DNA replication and repair Flashcards
The flow of genetic information
- REPLICATION (DNA SYNTHESIS)
- TRANSCRIPTION (RNA SYNTHESIS)
- TRANSLATION (PROTEIN SYNTHESIS)
The mammalian cell cycle
G0 - quiescent cells
M phase -mitosis
G1- rapid growth and preparation for DNA synthesis
S phase - ) phase of the cell cycle is when chromosomes are replicated. This requires DNA synthesis and histone synthesis (the latter to make the proteins that will package the newly replicated DNA).
Cell cycle time (exponential growth in rich media): E. coli ≈20-40 min; yeast 70-140 min; human cell line (Hela): 15-30 hours
DNA replication
The first step is separation of strands (unzipping)
Performed by helicase
That results in formation of the replication fork.
Each of the separated strands provide a template to create a new strand of DNA.
An enzyme called primase starts the process.
This enzyme makes a small piece of RNA called primer.
The primer is the starting point for the construction of the new strand of DNA
An enzyme called DNA polymerase binds to the new primer and starts the process.
DNA polymerase can only add DNA bases in one direction:
from the 5’ to 3’.
The other strand, the lagging strand, can not be made in this continuous way, because it runs in the opposite direction.
The DNA polymerase can only make the DNA in a series of small chunks called Okazaki fragments.
Each fragment is started by an RNA primer…
DNA polymerase than adds a short row of DNA bases in the 5’ to 3’ direction.
Once the new DNA has been made, the enzyme exonuclease removes all the RNA primers from both strands of DNA.
Another DNA polymerase fills all the gaps that are left with DNA.
Finally, another enzyme named DNA ligase seals up the fragments in both strands, to form continuous double strands.
DNA replication is described as semi-conservative , why ?
semi-conservative because each DNA molecule is made up of one old (conserved) and one new one.
Strand separation at the replication fork causes positive
supercoiling of the downstream double helix
DNA gyrase is a topoisomerase II, which
breaks and reseals the DNA to introduce negative
supercoils ahead of the fork.
Fluoroquinolone antibiotics target DNA gyrases in many
gram-negative bacteria: ciprofloxacin and levofloxacin (Levaquin)
The strand separation process (unwinding the complementary Watson-Crick DNA strands) causes overwinding ahead of the fork. Any DNA that is overwound (or underwound) is said to be “supercoiled.” Overwound DNA is positively supercoiled. The increasing torsional stress needs to be dissipated in order for the fork to continue to unwind so that replication can proceed. This is accomplished by DNA topoisomerases, which cut the DNA strands, unwind them and reseal the strands. As they do so they introduce negative supercoiling into the DNA to compensate for the positive supercoiling. Gram-negative bacteria, such as E. coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, can be killed by fluoroquinolone antibiotics, which inhibit DNA gyrase, a topoisomerase II. Topoisomerase II cuts both strands of DNA, swivels them and rejoins them. Topoisomerase I cuts only one strand, and relaxes negative supercoils.
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
Genome size: H. sapiens (human) ≈2.9 Gbp;
Mutation rate in DNA replication ≈10-8-10-10 per bp
Misincorporation rate:
transcription ≈10-4 per nucleotide;
translation ≈10-3-10-4 per amino-acid
Factors Influencing the Rate of Spontaneous Mutations
Accuracy of the DNA replication machinery.
Efficiency of the mechanisms for the repair of damaged DNA.
Degree of exposure to mutagenic agents in the environment.
Environmental agents causing damage to living cells
Induced mutations result from exposure or organisms to mutagens, physical and chemical agents that cause changes in DNA, such as ionizing irradiation, ultraviolet light, or certain chemicals.
Genome evolution – nucleotide substitutions
Basically two causes: damage, and copy errors during replication.
The two causes can be teased apart by comparing species with different generation times. More generations per unit of time mean more copying errors, while the rate of damage might stay relatively constant.
Errors are recognized and repaired by specific and highly efficient repair mechanisms.
Resulting error rate is low: about 3x10-8 per nucleotide per generation in humans.
The repair mechanism is extremely important: damage to this system increases the likelihood of getting cancer.
The rate of mutagenesis is higher in males than in females (see e.g. Berlin et al., J Molec Evol 62(2) 226-233), probably due to more cell divisions in the male germline. This results in low mutation rates on the X, and high mutation rates on the Y chromosome.
In mammals, the rate of transitions (pyrimidine-to-pyrimidine or purine-to-purine) is about twice higher than the rate of transversions (pyrimidine-to-purine or vice versa).
DNA repair defects cause disease
hereditary nonpolyposis corectal cancer - caused by DNA mismatch repair- Sensitivity to UV radiation , causes cancer in the colon and ovary
Xeroderma pigmentosum - affected nucleotide excision repair - sensitive to UV radiation , point mutations - causes skin carcinomas , melanomas .
