DNA entered the bacterial cells, not protein so DNA functions as genetic material
Primary structure of DNA
5' end starts with a phosphate; 3' is the other side
Sugar-phosphate is the backbone of DNA strand
Phosphodiester bond links deoxyribonucleotides
Bases (A,T,C,G) project from the backbone
Complementary base pairing
Sugar-phosphate backbone of dna
Complementary base pairs held together by hydrogen bonding
Antiparallel strands (their 5' -> 3' polarities run in opposite directions)
DNA replication
Specific base-pairing is functionally significant, but the actual mechanism is still a mystery
Three predictions from the Mendelson-Stahl Experiment
Semiconservative replication
Conservative replication
Dispersive replication
Semiconservative replication
In this model, the two strands of DNA unwind from each other, and each acts as a template for synthesis of a new, complementary strand. This results in two DNA molecules with one original strand and one new strand.
The results in DNA that are hybrids
Results after 2 generations from semiconservative replication
1/2 low-density DNA
1/2 intermediate-density DNA (hybrid)
Conservative replication
this model, DNA replication results in one molecule that consists of both original DNA strands (identical to the original DNA molecule) and another molecule that consists of two new strands (with exactly the same sequences as the original molecule).
This looks like 2 pairs of homozygous
Results of conservative replication after 2 generations
1/4 high-density DNA
3/4 low-density DNA
Dispersive replication
In the dispersive model, DNA replication results in two DNA molecules that are mixtures, or “hybrids,” of parental and daughter DNA. In this model, each individual strand is a patchwork of original and new DNA.
Results in molecules that are all hybrid (the spotted one)
Results after 2 generation in dispersive replication
All intermediate-density DNA (hybrid)
Semiconservative
Conservative
Dispersive
DNA replication
Begins at specific sites= origins of replication
-Bacterial chromosomes have one origin of replication
-Eukaryotes have multiple origins along chromosome (speeds up replication)
Replication proceeds in both directions from origin
Replication fork
DNA is unwound to form this in order to replicate
Helicase- untwists the double helix and separates the strands
Single-strand DNA- binding proteins (SSBPs)- bind to separated DNA strands to prevent reparing
Topoisomerase
Relieves strain ahead of replication fork caused by unwinding by breaking, swiveling and rejoining DNA strands
Where does DNA synthesis occur?
Enzymes which synthesize DNA cannot start a new chain. They can only add to an existing strand (need -OH group)
Primase
Lays down an RNA primer which is complementary to the template strand
Provides the initial chain so replication can proceed
DNA polymerases
Enzymes which catalyze the synthesis ob DNA by adding nucleotides to an existing chain
-Can only add dNTPs in 5' to 3' direction
DNA polymerase III
Adds a nucleotide to the RNA primer and then keeps adding complementary nucleotides to the growing strand
Why does orientation matter?
DNA strands are antiparallel because DNA polymerases can only add nucleotides to the free 3' end (they need the free OH to attach the next base)
So, a new strand can only be synthesized in the 5' -> 3' direction
Two strands of DNA that must be synthesized differently due to their different orientations
Leading strand
Lagging strand
Leading strand
Synthesized towards the replication fork
Occurs continuously
Requires one primer
Lagging strand
Synthesized away from the replication fork
Discontinuous
-New fragment cannot be started until fork moves forward and exposes template
Creates okazaki fragments
What does the lagging strand require for each fragment?
A primer such as DNA pol I and DNA ligase
DNA pol I
Replaces the RNA primer with DNA nucleotides
DNA ligase
Joins all of the fragments into a continuous strand
Replisome
Many enzymes involved in replication form this large macromolecular machine
Which of the enzymes synthesizes short segments of RNA?
C) Primase
If this is replicated what would it be 3'-AAGTCAGT-5'?
5'-TTCAGTCA-3'
Problems with copying the ends of linear chromosomes?
DNA unwinding completed
Leading strand completed
Lagging strand nears completion
Lagging strand is too short because there is no primer for DNA polymerase- unreplicated and is eventually lost shortening chromosomes
What is the solution for copying linear chromosomes?
Telomeres
Telomeres
Consist of a short nucleotide sequence (TTAGGG in humans) repeated between 100-1,000 times
"Buffers" at the ends of eukaryotic chromosomes
Do NOT encode any genes
Once they reach a critical limit the cell enters senescence
Telomerase
Active during embryonic development but shut off in most somatic cells at later stage.
Male germ cells, activated lymphocytes and some stem cells still have enzyme
Active in ~90% of tumors
What is telomerase composed of?
Composed of protein and RNA
Telomerase reverse transcriptase (TERT)
Telomerase RNA (TERC)
-CAAUCCCAAUC
The DNA of Telomeres has been highly conserved throughout the evolution of eukaryotes. What does this most probably reflect?
C) The critical functions of telomeres must be maintained
Proofreading and Repair
Overall error rate only 1 mistake per 1 billion nucleotides
Initial errors between incoming bases and the template strand are much more common
Why are initial errors between incoming bases and the template strand much more common?
Occurs 1 every 100,000 bases
DNA polymerases proofread each base as soon as it is added
If incorrectly paired, it is removed and synthesis resumes
Mismatch Repair (MMR)
Other enzymes remove and replace incorrectly paired nucleotides
Defects in these enzymes are linked to cancer
-Hereditary non-polyposis colon cancer (HNPCC)
What happens if the error arises after replication (sun, smoke, carcinogens, etc.)?
DNA is constantly monitored and repaired
Nucleotide excision repair (NER)
Nucleotide excision repair (NER)
Damaged segment is cut out by a nuclease and the gap is filled in by DNA polymerase and ligase using the undamaged strand as a template
Seroderma pigmentosum
Damage from UV light causes thymine dimers which interfere with replication
This disorder is caused by inherited defect in NER)
-Hypersensitivity to light
-Increased risk of skin cancer (1000x-2000x)
Benzopryene in cigarette smoke binds to DNA replication. Which repair mechanism would most likely be used to repair the damage caused by this chemical?
