Chapter 11: DNA Replication and Recombination Flashcards
This is the chemical affinity between nitrogenous bases as a result of hydrogen bonding.
Complementarity
Cytosine is a ______.
pyrimidine
Guanine is a ______.
purine
Thymine is a ______.
pyrimidine
Adenine is a ______.
purine
Why isn’t A-G bonding possible?
The double helix must be three rings across. A-G bonding would require four, because both are purines and have two rings each. That would make the DNA too big.
Why isn’t C-T bonding possible?
The double helix must be three rings across. C-T bonding would require two, because both are pyrimidines and have only one ring. That would make the DNA too small.
Why isn’t C-A or G-T bonding possible?
Though it’s purine-pyrimidine, the charges on the molecules would repel each other, and no hydrogen bonding could happen.
What’s the total diameter of a DNA strand?
20 Å
How does the cell know when the wrong base pair has been put in?
The DNA is too wide or too narrow.
This type of DNA makes up the majority of DNA in the body and is the original structure determined by Watson and Crick. It is a right-handed corkscrew.
B-DNA
This type of DNA is a right-handed corkscrew but is twisted a bit tighter and is found only in solution, not in living cells.
A-DNA
This type of DNA is a left handed corkscrew and is twisted a bit looser (12 base pairs per turn). It is found in the body.
Z-DNA
What type of sugar does DNA have?
deoxyribose
What type of sugar does RNA have?
ribose
Which base pairs differ between RNA and DNA?
RNA has uracil instead of DNA’s thymine.
Is RNA single stranded or double stranded?
It’s single stranded except for in some viruses, but it can hydrogen bond with itself to form shapes that are critical to its function.
This type of RNA is never translated into protein, but is an important structural component of ribosomes.
rRNA (ribosomal RNA)
This type of RNA carries information from DNA to the ribosomes, where translation occurs.
mRNA (messenger RNA)
This type of RNA carries amino acids to the ribosome during translation.
tRNA (transfer RNA)
This type of RNA helps process mRNA by getting rid of introns and splice exons.
snRNA (small nuclear RNA)
This type of RNA is involved in DNA replication and is an enzyme RNA that places the telomere on the end of the chromosome.
telomerase RNA
This type of RNA is a short strand that can base pair with an mRNA sequence to prevent expression of that particular gene.
antisense RNA
What does “semiconservative replication” mean?
It means each time DNA is replicated, each of the two original strands gets a new pair strand, so the new DNA molecule has one old strand and one new.
Because of the strength and stability of the DNA molecule, a lot of _____ needs to be used up to pull the two strands apart during replication.
ATP
Energy is used in DNA replication to:
Pull the two strands apart and create new covalent bonds.
Is base pairing during replication an active or passive process?
Passive.
Explain how base pairing is a passive process.
All four different nucleotides bombard the space where the next base pair belongs. They are present in high concentration. Polymerase locks them into place.
In this type of replication, two new strands of DNA are made from the parent strands. The new strands then associate with each other and the old strands reassociate.
Conservative
In this type of replication, new DNA strands are comprised of both old and new DNA.
Dispersive
What did the Meselson-Stahl experiment prove?
That DNA replication is semi-conservative.
In this technique, samples are forced by centrifugation through a density gradient through a heavy metal salt. Molecules of DNA reach equilibrium when their density equals the density of the gradient medium.
Sedimentation equilibrium centrifugation
In the centrifuge, which will reach equilibrium closer to the bottom of the tube –14N DNA or 15N DNA?
14N DNA
How did the Meselson-Stahl experiment prove that DNA replication was semiconservative?
E. coli cells were grown in 15N-labeled medium, labeling the E. coli DNA as 15N. The culture was then centrifuged. That E. coli culture was added to a 14N medium and the cells were allowed to replicate once and centrifuged, then centrifuged again after a second replication and then a third.
What was the ratio of 14N/14N to 14N/15N DNA in the Meselson-Stahl experiment at generation I?
All 15/14N DNA.
What was the ratio of 14N/14N to 14N/15N DNA in the Meselson-Stahl experiment at generation II?
1:1
What was the ratio of 14N/14N to 14N/15N DNA in the Meselson-Stahl experiment at generation III?
1:3
What would be the outcome of the Meselson-Stahl experiment if DNA replication were conservative?
You would just have one band of 14N DNA and one of 15N DNA.
How is semiconservative replication in eukaryotes tested?
