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DNA and Replication

DNA REPLICATION

• Remember in the S phase or Synthesis phase of Cell cycle chromosomes double or duplicate.

Remember?

• Chromosomes are made of DNA and histone proteins.

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History of DNA discovery

• Experiments on bacteriophage viruses by Hershey & Chase proved that DNA was the cell’s genetic material

Radioactive P32 was injected into E. coli bacteria!

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DNA (P32)

was detected

in bacteria

cell not

Sulfur (S35)…

DNA was the cell’s genetic

material

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Discovery of DNA Structure

• Erwin Chargaff showed the amounts of the four bases (A,T,C,G) on DNA

• In a body or somatic cell:

Adenine (A) = 30.3%

Thymine (T) = 30.3%

Guanine (G)= 19.5%

Cytosine (C)= 19.5%

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Chargaff’s Rule • Adenine must pair with Thymine

• Guanine must pair with Cytosine

• The bases form weak hydrogen bonds

G C T A

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DNA Structure • Rosalind Franklin took diffraction x-ray photographs of DNA crystals

• In the 1950’s, Watson & Crick built the first model of DNA using Franklin’s x-rays

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DNA

• Two strands coiled called a double helix

• Sides made of a 5C sugar deoxyribose bonded to phosphate (PO4) groups

• Center made of nitrogen bases bonded together by weak hydrogen bonds

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DNA Double Helix

Nitrogenous Base (A,T,G or C)

“Rungs of ladder”

“Legs of ladder”

Phosphate & Sugar Backbone

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Helix

• Most DNA has a right-hand twist with 10 base pairs in a complete turn

• Left twisted DNA is called Z-DNA or southpaw DNA

• Hot spots occur where right and left twisted DNA meet… producing mutations

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DNA • Stands for Deoxyribonucleic acid

• Made up of subunits called nucleotides

• Nucleotide made of: 1.Phosphate group 2.5-carbon sugar

3.Nitrogenous base

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DNA Nucleotide

O=P-O O

Phosphate Group

N Nitrogenous base (A, G, C, or T)

CH2

O

C1 C4

C3 C2

5

Sugar (deoxyribose)

O

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Nitrogenous Bases

• Double ring PURINES Adenine (A) Guanine (G) • Single ring PYRIMIDINES Thymine (T) Cytosine (C) T or C

A or G

• The pentose sugars (sides) of the double helix DNA molecule are anti-parallel to each other – Meaning the two linear strands are wound around each other and each going opposite directions.

• One side has carbon 5 of the pentose sugar pointing up…5’

• One side has carbon 3 of the pentose sugar pointing up…3’

CH2

O

C1 C4

C3 C2

5

Sugar (deoxyribose)

CH2 O

C1 C4

C3 C2

5

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DNA Replication • Enzyme helicase unwinds and separates the 2 DNA strands by breaking the weak hydrogen bonds

• Single-Strand Binding Proteins (SSB) attach and keep the 2 DNA strands separated and untwisted

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DNA Replication (DNA copying)

• After the two strands untwist and separate, they form a Replication Fork (Y-shaped region)

• Then new strands will begin to form at the fork.

Replication Fork

Parental DNA Molecule

3’

5’

3’

5’

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DNA Replication • Before new DNA strands can

form, there must be RNA primers present to start the addition of new nucleotides

• Primase is the enzyme that synthesizes the RNA Primer…it prepares the DNA molecule to receive the new nucleotides.

• DNA polymerase can then add the new complimentary base pairs (nucleotides).

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Synthesis of the New DNA Strands

• One nucleotide at a time…The Leading Strand receives the nucleotides from the point of origin toward the opening replication fork

RNA Primer DNA Polymerase Nucleotides

3’ 5’

5’

Point of origin

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• The Lagging Strand receives nucleotides from the opposite direction of replication and is done in MANY short segments (Okazaki fragments)

• It is replicated from the replication fork toward the origin

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PROOFREADING NEW DNA • DNA polymerase initially makes about 1 in 10,000 base pairing errors

• Enzymes proofread and correct these mistakes

• The new error rate for DNA that has been proofread is 1 in 1 billion base pairing errors

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SEMI CONSERVATIVE MODEL FOR DNA REPLICATION

• The two strands of the parental molecule separate, and each acts as a template for a new complementary strand

• New DNA consists of 1 PARENTAL (original) and 1 NEW strand of DNA

Parental DNA New DNA

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DNA DAMAGE & REPAIR • Chemicals & ultraviolet radiation damage the DNA in our body cells

• Cells must continuously repair DAMAGED DNA

• Excision repair occurs when repair enzymes remove damaged parts of DNA

DNA polymerase and DNA ligase replace and bond the new

nucleotides together