L6: Replication of DNA Flashcards
what are the substrates for DNA synthesis?
1) primer-template junction
2) dNTPs
substrates for DNA synthesis - primer-template junction
- provides a 3’-OH group to facilitate nucleotide addition
- needed bc enzymes which catalyze DNA synthesis cannot initiate strand synthesis de novo (on their own)
substrates for DNA synthesis - dNTP
- cleavage of phosphate groups provides energy for DNA catalysis
- has a sugar, three phosphates (for energy), and a nitrogenous base
what are DNA polymerases?
- class of enzymes which catalyze the synthesis of DNA
- require a template and primer (3’-OH)
- synthesis DNA in the 5’-to-3’ direction and in a processive manner
DNA polymerases - active site
- facilitates the addition of a correct nucleotide
- specific for correctly base-paired dNTP
- catalysis is far less efficient for incorrectly base-paired dNTPs or rNTPs
- resembles a hand that grips primer-template junction
DNA Polymerases - Active Site Specificity
DNA polymerases ensures active site specificity when incorporating nucleotides during DNA replication
DNA Polymerases: Active Site Specificity - correct dNTP
- it forms the proper base pair with the template strand
- 3’ OH group of the primer is positioned correctly
DNA Polymerases: Active Site Specificity - incorrect dNTP
- it does not form a proper base pair with the template strand
- This mismatch prevents proper alignment in the active site.
- polymerase cannot catalyze the reaction, blocking the addition of the nucleotide
DNA Polymerases: Active Site Specificity - rNTP
- a ribonucleotide present instead of a deoxyribonucleotide
- 2’-OH group preventing proper positioning with the active site
- steric gate prevents rNTPs from being incorporated into DNA
- ensures that only dNTPs (not rNTPs) are used for DNA synthesis.
DNA Polymerases: Active Site - resembles a hand
- fingers enclose DNA when correct base-pair is in place
- thumb interacts with negative charges and structure of phosphate backbone
- palm forms hydrogen bonds with minor groove
DNA polymerases - what is processivity
number of monomers (nucleotides) added by an enzyme to a growing polymer (nucleic acid) each time it binds
DNA polymerases: processivity - why is it important
- Every time the DNA pol get on DNA, synthesizes millions of nucleotides before falling off
- Important bc binding is slow and synthesis is fast so DNA pol only wants to do the binding step once
DNA polymerases - high vs low processivity
- high: makes millions
- low: jump on, make one/two, then fall off
- increased processivity correlates with faster DNA synthesis
DNA pol - proof-reading capacity
many have 3’ exonuclease activity to remove incorrectly incorporated base pairs at end of strand
DNA pol: proof-reading ability - exonuclease vs endonuclease
- endonucleases – cut within DNA strands,
- exocnulease - cuts nucleotides from the end of a DNA strand
DNA pol: proof-reading capacity - how is it done?
- error detection
- removal of mismatched nucleotide
- resumption of DNA synthesis
DNA pol: proof-reading ability - error detection
- DNA pol detects a mismatched base at the 3’ end of the growing strand (near the thumb)
- Instead of continuing synthesis, the DNA pol shifts the strand to the exonuclease active site (in the palm)
DNA pol: proof-reading ability - removal of mismatched nucleotide
3’ to 5’ exonuclease activity removes the incorrect nucleotide from the strand (in the palm)
DNA pol: proof-reading ability - resumption of DNA synthesis
- DNA pol moves the strand back to its polymerization active site
- DNA synthesis resumes
what is the Replication Fork
- junction between separated DNA template strands
- forms because both DNA strands are replicated simultaneously
replication fork - what problem does this create?
- DNA is always synthesized 5’-to-3’
- causes one strand to be synthesized in fragments
replication fork - leading vs lagging strand
- Leading DNA strand is synthesized continuously
- Lagging DNA strand is synthesized in a discontinuous fashion and leads to the formation of Okazaki fragments
what are the proteins at the replication fork?