We label a chromosome with tridiated thymidine and allow replication to occur. After replication, if you have one labeled and one unlabeled chromosome, there was no sister chromatid exchange; if there was, there will be reciprocal regions on both that are labeled.
Is the prokaryotic chromosome linear or circular?
Circular
How many origins of replication are there in prokaryotic DNA?
Only one.
Is bacterial DNA replication unidirectional or bidirectional?
Bidirectional (two replication forks)
How is torsional stress relieved when replicating the circular prokaryotic chromosome?
The chromosome is broken open into a linear strand, and then DNA gyrase spins the strand to relieve additional stress and to prevent tangling.
Where is DNA gyrase found?
Always ahead of the replication fork.
How many polymerases are involved in bacterial DNA replication?
Five.
In which direction does DNA replication occur?
5’ to 3’
What’s the role of polymerase III in bacteria?
5’ to 3’ polymerization and exonuclease proofreading
What’s the role of polymerases II, IV, and V in bacteria?
DNA repair
Is an RNA primer necessary for prokaryotic DNA replication?
Yes.
This bacterial polymerase is a holoenzyme comprised of multiple subunits.
Polymerase III
How does helical unwinding occur in prokaryotic DNA?
DnaA bonds to 9mers at the origin site, bending the DNA. The replication bubble forms, and DnaB and DnaC come to open the helix further. Single stranded binding proteins hold it apart. DNA gyrase comes in to prevent supercoiling.
Why is a primer needed for bacterial DNA replication?
Polymerase III needs a 3’ end to start adding nucleotides. The RNA primer does not, so it can start, and then it provides that 3’ end.
Is bacterial DNA parallel or antiparallel?
Antiparallel
Why is there a lagging strand?
Because DNA is antiparallel, one strand’s template runs 3’ to 5’, which won’t work. Therefore, many points of initiation are necessary along that template so that synthesis can occur in the proper direction.
What are the fragments of the lagging strand called?
Okazaki fragments
How are Okazaki fragments joined together?
DNA ligase
How can concurrent synthesis occur in bacterial DNA?
A dimer loops the template strand around, inverting it (but synthesis still occurs 5’ to 3’. The other part of the dimer is the beta subunit of the holoenzyme and prevents the core subunit from falling off the template.
How many origins of replication are there in eukaryotic DNA?
Multiple.
Why are there so many origins of replication in eukaryotic chromosomes?
The DNA is extremely long, so this speeds the process up.
Does replication always occur at a constant speed?
No. Embryonic cells, for example, replicate much faster.
How does the cell control speed of replication?
If replication is slower, it will use fewer replication bubbles/origins.
What is the function of DNA polymerase?
It links the nucleotides.
What are the four properties of DNA polymerase?
- Needs all of the four DEOXYnucleosides.
- Needs a template strand.
- Needs a primer.
- Has to go in the 5’ to 3’ direction.
What does the prereplication complex do?
It regulates the timing and sites of replication origin.
What does the origin recognition complex do?
It tags the origin as the site of initiation.
As each nucleotide is added, the last two phosphate groups are hydrolyzed to form _____.
pyrophosphate
What drives the addition/polymerization of each new nucleotide to the strand?
Exergonic hydrolysis of pyrophosphate into two phosphate molecules.
What does polymerase add onto?
The ‘3 OH.
Can polymerase add onto anything besides a 3’ hydroxyl group?
NO!
What do helicases do in DNA replication?
They open the helix and push the strands apart like a plow.
Where are helicases located in DNA replication?
Right at the replication fork.
These bind to the separated DNA strands and prevent the strands from bonding to each other again.
Single stranded binding proteins
This relieves torsional stress during DNA replication.
DNA gyrase
Where is DNA gyrase located during DNA replication?
A little bit ahead of the replication fork
This synthesizes the RNA primer.
RNA primase
Does RNA primase need a 3’ OH to lay down its nucleotides?
No.
This makes the phosphodiester bonds, extends the primer, and copies the template, then proofreads the sequence.
DNA polymerase III
How does polymerase III fix a bad nucleotide?
It backs up, chops off the bad one, and replaces it with the proper one.
Where do the helicases get their energy from?
ATP hydrolysis
This is when polymerase jumps up the fork, puts a primer there, and continues forward.
Discontinuous synthesis
Lagging strands have _____ synthesis.
discontinuous
Does the lagging strand synthesize toward or away from the fork?
Awayyy!!!
Does the leading, continuous strand synthesis toward or away from the fork?
