Chapter 8
How do X-rays damage DNA?
X-rays cause single and double strand breaks in DNA, and changes to the nucleobases. Double strand breaks often result in deletions that are lethal
Deinococcus radiodurans
an extremophillic bacterium that is one of the most known radiation-resistant organisms known
Replica plating
a clever method for indirect selection of auxotrophic mutants, devised by Joshua and Esther Lederberg in the early 1950s. In this technique, the bacterial culture is spread onto a nutrient agar plate. The mutants and nonmutant cells will create the master plate
What do phages consist of and how do they infect bacterium?
Consist of Dna or rna (never both) surrounded by a protein coat. A phage infects a bacterium by attaching to the cell and then injecting its nucleic acid into that cell
Give an example of a type of mutation that cells cannot repair?
Insertional inactivation caused by transposition
How does UV light damage DNA?
Irradiation of cells with UV light causes covalent bonds to form between adjacent thymine molecules on a DNA strand, producing thymine dimers. The dimer cannot fit properly into the double helix, distorting the DNA molecule. Replication and transcription stop at distortion, and as a result, the cells will die if the damage is not repaired
______ and ______ are replicons. What are not?
Plasmids and chromosomes are replicons, but fragments of chromosomal DNA are nto
What are the two types of transduction?
generalized and specialized
induced mutations
genetic changes that occur due to an influence outside of a cell, such as exposure to a chemical or radiation
Transducting particle
injects bacterial DNA
Base analogs
Structurally resemble nucleobases but have differnt hydrogen-bonding properties. The analog can be mistakenly incorporated by DNA polymerase
What is the master plate in replica plating?
The master plate is the medium that mutant and nonmutant cells grow on and form colonies
How can bacteriophages transfer bacterial genes from a donor to a recipient?
(bacterial viruses)- can transfer genes by transduction
What are the three general groups of chemical mutagens?
1. Chemicals that modify the nucleobases in DNA, changing their base-pairing properties 2. Base analogs 3. intercalating agents
What three mechanisms are used to transfer genes from a donor to a recipient?
1. Transformation: naked DNA uptake by bacteria 2. Transduction: bacterial DNA transfer by viruses 3. Conjugation: DNA transfer during cell to cell contact
What's an example of a base analog?
5-bromouracil, resembles thymine, but it often base pairs with guanine instead adenine 2-amino purine, resembles adenine but often base pairs with C instead of T
What is naked DNA?
DNA that is free in the cells' surroundings; it is not conatined within a cell or a virus
What happens if damaged DNA is not repaired?
It can lead to cell death; cancer in animals For example, in humans, two genes associated with breast cancer code for DNA pair enzymes; mutation in either result in 80% probability of breast
What do chemicals that modify nucleobases increase the chance of?
It increases the chance that an incorrect nucleotide will be incorporated during DNA replication.
How do mircroorganisms commonly acquire genes from other cells.
The process of horizontal gene transfer
What will happen if a chromosomal fragment is transferred?
Then it must become integrated into a replicon to be maintained in a population. This involves a process called homologous recombination. which can happen only if the donor DNA is similar in nucelotide sequence to a region in the recipients cell's genome
Following gene transfer, how are recipient cells able to replicate DNA to pass it on to daughter cells?
This can happen only if the DNA is a replicon, meaning it has an origin or replication
Why are transposons introduced intentionally into a cell?
To generate (induce) mutations. The transposon, which cannot replicate on its own because it lacks an origin of replication, inserts into the cells genome. This generally inactivates the gene into which it inserts
intercalating agents
increase the frequency of frameshift mutations. They do this because they are flat molecules that can insert (intercalate) between adjacent base pairs in a strand of DNA. This pushes the nucleotides apart, producing a space between bases that allows errors to be made during replication
What is the role of methylation in the process of nucleotide incorporation by DNA polymerase?
methylation helps us understand why cells can detect which strand is new. Soon after a DNA strand is synthesized, an enzyme adds methyl groups to certain nucleobases. This takes time, so the new strand is still unmethylated immediately after it is synthesized. Therefore, the template strand is methylated, whereas the new strand is not, allowing the repair enzyme to distinguish between the two strands. After the nucleotides are removed from the new strand, the combined actions of DNA polymerase and DNA ligase then fill in that section and seal the gap.
What happens if intercalating agents insert into the template strand? What if was inserted into the strand being synthesized?
template stand> a base pair will added as the new strand in synthesized Strand being synthesized> a base pair will be deleted. Often results in premature stop codon
What are the steps in replica plating
1. Create master plate and press it onto a sterile velvet, which picks up cells of every colony 2. Cells adhering to the velvet are transferred to the nutrient agar and glucose-salt agar, resulting in exact replicas Protrophs grow on both types of media, but auxotrophs grow only the nutrient agar. The plates are exact replicas, so auxotrophs on the master plate that cannot grow on glucose salts can be identified
What technique is used to overcome the problem in indirect selection?