- primase
- helicase
replication fork proteins - primase
- an RNA Pol that can initiate polymerization de novo (on its own)
- forms short RNA primers (3’ -OH group) to “prime” DNA Pol activity
- primers must be removed to complete DNA synthesis
replication fork proteins: primase - lagging vs leading strand
- leading DNA strand: requires only one primer
- lagging DNA strand: requires multiple primers (one for each Okazaki fragment)
replication fork proteins: primase - primer removal
- Rnas H
- DNA Pol fills in the spaces
- DNA ligase repairs final nick to make the strand continuous
replication fork proteins: primase - Rnas H
- cleaves bonds between two ribonucleotides
- therefore removes all rNTPs except last one bound to DNA, which is removed by a 5’ exonuclease
replication fork proteins - helicase
- its a ring-shaped hex dimer
- uses ATP to catalyze the separation of DNA strands
replication fork proteins: helicase - what problem does it create?
- single stands can perform intramolecular base pairing
- prevents DNA Pol from synthesizing DNA
helicase problem - what is the solution?
- single-stranded DNA-binding proteins (SSBs)
- cooperate bind and stabilize the separated DNA strand
- ssDNA is held in an elongated state suitable for replication
What problem does unwinding of the DNA strands create?
- positive supercoiling is created upstream of the replication fork
- prevents DNA from unwinding further
positive supercoiling during DNA separation - how is this solved?
- topoisomerase
- relieves positive supercoiling of dsDNA upstream of the replication fork
- it cleaves both DNA strands, moving them around, and reseals them to create negative supercoiling upstream
topoisomerase: positive supercoiling -> negative supercoiling - why is it important for this to happen
- positive supercoiling prevents replication
- negative supercoiling promotes replication bc it can be used as energy
what are the main DNA Pol in E. coli?
- 5 DNA Pol:
1. DNA Pol III
2. DNA Pol I
3. 3 other DNA Pol involved in DNA repair
what are the main DNA Pol in E. coli? - DNA Pol III
- primary enzyme in chromosome replication
- processive and has proof-reading capacity
what are the main DNA Pol in E. coli? - DNA Pol I
- has 5’ exonuclease activity to remove RNA-DNA linkage resistant to RNase H cleavage
- synthesizes DNA in its place
- not highly processive, but has proof-reading capacity
what are the main DNA Pol in eukaryotes?
- 3 essential to replicate the genome:
1. DNA Pol α (alpha)/primase
2. DNA Pol ε (epsilon)
3. DNA Pol δ (delta)
4. other DNA pol: DNA repair
DNA Pol in eukaryotes - DNA Pol α (alpha)/primase
- primase synthesizes RNA primer
- DNA Pol α initiates DNA synthesis
- has low processivity, so is replaced by other DNA Pol
DNA Pol in eukaryotes - DNA Pol ε (epsilon)
synthesizes leading strand
DNA Pol in eukaryotes - DNA Pol δ (delta)
synthesizes lagging strand
what is the sliding clamp?
- doughnut-shaped complex that binds to DNA Pol and increases processivity
- it encircles DNA but leaves space for water molecules to facilitate smooth sliding
- placed onto DNA via sliding clamp ladder
sliding clamp - why is it important?