Toward
This takes out the RNA primer once polymerase III hits it and falls off.
DNA polymerase I
This guy bites off all the RNA primer nucleotides like a PacMan.
exonuclease subunit
The exonuclease subunit and the polymerase subunit are part of _____.
DNA polymerase I
This synthesizes the DNA nucleotides to replace the RNA ones from the primer.
polymerase subunit
This makes the last phosphodiester bond to put together the Okazaki strands.
DNA ligase
How can DNA polymerase III work on both the lagging strand and the leading strand at once, if one is farther ahead than the other?
Like cooked spaghetti, the DNA bends around into a loop, and the Okazaki strand follows.
What limits the length of the Okazaki strand?
How much the DNA can bend
Do the lagging strands require a new primer each time or just one?
A new one for each fragment
What is the problem at the end of linear DNA replication?
The lagging strand still has a bit of primer at the 5’ end that needs to be turned into DNA.
What replaces the primer at the 5’ end of linear DNA?
A telomere
These protect the end of the DNA from unraveling and put in a bit of DNA.
Telomeres
Telomeres are put into place by _____.
Telomerase
Telomeres are comprised of what?
Repeating sequences of six nucleotides
After another primer is placed on the end after the telomere, why doesn’t it need to be removed?
It’s just extra nucleotides.
What is telomerase’s connection with aging?
When telomeres get too short, cells die.
What is telomerase’s connection with cancer?
Cancer cells have plenty of telomerase, so their telomeres never shorten. Embryonic cells also have a lot of telomerase. This allows for lots of replication.
This protein oversees the tucking in of the single stranded end of the DNA as it loops and base pairs.
Shelterin
This process is the part of recombination where one phosphodiester bond is broken.
Endonuclease nicking
This part of recombination is where the complimentary strands that were nicked peel away and base pair with each other’s complimentary strands.
Strand displacement
The spot where the strands of recombinant DNA physically cross over each other is called the _______.
heteroduplex region
After two recombinant strands have crossed over and base paired, this enzyme comes in and seals the nick, stabilizing the molecule.
Ligase
This term refers to the way that the heteroduplex region can move around after ligase has sealed the nick, as base pairs peel up.
Branch migration
This is what accounts for differences the sequences of alleles, which is what makes them different from one another.
Branch migration
The X shape that is formed right after branch migration when the two recombinant chromosomes try to pull apart is called the _____ configuration.
cis
When the bottom half of the cis configuration flips around, a _____ structure is formed.
Holliday
When endonuclease nicks the Holliday structure, in order to get a visibly recombinant chromosome, it must nick at the ____ and ____ spots.
top/bottom, or north/south
If endonuclease were to nick at the east and west spots of the Holliday structure, what would happen?
You would get two seemingly nonrecombinant chromosomes.
This enzyme seals the nicks up after recombination is complete.
Ligase
This is where a base pair mismatch occurs in a recombining chromosome, making an ugly bubble that is repaired by polymerase II.
Gene conversion
What enzyme corrects for gene conversion?
polymerase II
How does polymerase II correct a gene conversion?
It chops one of the mismatched nucleotides out and replaces it with the complimentary one.
This is where polymerase II replaces the bad nucleotide with a matching one.
Excision
How does polymerase II know which strand’s nucleotide to take out?
It doesn’t. It’s totally random.
How do you know that gene conversion has occurred?
Number of alleles changed (a new one was introduced with that new base pair, which changed the sequence).
When will gene conversion occur?
Every time there’s a region of heteroduplex.
Can gene conversion occur in somatic cells too?
Yes.
Is recombination completely random?
No, there are certain hotspots, and it occurs more often in females.
Why does recombination occur more often in females?
Probably due to the fact that meiosis arrests, leaving chromosomes in contact for years and years.
This hypothesis states that the exposure to a phage “induces” resistance in bacteria.
Adaptation hypothesis
Mutations that occur regardless of the presence or absence of a T1 bacteriophage are called _____.
spontaneous mutations
The experiment that showed that bacteria are capable of spontaneous mutation is called the _____.
fluctuation test
Bacteria that can grow on a minimal medium, synthesizing all of their own essential organic compounds are called ______.
prototrophs
These bacteria are wild types for all growth requirements.
prototrophs
Bacteria that have lost the ability to synthesize one of their essential organic compounds, and must therefore get it from their plate, are called _____.
auxotrophs
During this phase, bacteria growth is slow.
