catalyzes the formation of phoshodiester between 3’OH

    of terminal nucleotide with interior phosphate (5’) of incoming nucleotide Figure 11.12

    2. Proofreading function: excises wrong nucleotide (5-3’ exonuclease OR 3’-5’ exonuclease) Figure 11.17

    3. Multiple polymerases in both E and P

    Also see Figures 11.15 and 11.16


    Replication10 l.jpg
    Replication

    • DNA POLYMERASE

    • 1. enzyme which adds new nucleotides

    • complementary bases A-T, G-C

    • new nt attached to 3’-OH of sugar of preceding nt

    • phosphodiester linkage

    • ONLY in 5’-3’ direction

    • 2. Corrects nucleotide mistakes (Proofreading, exonuclease)

    • 3. Removes primer

    • Both sides serve as template to make two additional strands

    • Bidirectional Replication


    Continuous vs discontinuous l.jpg
    Continuous vs Discontinuous

    1. Leading strand

    5’-3’direction - Figure 11.18

    2. Lagging strand (discontinuously)

    overall 3’-5’ direction

    shorter strands ‘Okazaki fragments’

    Okazaki fragments made in 5’-3’

    LIGASE - Figure 11.19

    enzyme used to seal fragments together

    catalyzes covalent bond formation between

    sugar and phosphate to repair ‘nicks’

    NICK

    broken phosphodiester bond


    Primase l.jpg
    Primase

    • DNA can NOT start new DNA strand without pre-existing nucleic acid

      • Requires a free -OH end

    • Enzyme which adds RNA primer

    • Primer is excised after used and gap is filled with deoxyribonucleotides (DNA polymerase)

    • Figure 11.21


    Topoisomerase fig 11 23 l.jpg
    Topoisomerase Fig 11.23

    • Problem:

    • unwinding DNA (HELICASE) would cause

    • positive supercoils in circular DNA

    • (in front of rep fork)

    • Solution:

      • Topoisomerase nicks (cuts) DNA to relieve pressure

      • Nicks one or both strands of DNA

      • provides a swivel point or axis of rotation Fig. 11.24

    GYRASE topoisomerase II

    introduce negative supercoils in E coli chromosome

    when DNA unwinds; chromosome becomes relaxed

    Figure 11.25


    Telomerase figure 11 34 l.jpg
    Telomerase Figure 11.34

    Eukaryotes need to add TELOMERES

    ends of chromosomes/G rich

    1. Primer is removed on lagging strand

    2. Can not fill gap because no free 3’-OH and

    can not move in 3’-5’ direction

    3. Telomerase adds G rich sequence at 3’ end of chromosome (LEADING)

    (has RNA template)

    4. Complementary strand synthesis


    Prokaryotes vs eukaryotes l.jpg
    Prokaryotes vs Eukaryotes

    • 1 Origin of Replication

    • (ori)

    • 1000nt/sec

    • Multiple sites of initiation

    • 50 nt/sec

    • Telomerase

    • enzymes which completes

    • ends of chromosomes

    • Nucleosomes

    FIGURES 11.26, 11.28


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