DNA Replication 1 Flashcards
central dogma of biology (3)
- genetic information moves to proteins
- once information is transferred to protein, it cannot be transferred back to RNA/DNA
- RNA and DNA can pass information back and forth and replicate
what are the challenges that need to be resolved to replicate DNA? (5)
- DNA copy must be accurate
- must be able to free DNA strands from protein and from each other
- must be repair/proof-reading mechanisms
- must be fast
- eukaryotes only: issue with replicating telomeres
telomeres
- the ends of linear DNA
semi-conservative
- describes how DNA molecules contain one strand from original parent DNA molecule and one from newly synthesized strand after replication
DNA polymerase (2)
- function
- what does it require
- enzyme that uses DNA as a template to make new DNA copies
- requires primer and magnesium 2+
DNA polymerase primer
- short strand oligonucleotide with free 3’-OH
what is magnesium’s function in DNA replication? (2)
- acts as a co-factor for DNA polymerase by coordinating dNTP in the active site
- aspartate forms ionic bond with magnesium in the active site
what direction does DNA polymerase catalyze the synthesis of new DNA?
- ONLY in the 5’ -> 3’ direction
fidelity
- the degree of exactness with which something is copied or reproduced
how does DNA polymerase achieve high fidelity (3)
- Mg2+ ensures proper orientation of dNTP
- geometry of active site ensures only correct base pairing can occur
- alpha-helix “lid” closes on active site when correct base pairing forms to trap dNTP so catalyses can occur
how much does magnesium, active site geometry and alpha-helix allow mistakes in DNA?
- allows a mistake every 10^4 - 10^5 nucleotides
how does DNA polymerase correct mistakes that do occur?
- 3’ -> 5’ exonuclease activity removes nucleotides one by one from DNA strand
what are the steps involved in DNA polymerase’s exonuclease activity? (3)
- if mismatch base is incorporated, DNA polymerase will shift to 3’ -> 5’ exonuclease site
- mismatch is removed
- DNA polymerase will switch back to 5’ -> 3’ active site and continue polymerase activity
what is a downside of DNA polymerase exonuclease activity and why is it still used anyway? (2)
- it is wasteful as it can remove pairings that are not mismatched
- still used because it does ensure high fidelity
how much does DNA polymerase exonuclease activity allow mistakes in DNA?
- allows a mistake every 10^6 - 10^8 nucleotides
what happens if the exonuclease activity fails to detect a mismatch?
- DNA repair enzymes can correct error after replication
what is the final error rate considering all of the mechanism used to ensure high fidelity in DNA replication?
- a mistake every 10^10 nucleotides
describe the E.coli genome
- circular genome with one oriC
oriC definiton
- origin of replication
oriC structure (2)
- 4-5 repeats of a sequence recognized by DnaA protein
- DNA unwinding elements (DUE)
DNA unwinding elements (DUE) (2)
- 3 tandem repeats (~13 nucleotides each) rich in A and T
- where first strand separation occurs due to weaker H-bonding (only 2 bonds) between A-T
replication fork
- point where DNA is being synthesized
- replication occurs bidirectionally: 2 replication forks per circular genome
does DnaA require ATP (2)
- yes, it required ATP
- ATP is a co-factor for DnaA because it is not hydrolyzed
DnaB (2)
- helicase: separates the DNA helix
- one DnaB per replication fork, so there are 2 DnaB proteins total during DNA replication
DnaC
- ATPase: it hydrolyzes 1 ATP to load DnaB
how does DnaB bind to the replication fork?
- with the help of DnaC, which hydrolyzes an ATP
what Dna proteins stay/leave the DNA helix? (2)
- DnaB stays in front of DNA polymerase in each replication fork to separate strands
- DnaC and DnaA dissociate from DNA after initiation
what enzyme is located in front of DnaB during DNA replication? (2)
- function
- topoisomerase, specially DNA gyrase
- remove supercoils formed by DNA unwinding the helicase/DnaB
ss binding proteins
- bind to ssDNA during DNA replication to prevent DNA renaturation
leading strand (2)
- synthesized continuously
- synthesized in direction that DNA unwinds
lagging strand (2)
- synthesized discontinuously in short stretches
- synthesized in the direction opposite to DNA unwinding
okazaki fragments
- short stretches of DNA synthesized on lagging strand
DNA polymerase I (3)
- where is it found
- different activities
- main function
- DNA polymerase found in E. coli
- has 3 activities: polymerase, proof-reading (3’ -> 5’ exonuclease), and 5’ -> 3’ exonuclease
- nick translation: using 5’ -> 3’ exonuclease it removes RNA primers and substitutes them with DNA
what does each synthesized DNA segment start with?