A technique called replica plating, sometimes preceded by penicillin enrichment of mutants
The recipient cell must be ______ in order for transformation to occur?
Competent- a specific physiological state that allows the cell to take up DNA
Name two types of intercalating agents
Ethidium bromide- a chemical commonly used to stain DNA, should be used with great care because it may cause cancer chloroquine- used to treat malaria
Where does naked DNA originate from?
It orginates from cells that have either burst or secreted some DNA
direct selection
Mutants that can grow under conditions in which the parent cells cannot; usually easy to isolate by direct selection - In this method, cells are inoculated onto an agar medium that supports the growth of the mutant, but does not allow the parent to grow
How can UV light be mutagenic if it damages DNA?
UV causes mutations indirectly. Its major mutagenic action results from the cells attempt to damage by SOS repair
What two kinds of radiation are commonly used as mutagens?
UV light and X rays
When can the wrong nucleotide be incorporated as the complementary strand be synthesized?
When a DNA strand has a base analog
Penicillin enrichment
sometimes used before replica plating to increase the proportion of auxotrophs in a broth culture. By increasing the proportion of mutant cells, it is easier to isolate them. Penicillin kills only growing cells: prototrophs die, auxotrophs survive. Pencilillinase is then added to destroy the penicillin, and the cells plated on nutrient agar
in the several dozen prokaryotic species that can become competent in nature, the process is ________
tightly controlled
Generalized transduction
transfers any genes of the donor cell; results from the rare error that sometimes occurs during the construction of phage particles; transfer of DNA to new bacterial host
indirect selection
used to isolate an auxotrophic mutant from a prototrophic parent strain. -This process is more difficult than direct selection because any medium that supports the growth of the mutant also allows the growth of the parent
Explain how modified nucleobases can be repaired
Modified nucleobases can result in base subtitutions if they are not repaired before the DNA is replicated A repair mechanism involves oxidized guanine. DNA glycolase removes that oxidized nucleobase from the sugar phosphate backbone. Another enzyme then recognizes that the nucleobase is missing and cuts the DNA at this site. DNA polymerase degrades a short section of this strand to remove the damage. This same enzyme synthesizes another strand with the proper nucleotides, and DNA ligase seals the gap in the single stranded DNA
Do all competent bacteria take up DNA regardless of its source?
No, most do but some species accept DNA only from closely related bacteria
Describe three mechanisms cells use to repair thymine dimers
1. photoreactivation- an enzyme used the energy of visible light to break the covalent bonds of the thymine dimer, restoring the DNA to its original state. This enzyme is only found in microbes. 2. Excision repair (dark repair)- the enzyme removes the DNA strand with damaged region. DNA polymerase and ligase then fill in and seal the gap left by the removal of the segment 3. SOS repair- the last attempt that bacteria use to repair extensively damaged DNA. These enzymes are induced when DNA is so heavily damaged by UV light that photoreactivation and excision repair may not be able to correct all of the damage. DNA and RNA polymerases stall at sites of unrepaired damage, so the cells cannot replicate or transcribe their DNA. WIthout SOS repair, the cells would die.
Why are mutations rare?
Because changes that disrupt the structure of DNA are generally repaired before they can be passed on to progeny
How can the fact that DNA is naked be demonstrated?
By adding DNase (enzyme that degrades DNA) to the medium; this prevents transformation, indicating that the process requires naked DNA
mutagen
an agent that induces the genetic changes in induced mutations
Alkylating agents
chemicals that add alkyl groups onto nucelobases
Explain how nucelotide incorporation errors by DNA polymerase can be repaired?
It can be repaired by 1. Poofreading by DNA polymerase- these enzymes check the accuracy of their actions. The enzymes can back up and excise (remove) a necleotide not correctly hydrogen bonded to the opposing nucleobase in the template strand. The DNA polymerase then inserts the correct nucleotide. Very efficient but not flawless-error rate during DNA replication is as low as 10^-9 to 10^-11 errors per base 2. Mismatch Repair- fixes errors missed by the proofreading of DNA polymerase. A specific protein binds to the site of the mismatched nucleobase, directing an enzyme to cut the sugar phosphate backbone of the new DNA strand. Another enzyme then degrades a short region of that DNA strand, therby removing the misincorporated nucleotide. The results of both of these mechanisms is the potential mutation is eliminated
