Topic 5 Flashcards
It is crucial for eukaryotic chromosomes to replicate at the S phase once and only once during each cell cycle. Why is ensuring this challenging?
It i challenging because of multi-origin of replications
Why does the formation of the replication bubble have to be well-coordinated with spindle fiber formation?
Chromosomal damage can occur if segregation occurs before replication.
Describe the 3 steps and results from the Meselson and Stahl experiment
- Labelled DNA with heavy nitrogen isotope
- Transferred heavy DNA to light nitrogen isotope medium and allowed DNA to replicate
- Bands appeared halfway between heavy and light nitrogen bands which indicated semi-conservative replication
(DNA/RNA) primer is used in PCR, while (DNA/RNA) primer is used in vivo
DNA primer is used in PCR, while RNA primer is used in vivo
What are the 2 main steps in adding a nucleotide to a growing strand?
- Recognition of proper dNTP and attacking dNTP with a hydroxyl group
- dNTP has to be able to base pair with the base on the template strand
Processivity
An enzyme’s ability to catalyze consecutive reactions without releasing its substrate
- e.g. the average number of nucleotides added each time when an enzyme binds a primer:template junction
True or false: DNA polymerase has separate active sites for each of the 4 dNTPs
False
- DNA polymerase is a processive enzyme that catalyzes the DNA synthesis using a single active site for any of the 4 dNTPs
What 3 things is DNA polymerase able to do that makes it a processive enzyme??
- Sterically distinguish between dNTPs and its rNTPs precursors
- Show kinetic selectivity for adding correct base-paired dNTP (not just recognizing the dNTP but also recognizing the nucleotide on the template)
- Initiate synthesis using either RNA or DNA primer annealed to the template
ddNTPs serve as…
Terminators
What component of the DNA polymerase recognizes dNTPs over rNTPs?
There are discriminator amino acids that recognize the proper structure of the incoming nucleotide
- rNTP can’t fit into the polymerase due to its 2’OH that isn’t recognized by the discriminator amino acids
DNA polymerase forms a…
Pocket that fits the 3 phosphate groups of the incoming nucleotide
How does the DNA polymerase grip the primer-template junction?
Like a palm
Function of thumb domain in DNA polymerase (2)
- Maintaining the correct position of the primer and the active site
- Maintaining a strong association between DNA polymerase and its substrate
Function of palm domain in DNA polymerase
Catalytic site and monitor base pairing of the most recently added nucleotides
- Metal ions are present in the palm and are important in making the 3’OH nucleophilic
Function of fingers domain in DNA polymerase
Bind and enclose dNTP with the catalytic metal ions in the palm
What component of the DNA polymerase proofreads newly synthesized DNA?
3’ to 5’ exonuclease
Difference between endonuclease and exonuclease
Endonuclease: cuts DNA in the middle
Exonuclease: cuts DNA at the ends and can chow through the strands towards the middle of the DNA molecule
Helicase
Unwinds DNA at the replication form to create ssDNA template for primase
Primase
A specialized RNA polymerase that makes short RNA primers used to anneal to an ssDNA template to serve as a starting point during DNA replication
RNase H
Removes RNA primers from RNA:DNA hybrid to complete DNA synthesis
- DNA polymerase fills the gap and DNA ligase repairs the nick
What are the three essential DNA pol in eukaryotes?
- Pol α
- Pol δ
- Pol ε
Pol α function
Primer synthesis during DNA replication
Pol δ function
Extend lagging strand
Pol ε function
Extend leading strand
DNA pol __ has relatively low processivity in comparison to DNA pols _______
DNA pol α has relatively low processivity in comparison to DNA pols δ and ε
Polymerase switching
Replacement of DNA pol α with DNA pol ε at the leading strand and pol δ at the lagging strand
Sliding clamp protein
Keeps the polymerase in close proximity to the DNA template which increases the processivity of the DNA polymerase
Origin of replication
Sites at which DNA unwinding to initiate synthesis
Replicator
The DNA sequence that direct the initiation of DNA replication
- Binding sites for the initiator protein
- AT-rich DNA sequence that unwinds easily
Initiator protein
Sequence-specific DNA binding protein involved in the initiation of replication
Replicon
The DNA replicated from a particular origin of replication
Origin Recognition Complex (ORC)
6 protein complex that recognizes replicators
- Required to recruit other replication proteins, such as DNA helicase, to the replicator
True or false: ORC:DNA interactions require ATP hydrolysis
True
True or false: ORC binding leads to strand separation
False
On S. cerevisiae, B1 and A make up the…
ORC binding site
On S. cerevisiae, B2…
Facilitates DNA unwinding when the ORC binds
Describe how replicators are activated to initiate replication, and then how they prevent multiple replications (4 steps)
- Activation of replicator 3 and 5
- Replicator 2 and 4 are passively activated by extension of origin 3 and 5
- Replicator 1 activates independently
- Each origin becomes inactive after it is replicated to prevent multiple replications.
Describe the temporal control of the initiation of replication in eukaryotic cells (2 things)
- Replicator selection (G1): process to identify sequences that will direct the initiation of replication
- Origin activation (S): Triggers the replicator associated complex to initiate DNA unwinding and DNA pol recruitment
When does helicase loading occur? What about replicator/origin activation?