Lag phase
During this phase, bacterial growth becomes very rapid.
Log phase
During this phase, nutrients become limited and cells stop dividing on the plate.
Stationary phase
This is when transfer of genetic information occurs between members of the same species.
Vertical gene transfer
This is when transfer of genetic information occurs between members of related but different bacterial species.
Horizontal gene transfer
DNA uptake from the environment by bacteria is called _____.
transformation
Transfer of DNA from one bacterium to another by direct contact is called _____.
conjugation
Transfer of DNA from one bacterial cell to another by a virus in a phage is called _____.
transduction
How was conjugation discovered?
Two strains of bacteria were colonized. One can make all enzymes except met and bio, and one can only make met and bio. The strains were allowed to grow on separate petri dishes as controls, and then they were also combined and grown that way. The baby cells in the dish could make all the enzymes because the ones that could make only met and bio shared their DNA with those that couldn’t make those.
What happens if you try to do the conjugation experiment in a U shaped tube with a filter that only allows media to pass through, then place them on petri dishes that do not have all the nutrients they need?
Nothing. They can’t grow because they didn’t have conjugation and can’t survive without their medium to feed them.
This is a sort of “rope” or bridge that one cell puts out to pull another towards it for conjugation.
Pilus
The donor cell, which has the genes necessary to make a pilus and donate its DNA, is called the _____ cell.
F+
The recipient cell in conjugation is called the ______ cell.
F-
These are extra circles of DNA (smaller than the main chromosome) that contain the genes for conjugation in the F+ cell.
Plasmids
This type of cell is one where a region on the plasmid matched spots on the main chromosome and crossover occurred, making the plasmid part of the main chromosome.
Hfr cell
What is special about Hfr cells?
Because they contain part of the plasmid in the main chromosome, they can transfer genes from the main chromosome.
Why aren’t the F- cells who receive DNA from an Hfr cell considered Hfr or F+ cells after the process ends?
The main chromosome is very large, and it would take hours to transfer the whole thing. But the bridge can only stay intact for 20-30 minutes, and so a lot doesn’t get transferred. The other cell only has part of the plasmid sequences, not the whole plasmid.
What’s the interrupted mating technique, and how is it done?
It helps you create a map for bacteria. You put the cells together in a mixture and let them start their bridge thing, then put them in a blender and break up their bridges. Stop one batch at five minutes, another at ten, ect, and you can see which genes got transferred in what order.
This type of cell has had its plasmid pop back out via recombination, pulling a few main chromosome genes with it.
F’ cell
What happens when an F’ cell undergoes conjugation?
You end up with an F- cell that’s also F’, and is partially diploid.
What is an F- cell that’s also F’ and is partially diploid called?
A merozygote
These bacterial cells can have dominant and recessive genes.
Merozygotes
This protein facilitates recombination between a single complementary strand and its homologous region on the host chromosome.
RecA
This is a form of recombination in which a single complementary strand ends up with its homologous region on the host chromosome.
Single stranded displacement
This protein unwinds the helix, facilitating recombination involving RecA.
RecBCD protein
Plasmids that can exist autonomously or integrate into the chromosome are called _____.
episomes
In R plasmids, this encodes genetic information essential to transferring the plasmid between bacteria.
Resistance transfer factor (RTF)
In R plasmids, these are genes conferring resistance to antibiotics or heavy metals like mercury.
r-determinants
This plasmid encodes one or more proteins that are toxic to bacterial strains that don’t have that plasmid.
Col plasmid
Special proteins within the Col plasmid are called _____.
colicins
In this assay, a bacterial lawn is sprinkled with virus particles, then the clear spots made by the virus killing the bacteria is counted so that viral density can be counted.
plaque assay
Why doesn’t transduction require physical contact?
Virus particles are very small and can get through the filter or a cell wall.
How can a virus transfer bacterial DNA?
A piece of chopped up bacterial DNA ends up in a phage’s head and gets injected into a host cell.
In this process, viral DNA inserts itself into the bacterial genome rather than kill the cell. The bacteria keeps it silent, but eventually the virus pops back out, pulling a bit of the bacterial DNA with it and leaving some of itself behind, rendering it a defective virus.
Specialized transduction
HIV is an example of _____.
specialized transduction
Viral DNA integrated into the bacterial chromosome is called a _____.
prophage
Viruses that can either lyse the cell or behave as a prophage are called _____.
temperate phages
Viruses that only want to lyse the cell are called _____.
virulent phages