- w/o clamp: DNA Pol dissociates after 20-100 bp synthesized
- w/ clamp: DNA Pol dissociates after thousands-millions bp synthesized
what does DNA pol and the sliding clamp do upon reaching dsDNA
- DNA Pol undergoes a confirmational change that reduces affinity for the clamp
- the sliding clamp is left behind to recruit proteins (like those for Okazaki fragment repair)
DNA synthesis at the replication fork
- holoenzyme
- trombone model in E. coli
DNA synthesis at the replication fork - holoenzyme
a multiprotein complex in which core enzyme activity is associated with additional components that enhance function
holoenzyme - DNA Pol III holoenzyme
- a holoenzyme that has these components:
1. three DNA Pol III enzymes
2. one sliding clamp loader with three τ (tau) proteins
DNA synthesis at replication fork - trombone model in E. coli
- helicase is linked to the lagging strand and separates DNA
- one DNA Pol III works continuously on leading strand
- lagging strand is “spooled out” before Primase synthesizes the primer
- sliding clamp is loaded onto lagging strand and DNA Pol III unit begins
- DNA Pol III reaches preceding Okazaki fragment, dissociates (leaving sliding clamp) and is recruited to next lagging strand primer
initiation of DNA replication
- origin of replication
- replicator
- initiator
initiation of DNA replication - origin of replication
physical site at which DNA unwinding and replication is initiated
initiation of DNA replication - replicator
- cis-acting DNA sequences that directs replication initiation
- usually AT-rich and therefore unwinds readily
initiation of DNA replication - initiator
- trans-acting protein that binds to replicator and initiates replication
- humans: ORC, E. coli: DnaA
initiation of DNA replication - difference between cis-acting and trans-acting
- cis-acting: bound to the DNA
- trans-acting: can move around and binds to the cis-acting protein
initiation of DNA replication in E. coli
- DnaA binds
- DnaC places DnaB on ssDNA
- primase is recruited
- replication bubble formed (only one)
initiation of DNA replication in E. coli - DnaA binds
- DnaA initiator protein directly binds 9-mer replicator sequences
- when associated with ATP, DnaA induces strand separation at 13-mers
initiation of DNA replication in E. coli - DnaC places DnaB on ssDNA
- DnaC: DNA helicase loader
- DnaB: helicase
initiation of DNA replication in E. coli - primase is recruited
primase is recruited followed by the assembly of DNA Pol III holoenzyme
initiation of DNA replication in E. coli - forms replication bubble
initiation of DNA replication in E. coli - in rapidly growing cells
- origin of replication reinitiate DNA replication before cell division
- results in some DNA being replicated twice
- allows fast division but means multiple rounds of replication are occurring simultaneously
- typically good for prokaryotes
initiation of eukaryotic DNA replication
- eukaryotic chromosomes replicate only once per cell cycle
- it is unable to reinitiate DNA replication before the cell divides
initiation of eukaryotic DNA replication - explain how DNA is unable to reinitiating DNA
helicase activity is tightly controlled:
- helicase loading occurs at G1 phase of cell cycle
- loaded helicase are only activated by two protein kinases
- these kinases are active at S phase of cell cycle
initiation of eukaryotic DNA replication - timing of helicase loading and activation
- allows only one round of DNA replication per cell division
- helicase loading is regulated by CDK (protein kinase) levels
initiation of eukaryotic DNA replication: timing of helicase loading and activation - CDK (protein kinase levels)
- decrease during G1, helicase is loaded but not activated
- increase during S-G2-M, inhibits helicase loaded and activates previously loaded helicase
end replication problem - telomeres
- region at the end of a eukaryotic chromosome
- does not contain genes
- instead contains short DNA repeats
end replication problem - replication of telomeres
- leading strand synthesis: continuous and complete
- lagging strand: discontinuous and shortened
end replication problem: replication of telomeres - why is this a problem?
- results in telomere shortening
- DNA gets shorter with every cell cycle
- it is fine if its just the telomeres but can effect DNA and genes
end replication problem - telomere shortening
- the replication fork will reach the end of the linear chromosome
- but the last RNA primer on the lagging strand has been removed
- the primase needs something to sit on but the DNA is too small so it cannot be primed or replicated
- ssDNA will be degraded or lost
solution to end replication problem of lagging strand
telomerase
solution to end replication problem of lagging strand - telomerase
- a novel DNA Pol that does not require an exogenous template
- it is a ribonucleoprotein with an RNA component (telomerase RNA or TER) that can act as a template
- it will then exploit head-to-tail repeats of TG-rich sequences at telomeric ends of linear DNA
- has a reverse transcriptase subunit (TERT) that synthesizes DNA
telomerase - how does it work?
- unreplicated strand has 3’ overhang
- telomerase binds to overhand and synthesizes DNA
- telomerase catalyzes repeated additions of the same shirt sequence to terminus
- DNA Pol can then replicate DNA and complete the lagging strand
telomerase - how does it relate to aging and gametes
- not all cells have telomerase and DNA gets shorter as we age
- gametes have high concentrations of telomerase (don’t want to pass shortened DNA to progeny)