Helicase loading: G1
Receptor/origin activation: S
What are the 7 steps involved in helicase loading?
- Recognition of the replicator by the eukaryotic initiator, ORC.
- ORC recruits helicase loading proteins (Cdc6 and Cdt1) and Mcm2-7 helicase in the expense of ATP (head-to-head binding of Mcm2-7
- The assembly of this pre-replicative complex does not unwind the DNA or initiate replication after cells enter the S phase. - 2 kinases, CDK and DDK, phosphorylate Sld2/3 and Mcm2-7, respectively
- Formation of a complex with phosphorylated sld2/3 as well, but these molecules leave and result in the formation of CMG (Cdc45/Mcm2-7/GINS) complex
- The leading strand DNA pol ε is recruited before DNA unwinding
- DNA begins to unwind (Mcm2/7 subunits start separating and going opposite directions)
- DNA pol α/primase and DNA pol δ are recruited after DNA unwinding
Helicase structure before activation
Helicase encircle dsDNA as head-to-head double hexamer
Helicase structure after activation
Helicase encircle ssDNA as a single hexamer (and travel in opposite directions)
How does CDK activity regulate eukaryotic helicase activation?
- Low CDK activity is required for the loading of eukaryotic DNA helicase
- High CDK activity is required to initiate DNA replication (activation of helicase)
- After S phase, all loaded Mcm2-7 complexes will be removed from the DNA
G1 in terms of helicase activity
Loading phase
S in terms of helicase activity
Activation phase
CDK levels in G1 and its impact
In G1, CDK activity is low and helicase is loaded, BUT cannot be activated
CDK levels in S and its impact
In S, CDK activity is high and the helicase is activated to initiate DNA replication
What happens to used helicase after DNA replication?
Used helicase is disassembled
What happens to CDK levels in S, G2 and M? Why?
CDK activity remains high is S, G2 and M to prevent loading new helicase; thereby restrict one DNA replication/cell cycle
(Circular/Linear) DNA can be completely replicated
Circular
After circular DNA replication, the new DNA and the parental DNA are linked together (catenation). What separates the two daughter DNA molecules?
Topo II
Describe the “end replication problem” of linear chromosomes
- Because a primer is needed for the lagging strand, a little bit of DNA with be lost from the end of the chromosome during each replication cycle.
- Shortening of one of the two daughter DNA molecules is resulted from each round of DNA replication.
- Slowly disrupt the complete propagation of the genetic material from generation to generation
What are the 2 possible solutions for the end replication problem?
- To use a protein, instead of an RNA, as the primer for the last Okazaki fragment at each end of the chromosome.
- Use of telomere sequence
Describe the use of a priming protein instead of a primer to solve the end replication problem
- Used for some linear bacterial chromosomes, and some viruses
- An OH of an amino acid substitutes for the 3’-OH of an RNA primer
- The protein allows priming at the last nucleotide; therefore, no sequence is lost.
- The protein binds to the 3’ end of the lagging template strand (5’ end of the lagging strand itself)
Describe the use of a telomere sequence to solve the end replication problem
- Head-to-tail repeats of a TG-rich DNA sequence (NOT TA-RICH)
- Serves as an origin of replication for 3’ end of each chromosome
- Use telomerase as the DNA polymerase
- Telomeres shorten instead of DNA sequence
Describe the structure of telomerase
- A ribonucleoprotein complex that consists of:
1. An RNA subunit: serves as a template and no exogenous template is needed
2. Reverse transcriptase subunit: A DNA polymerase that uses the RNA subunit as a template.
What are 4 similarities between telomerase and DNA polymerase
- Require a template to direct nucleotide addition
- Extend at 3’OH end
- Use the same nucleotide precursors
- Act in a processive manner
What are 4 differences between telomerase and DNA polymerase?
- Telomerase has an RNA component
- Telomerase does not require an exogenous template (because the RNA itself is a template)
- Telomerase is able to use ssRNA substrate to make ssDNA (i.e. able to reverse transcribe)
- Telomerase has RNA:DNA helicase activity to displace its RNA template from the DNA for repeated rounds of synthesis
Which solution to the end replication problem is used by most eukaryotes?
Telomerase
True or false: repetitive telomeric DNA is protein coding
False
- Repetitive telomeric DNA is non-protein coding; thus, the extra sequence does not interfere with the cellular functions
Functions of telomere-binding proteins
Longer telomeres inhibit telomerase activity by recruiting telomere-binding proteins
- Telomere-binding proteins also protect the chromosome ends from recombination and chromosome fusion (repair processes that can fix the break)
Cdc13 function
Telomerase recruitment
Rif1 and Rap1 function
Inhibits telomerase activity
POT1 function
Inhibits telomerase activity
Shelterin
Protein complex that protects telomeres from DNA repairing enzymes
What proteins make up shelterin? (6)
- POT1
- Rap1
- TPP1
- TIN2
- TRF1
- TRF2
Telomere structure looks very similar to the ends of chromosomes that have been accidentally broken. How to telomeres prevent inappropriate attempts to fuse or repair the telomere?
Human telomeres form t-loop structures to prevent DNA repairing.
