Chapter 13: DNA and its Role in Heredity

Components of DNA (3)

nitrogenous base, phosphate group, sugar (ribose, deoxyribose)

Which bases are purines and pyrimidines?

Purines: Adenine, Guanine
Pyrimidines: Cytosine, Thymine, Uracil

Which bases form what number of bonds?

Adenine and Thymine: two bonds

Cytosine and Guanine: three bonds

Bacterial transformation

• some bacteria can take up foreign DNA from the environment

• foreign DNA enters cell and alters the bacteria’s genetic makeup

Significance of Griffith’s experiment

bacterial transformation was caused by a certain molecule

Significance of Avery’s experiment

bacterial transformation is caused by DNA

Significance of Hershey and Chase’s Experiment

DNA, and not protein is associated to genetic material

Describe Griffith’s experiment

• pneumococcus

• S strain (virulent) = dead mouse

• R strain (not virulent) = live mouse

• killed S strain = live mouse

• Live S strain + live R strain = dead mouse

• conclusion: chemical substance from one cell can genetically transform another

RNase, Protease, DNase

RNas: destroys RNA

Protease: destroys proteins

DNase: destroys DNA

Describe Avery’s Experiment

• A dead s strain was killed and divided into 3 samples

• treated with RNase, Protease, DNase

• each added to R strain cells

• R strain became virulent with RNase and Protease

• Means DNA must be the transforming substance

Describe Hershey and Chase’s Experiment

• bacteriophage T2 infects E. coli

• bacteriophage: outer protein coat, DNA inside

• E. Coli: genetic material inside

• virus DNA labeled with radioactive ³²P (phosphorus)

• virus Proteins labeled with radioactive ²⁵S (sulfur)

• virus allowed to infect bacteria

• bacterial cells separated from virus on outside

• after centrifuge DNA was found in the pellet meaning it was inside the bacteria

• protein was found in supernatant fluid which was outside the cell

• concludes that DNA is associated with genetic material, not protein

What was DNA labeled with in the Hershey-Chase Experiment?

radioactive ³²P

What was protein labeled with in the Hershey-Chase Experiment?

radioactive ³⁵S

Circumstantial evidence genetic material is in DNA

• DNA is located in nucleus and chromosomes

• somatic cells have twice as much DNA as reproductive cells

• amount of DNA is different between species

X-ray diffraction significance

Rosalind Franklin, suggested double helix structure

Watson and Crick significant discovery

built DNA structure

• 2 antiparallel strands

• nucleotide bases on interior

• sugar phosphate backbone exterior

• complementary base pairing

What IMFs hold DNA structure together?

hydrogen bonds between base pairs

van der waals forces between stacked adjacent bases

phosphodiester bonds link nucleotides

what is at the 5’ end and 3’ end?

5’ end: phosphate group attached to 5’ carbon
3’ end: hydroxyl group attached to 3’ carbon

DNA Grooves and Direction

Major and Minor Group: allows for interactions/hydrogen bonds with other molecules

Right handed helix, strands twist clockwise

5 key features of DNA structure

• double-stranded helix with sugar phosphate backbone

• right handed helix

• strands are antiparallel

• held together by complementary base pairing

• major and minor grooves where nitrogenous bases exposed

4 DNA functions and their structures

• stores genetic information: through base pair sequences

• susceptible to mutations: permanent changes in the sequence, and therefore the information encoded

• replication in the cell: using the complementary base pairing

• codes for a phenotype: proteins

3 Possible DNA replication patterns

• Seminconservative replication: each parent strand serves as template

• Conservative replication: parent strands are a template but are not a part of the daughter strand

• Dispersive replication: parent strands are template and daughter strands have parts from the parent and parts that are new

Describe Meselson and Stahl Experiment, What medium separates the DNA?

• ¹⁵N: heavier isotope used to label DNA strands

• ¹⁴N: lighter isotope used to label DNA strands

• Salt gradient separates DNA with two isotopes

• DNA placed in ¹⁵N at the bottom of salt solution

• then DNA placed in ¹⁴N for one round of replication and was at intermediate point

• Second round of replication created lightest DNA, contains none of the original DNA

• conclude DNA replication is semiconservative because DNA must contain a template strand from parent DNA

Which isotope is heavier and which one is lighter?

heavier: ¹⁵N

lighter: ¹⁴N

3 Main Steps of DNA replication

• initiation: double helix unwound by DNA helicase, single-stranded binding proteins keep strands separated, RNA primers made by primase

• elongation: adding complementary nucleotides, starts on 3’ end of template strand, DNA polymerase

• termination: RNA primers replaced by DNA via DNA polymerase, fragments connected by DNA ligase

how are DNA fragments linked?

DNA ligase

dNTP and dNMP

deoxynucleotide triphosphate

dinucleotide monophosphate

How many phosphates released from each dNTP?

2 phosphates released to release energy for the reaction

How does DNA replication start?

when the pre-replication complex binds at the origin of replication (ori), then initiation begins

Ori in Prokaryotes vs Eukaryotes

• prokaryotes: one single ori in circular chromosomes

• eukaryotes: multiple ori in linear chromosomes

RNA primer

• short single strand of RNA made by primase

• creates a starting point for DNA polymerase

Replication vs Building Directions

• read template strand from 3’ to 5’

• build new strand from 5’ to 3’

How is the sugar phosphate backbone built?

DNA polymerase turns dNTPs into dNMPs into phosphodiester bonds

Which protein keeps the template strands separated?

single-stranded binding proteins

leading strand vs lagging strand

• leading strand grows continuously and lagging strand grows "backwards” discontinuously

• one primer needed for leading strand, multiple primers needed for lagging strand

What are the short strands synthesized on the lagging strand?

Okazaki fragments

How do Okazaki fragments get connected? What is the gap called?

DNA ligase connects two neighboring DNA fragments and adds a phosphodiester bond

The gap is called a nick

What stabilizes the DNA polymerase to the template strand?

sliding DNA clamp holds polymerase to DNA strand

Telomeres

protective caps at the tips of DNA chromosomes

• helps DNA from getting damaged

• ensures DNA gets copied correctly

Issue with telomeres

• telomeres could be recognized as errors

• special proteins bind so they are not recognized as breaks in the chromosome

• chromosomes lose part of their telomeres every cell division

How do chromosomes lose part of their telomeres?

• after RNA primer removed the end of the lagging strand does not have a 3’ OH group

• overhanging fragment is removed

• after many cell divisions genes become lost and the cell dies

How do some cells avoid losing telomeres?

• telomerase

• expressed in stem cells, divide constantly

• adds missing parts of the telomeres back on

Types of DNA errors (4)

• mismatches: incorrect base pair

• bases change spontaneously (also mismatch)

• external mutagens damage DNA

• mutations from mismatches not being repaired

DNA correcting mechanisms (3)

• proofreading: done by DNA polymerase during replication

• mismatch repair: immediately after replication, keeps track of template strand

• excision repair: replaces damaged bases during the life of the cell

How does DNA polymerase conduct proofreading?

recognizes distorted shape of DNA from mismatch and exonuclease enzyme removes nucleotides one by one

How does DNA conduct mismatch repair?

• occurs right after replication and keeps track of template DNA

• mismatch detected and new DNA strand is cut, section with mismatch is removed and replaced with correct section

• DNA ligase seals the gap

How does DNA conduct excision repair?

• occurs during life of cell

• mostly damages not associated with replication ex. environmental mutagenic factors

• wrong base is detected and removed, hole is filled by DNA polymerase and gap is sealed by ligase