- RNA primer
what must be done before Okazaki fragments can be stitched together
- RNA primers must be removed
how are RNA primers removed form Okazaki fragments (2)
- DNA polymerase I 5’ -> 3’ exonuclease activity removes nucleotides in front of moving enzyme
- results in nick translation
nick translation
- due to removal of RNA primer by DNA polymerase I, ss break in DNA will be moved further down the strand
function of DNA polymerase I 5’ -> 3’ exonuclease activity (2)
- removal of RNA primers from Okazaki fragments
- DNA repair
DNA polymerase III (4)
- where it is found
- physical characteristics
- function
- physical features and characteristics
- DNA polymerase found in E. coli
- large protein with many polypeptide chains
- one part of enzyme synthesizes leading strand while another part synthesizes lagging strand at the same time (2 active subunits at the same time) using RNA primers as a starting point
- has high fidelity and processivity due to beta-clamp and clamp-loading complex
how does DNA polymerase III have high fidelity and processivity
- it can catalyze many reactions without releasing the substrate (DNA)
beta-clamp (2)
- made of 2 beta-subunits that can close on DNA to allow DNA polymerase III to have high processivity
- 1000 nt/s vs 10 nt/s in DNA polymerase I
clamp-loading complex
- δ-loader that can open beta-clamp and thread DNA through
what DNA polymerases are used in E. coli
- DNA polymerase I and III
what primase is used in E. coli for DNA replication
- DnaG
DnaG (2)
- primase used for DNA replication in E. coli
- synthesizes RNA primers (4-5 nt long) on both leading and lagging strand
describe discontinuous synthesis in E. coli DNA replication
- every 1000-2000 bp, DNA polymerase III releases lagging strand of DNA and binds it further down the replication fork where primase has synthesized a new primer
does clamp loading require ATP?
- yes, it requires 3 ATP molecules
DNA ligase (2)
- connects ss breaks and seals nicks between Okazaki fragments
- requires 1 ATP (2 ATP equiv) molecule per nick
ter sites
- special sequences in E. coli that are located opposite to the oriC
Tus protein
- bind to ter site sequences and “slow down” the replication fork
- 2 sets of Tus-ter complexes
role of topoisomerase in E. coli DNA replication
- topoisomerase IV resolves catenated DNAs after termination
what is happening during the same time as DNA replication in prokaryotes
- cell division
how does replication in eukaryotes differ from replication in prokaryotes (9)
- more complex and not fully understood
- larger genomes
- linear chromosomes
- movement of replication fork is slower
- presence of histones
- multiple origins of replication per chromosome
- okazaki fragments are shorter
- different main polymerase enzyme used
- primers made and removed by different enzymes
how much slower is the movement of the replication fork between eukaryotes and prokaryotes (2)
- why is it slower in eukaryotes
- 50 nt/s vs 1000 nt/s
- mistakes are more detrimental in eukaryotes
how much shorter are Okazaki fragments between eukaryotes and prokaryotes
- 100-200 nt vs 1000-2000 nt
what is the main enzyme of replication in eukaryotes?
- polymerase δ
what makes RNA primers in eukaryotes
- primase: polymerase alpha
what removes RNA primers from Okazaki fragments in eukaryotes?
- combination of RNase H and MF1
RNase H
- degrades RNA-DNA duplexes
MF1
- has 5’ -> 3’ exonuclease activity
what problem does DNA polymerase δ have with replicating telomeres
- DNA polymerase δ can only synthesize in 5’ -> 3’ direction, so DNA telomeres are left unreplicated after primer is removed
- ss overhang is degraded so DNA gets shortened
telomere
- linear chromosome ends
what is the solution to replicating telomeres?
- telomerase enzyme uses reverse transcription
telomerase (2)
- special enzyme in eukaryotes capable of reverse transcription
- can extend DNA ends by using RNA as a template
