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DNA Replication and Repair Monday, August 4 Figure 16.7 A model for DNA replication: the basic concept Figure 16.7 A model for DNA replication: the basic concept The hydrogen bonds are broken, and the two strands unwind and separate

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figure 16 7 a model for dna replication the basic concept3
Figure 16.7 A model for DNA replication: the basic concept

The hydrogen bonds are broken, and the two strands unwind and separate

figure 16 8 three alternative models of dna replication
Figure 16.8 Three alternative models of DNA replication

Dark blue = original parent strand

Light blue = newly synthesized strand

figure 16 9 the meselson stahl experiment tested three models of dna replication
Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication

Heavy isotopes of nitrogen were used to label the nucleotides which were then incorporated into DNA

figure 16 9 the meselson stahl experiment tested three models of dna replication8
Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication

Any newly synthesized DNA at this point would incorporate the lighter more common nitrogen isotope

figure 16 9 the meselson stahl experiment tested three models of dna replication9
Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication

The DNA could then be separated on the basis of density by centrifugation

The results from the first replication yielded a band of hybrid (15N-14N) DNA

figure 16 9 the meselson stahl experiment tested three models of dna replication10
Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication

The results from the second replication yielded a band of hybrid (15N-14N) DNA and one of light (14N) DNA

dna replication occurs with remarkable speed and accuracy
DNA Replication occurs with remarkable speed and accuracy
  • Bacteria have a single, circular chromosome
    • E.coli contains 5 million bps and can replicate and divide is less than an hour
  • Humans have 46 chromosomes of large molecules of DNA representing about 6 billion bps
    • Replication of this DNA is achieved in a few hours with very few errors
figure 16 10 origins of replication in eukaryotes
Figure 16.10 Origins of replication in eukaryotes

Replication begins at many specific sites that have a specific sequences recognized by proteins that bind and separate the strands to form replication bubbles

Bubbles expand laterally as DNA replication proceeds in both directions (bidirectional)

Eventually the bubbles fuse and synthesis of the daughter strands is complete

Visualization of 3 replication bubbles along DNA (arrows indicate direction of replication

figure 16 11 incorporation of a nucleotide into a dna strand
Figure 16.11 Incorporation of a nucleotide into a DNA strand

Rate of elongation:

Bacteria = 500 nt/sec

Humans = 50 nt/sec

DNA polymerase

Triphosphate monomers are chemically reactive

Exergonic reaction – unstable cluster of negative charges

figure 16 12 the two strands of dna are antiparallel
Figure 16.12 The two strands of DNA are antiparallel

DNA strands have polarity: 5’  3’

The phosphate group is attached to the 5’ carbon of the deoxyribose sugar

The phosphate group of one nucleotide is attached to the 3’ carbon of the deoxyribose of the adjacent nucleotide

5’-P  3’-OH

Antiparallel arrangement:

The sugar-phosphate backbones run in opposite directions

DNA replication only proceeds in the 5’  3’ direction. DNA polymerases only adds nucleotides to the 3’ end.

figure 16 13 synthesis of leading and lagging strands during dna replication
Figure 16.13 Synthesis of leading and lagging strands during DNA replication

The leading strand is synthesized continuously as the replication fork proceeds.

The lagging strand is synthesized in 100-200nts fragments (Okazaki) which are joined by DNA ligase.

figure 16 14 priming dna synthesis with rna
Figure 16.14 Priming DNA synthesis with RNA

DNA polymerases cannot begin synthesis of polynucleotides, they can only elongate an existing chain bound to the template strand.

The start of a new DNA molecule begins with a short (10nt) RNA primer. An enzyme, primase, joins RNA nucleotides to make the primer.

Another DNA polymerase can later replace the RNA primer with DNA.

figure 16 15 the main proteins of dna replication and their functions
Figure 16.15 The main proteins of DNA replication and their functions

Open strands

Hold strands apart

figure 16 16 a summary of dna replication19
Figure 16.16 A summary of DNA replication

SSB stabilize unwound DNA

Helicase unwinds DNA

DNA pol replaces RNA

figure 16 16 a summary of dna replication20
Figure 16.16 A summary of DNA replication

Leading strand synthesized 5’3’

SSB stabilize unwound DNA

Helicase unwinds DNA

DNA pol replaces RNA

figure 16 16 a summary of dna replication21
Figure 16.16 A summary of DNA replication

Leading strand synthesized 5’3’

SSB stabilize unwound DNA

Lagging strand synthesized in fragments

Helicase unwinds DNA

Joins fragments

DNA pol replaces RNA

figure 16 16 a summary of dna replication22
Figure 16.16 A summary of DNA replication

Proteins involved in DNA replication form a single, large complex that is stationary during replication. This complex may be anchored to the nuclear matrix (framework of fibers extending through the nucleus). DNA is reeled into the complex and daughter strands are extruded.

Leading strand synthesized 5’3’

SSB stabilize unwound DNA

Lagging strand synthesized in fragments

Helicase unwinds DNA

Joins fragments

DNA pol replaces RNA

maintaining the integrity of dna
Maintaining the Integrity of DNA
  • Accuracy in DNA replication is essential to prevent errors or mutations in the newly synthesized DNA
    • DNA polymerase has proofreading ability
      • The enzyme checks each nucleotide as it is incorporated into the chain
      • Any incorrectly paired base is removed by its 3’  5’ exonuclease activity and synthesis is resumed
    • The error rate of DNA pol is 1 in 10,000
    • Cells rely on DNA repair pathways to monitor DNA and correct mismatched bases or repair damaged DNA
      • 130 DNA repair enzymes have been identified in humans
      • DNA damage occurs by X-rays, UV, spontaneous chemical changes
dna repair
DNA Repair
  • Mismatch Repair
    • Nucleotide excision repair
      • Damaged DNA is removed by a nuclease
      • Gap is filled with correct nucleotides by DNA pol and ligase
    • Example: thymine dimers
      • Exposure to UV rays (sun light) can cause the covalent linking of adjacent thymine bases
      • This creates a kink in the DNA strand which interferes with DNA replication
replication termination
Replication Termination
  • DNA pol can only synthesis 5’  3’
    • Incapable of completing the 5’ ends of daughter DNA strands
    • Repeated rounds of replication result in shortening DNA molecules
  • Solution: telomeres
    • chromosomal DNA has special nucleotide sequences at their ends consisting of multiple repetitions (100-1000) of one short sequence: TTAGGG
    • Telomeres are restored by telomerase, which catalyzes the lengthening of these sequences
replication termination28
Replication Termination
  • DNA pol can only synthesis 5’  3’
    • Incapable of completing the 5’ ends of daughter DNA strands
    • Repeated rounds of replication result in shortening DNA molecules
  • Solution: telomeres
    • chromosomal DNA has special nucleotide sequences at their ends consisting of multiple repetitions (100-1000) of one short sequence: TTAGGG
    • Telomeres are restored by telomerase, which catalyzes the lengthening of these sequences
figure 16 19b telomeres and telomerase
Figure 16.19b Telomeres and telomerase

Telomerase is a complex of protein and RNA:

RNA contains a sequence that serves as the template for new telomere segments

Telomerase is not present in somatic cells – telomeres are shorter in older individuals

Telomerase is present in germ-line cells and cancerous cells

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