Week 4: DNA Replication Part II Flashcards
What are the roles of initator proteins for replication in e.coli
- Binds to origin
- Helps helicase beind
- Requires ATP
What unwinds DNA?
Helicase
What direction does the predominant DNA helicase move in?
5’-3’ along the lagging strand template
True or False: Helicase is on the lagging strand not the double helix
TRUE
What are SSB proteins and what does it do?
SSB proteinss are single strand binding proteins which keep DNA strands separated by preventing strands from H-bonding
What is the importance of SSB proteins?
SSB proteins keep DNA strands separated since they have the ability to stick back together after separation by helicase
Before DNA replication begins, what is required? Why is it needed?
An RNA primer made by primase. The primer is needed because DNA polymerase cannot start building from nothing.
What is RNA primer made of?
A short sequence of nucleotides with free 3’OH end that will be used by DNA polymerase
What direction does RNA primase read the the template strand in and what direction does it create the RNA primer in?
RNA primase reads the template strand in the 3’-5’ direction but makes RNA primer in the 5’-3’ direction
True or False: RNA primer does not get removed later on in DNA replication
False. It is later replaced because RNA primer is made of RNA.
What are the overall steps in bacterial DNA replication?
- Origin of replication
- Binding of initiator proteins
- Unwinding by helicase
- Binding of single-strand binding proteins
- RNA primers made by primase
- DNA polymerase
- Sliding clamp holds polymerase onto DNA
- Nick sealing by DNA ligase
Helicase + primase = ?
primosome
What direction does the new DNA grow in (i.e. what direction does DNA polymerase add nucleoside triphosphates?)
DNA adds nucleoside triphosphates in the 5’-3’ direction
How are nucleoside triphosphates added onto the growing DNA strand?
Two phosphates (pyrophosphate) are removed and the remaining phosphate bonds to the OH on the 3’ end of the growing strand and the base pair matches up
What are sliding clamps?
Sliding clamps helps to hold DNA polymerase in place as it synthesizes a new DNA strand (it tells DNA pol. to get its shit together)
What are okazaki fragments?
Okazki fragments are incomplete strands of DNA that are synthesized on the lagging strand
How are the Okazaki fragments on the lagging strand liked together?
The space (nicks) left by the removal of RNA primer is sealed by DNA ligase, from the 3’ end of one fragment to the 5’ end of another
What is used to make new DNA strands?
dATP (NOT ATP)
True or False: DNA helicase and primase always work together side by side
False. They work in conjuction but not always together.
What is the whole machine of bacterial DNA replication called?
the replisome
True or False: Directionality never changes
True.
What tends to happen as helicase uwinds DNA?
As DNA is being unwound by DNA helicase, it begins to want to spin, but it cannot, so supercoiling & torsional strain increases
What solves the supercoiling problem when DNA helicase unwinds DNA?
DNA topoisomerase cuts the DNA, lets it spin, then binds it back together. This is done in order to relieve tension
Although primase creates RNA primers to begin DNA replication, what is is not good at?
Creating primers at the ends of chromosomes
At the end of DNA replication, why is there an issue at the end of the lagging strand?
When the RNA primer is removed from the very last okazaki fragment, there is no way to add onto it (it’s a 5’ end) so the lagging strand ends up shorter than the leading strand.
What solves the issue presented by the lagging strand at the end of DNA replication? How does it solve the problem?
Telomerase solves the problem by adding a repetitive sequence (RNA template) to the lagging strand template allowing for more space to continue DNA replication, enough to make up for the loss of sequence information caused by the shorter lagging strand, and causes G-rich ends
What does telomere replication resemble?
Reverse transcriptase, because instead of using DNA to create RNA, you’re using RNA to create DNA
Telomerase abundant in stem and germ-line cells but not in _________ ______
Somatic cells
What is the issue with telomeres in cancer cells?
Most cancer cells produce high levels of telomerase
Why is it an issue for there to be a mistake in DNA replication (i.e. a strand with a wrong base pair)?
In future replications there will be a permanent mutation
What are the rates of error in RNA polymerase and DNA polymerase? What does this mean for the human genome?
RNA polymerases have an error rate of appox. 1 in 10^4, DNA polymerases have an error rate of approx. 1 in 10^9. This means that the human genome is only changed about 3 nucleotides every time a cell divides
What are the two mechanisms for DNA proofreading & repair?
- 3’-5’ exonuclease (“backspace”)
- Strand-directed mismatch repair
How does DNA polymerase proofread the newly synthesized DNA strand it creates?
It scans the strand as it makes it and removes the misincorperated nucleotide. This is the 3’-5’ exonuclease. It has two active sites, one for polymerizing and one for editing.
How does strand-directed mismatch work in DNA repair in eukaryotes?
If proofreading fails this is what is typically relied on.
* MutS protein recognizes and locks onto DNA mismatch
* proteins recuits MutL and scans DNA
* sliding clamp aids in positioning structure onto the new strand via identification of a nick
* MutL nuclease is then activated and removes the part of the strand that contains the error
True or False: DNA can’t get damaged after synthesis
False.
Which human diseases can be linked to defects in repair mechanisms?
A. Breast
B. Colon
C. Skin
D. All of the above
D.
What are some ways DNA can be damaged through stressors?
- oxidation
- radiation
- heat
- chemicals
What is a pyrimidine dimer?
When pyrimidines (i.e. thymine) that are stacked on top of each other form a bond (you do not want this!!)
What are some ways DNA can be damaged spontaneously?
- Depurination: the removal of a purine as a result of water
- Deamination: specific to cytosine, the removal of an amine group due to water resulting in uracil (DNA will have RNA in it because of this) – however this is common!
What can be caused by spontaneous damage to DNA?
Deamination changes C to U which causes switched base pair mutations in DNA replication. Depurination removes a purine which results in a missing nucleotide when DNA replicates.
Name the two general mechanisms of DNA repair (not proofreadings, so after synthesis)
- Base excision repair (BER)
- Nucleotide excision repair (NER)
Differentiate BER from NER
Base exicion repair replaces one nucleotide, nucleotide excision repair cuts out a whole section of the strand with the error and with the help of DNA polymerase and DNA ligase replaces the cut section and seals it together.
What are the two ways of DNA repair for double-stranded breaks?
- Nonhomologous end joining (NHEJ): when both strands break DNA nuclease processes it which causes some lost info/nucleotides, but then both ends are joined by DNA ligase (“quick and dirty” type of solution)
- Homologous recombination HR: when both strands break but there are undamaged homologous DNA strands available, the double strand break will be accurately fixed by using the undamaged DNA strands as a template (slower, but more accurate and best case solution)
