DNA replication Flashcards
the central dogma of biology
DNA to RNA = transcription
RNA to protein = translation
importance of DNA replication
- ensures an exact copy of the species’ genetic info is passed from cell to cell
- if DNA failed to replicate itself meiosis and mitosis would pause
features of DNA structure
- right-handed and asymmetrical
- complimentary base pairs (A with T and G with C)
- 10 base pairs per turn
- 0.34 nm between stacked basses and 3.4 nm per helical turn
- anti-parallel 5’ to 3’ strands
why do AT and GC pair
- the space between ladders can only fit 3 rings
- A and T are able to make 2 H-bonds
- C and G are able to make 3 H-bonds
semi-conservative copying mechanism
- after one round of DNA replication each DNA contains 1 parental and 1 newly-synthesized strand
conservative replication
- after first replication one new and one parental strand
- after second replication one parental and 3 new strands
dispersive replication
nothing is conserved
- after first replication 2 half strands
- after second replication 4 half strands
semiconservative replication products
- after first replication 2 half strands
- after second replication 2 half strands and 2 new strands
Meselson and Stahl experiment
used cesium chloride equilibrium-density gradient centrifugation to separate double-stranded DNA molecules of different densities
- heavier DNA sediments near bottom and lighter ones near the top
- concluded that DNA replication in E. coli is semiconservative
Theta replication
- replication that occurs in most circular DNA (bacterium)
- replication is bidirectional
- unwinding at replication origin produces single-stranded templates (making a replication bubble) with replication forks at the end proceed around the circle
- eventually 2 circular DNA molecules are produced
rolling circle replication
- occurs on the F factor of some viruses
- replication is unidirectional
- break in a nucleotide strand causes DNA synthesis to begin at 3’ end of the broken strand, inner strand is used as the template, 5’ broken end is displaced
- cleavage releases a single-stranded linear DNA (which may circularize) and double-stranded circular DNA
- products are multiple circular DNA’s
Linear chromosome replication
- occurs in the linear chromosomes of eukaryotic cells
- replication is bidirectional
- numerous origins of replication where DNA unwinds producing a replication bubble
- DNA synthesis takes place on both strands at each end of the bubble, replication fork proceeds outward and eventually reaches another where DNA segments will fuse
- product is 2 identical linear DNA
RNA primase
helps DNA polymerase
- synthesizes a bit of RNA where you want DNA polymerase to start and contains an -OH group
DNA replication requirements
- magnesium (Mg2+)
- DNA dependent DNA polymerase
- 4 dNTPs
- a template DNA to be copied
- an RNA primer (provides 3’-OH end to initiate DNA synthesis by DNA polymerase)
features of DNA replication
- always synthesized 5’ to 3’
- ## newly synthesized strand is complementary and anti-parallel to parent strand
how is new DNA synthesized from dNTPs
- 3’-OH group of the last nucleotide on the strand attacks the 5’ phosphate of the incoming dNTP
- two phosphates are cleaved off and a phosphodiester bond forms between the 2 nucleotides
- the last incorporated nucleotide bonds with the alpha phosphate of the incoming nucleotide
DNA chains and nuclease cleavage
- single and double-stranded DNA is susceptible to cleavage by nucleases
- chain cleavage can leave the alpha phosphate group attached to the 5’ or 3’ carbon of the sugar
leading strand
- continuous DNA synthesis
- helices and polymerase are moving in the same direction so the polymerase doesn’t need to pause
lagging strand
- discontinuous DNA synthesis
- made in segments since the polymerase needs to wait for another section of the DNA to get unwound by helicase
DNA synthesis on leading and lagging strands
- DNA synthesis proceeds 5’ to 3’, same direction as unwinding
- on lagging strand, synthesis proceeds in the direction opposite of unwinding and runs out of template. it starts again, each time proceeding away from the replication fork which creates Okazaki fragments
- on the leading strand, synthesis proceeds in the same direction as unwinding making one long strand
replication fork
where the helices unwinds the DNA and the polymerase follows it
what makes the rolling circle model of replication different from the others
- replicates one strand at a time and doesn’t make a replication bubble
- no lagging strand because polymerase and helicase go the same way
origin of replication: theta model
- DNA unwinds at the origin
- at each fork synthesis of the leading strand proceeds continuously
- synthesis of the lagging strand proceeds discontinuously
origin of replication: rolling circle model
- continuous DNA synthesis begins at 3’ end of broken nucleotide strand
- as the DNA molecule unwinds, the 5’ end is progressively displaced
origin of replication: linear eukaryotic
- at each fork the leading strand is synthesized continuously in the same direction of unwinding
- the lagging strand is synthesized discontinuously in the direction opposite of unwinding
proteins involved in prokaryotic DNA
- one 13-base pair tandem sequence (AT rich - easier to separate than GC)
- four 9-base pair initiation protein binding sites
priming of oriC
- initiator proteins (DnaA) bond to ori C, the origin of replication causing small stretch of the DNA to unwind
- unwinding allows helicase and other single-stranded-binding proteins to attach to the single-stranded DNA
DNA helicase in prokaryotic replication
- helicase unwinds the DNA 5’ to 3’ and travels on the lagging strand breaking H-bonds and moving the replication fork
- unwound single-stranded DNA is coated with single-stranded binding protein to keep it single-stranded
true or false: no DNA has free ends
true
DNA gyrase
-released the stress caused by helicase by cutting one strand and letting it unwind a bit and wind back up again so it doesn’t create tension as the replication bubble gets bigger
what happens when there is more positive supercoiling
more resistant the strand becomes yo being unwound
DNA primase
synthesizes short RNA primer that provides the 3’-OH end for DNA polymerase to begin synthesis
- leading strand started first followed by lagging strand
5 DNA polymerases in E.coli
1 and 3: chromosomal DHA replication
2,4 and 5: DNA repair functions
replicative polymerases in prokaryotes
Pol 1:
- aids removal of RNA primers
- short tract synthesis
- has 5’ to 3’ and 3’ to 5’polymerase and exonuclease activity
- proofreading 5’ to 3’ exonuclease activity
Pol 3:
- main replicative polymerase
- highly processive (synthesizes lots of reactions without pausing)
- has 5’ to 3’ polymerase activity
- proofreading 5’ to 3’ exonuclease activity
beta sliding clamp
a ring-shaped polypeptide that encircles the DNA and interacts with DNA polymerase 3 to enhance processive DNA synthesis
- helps keep polymerase on the DNA
lagging strand and DNA polymerase
when Pol3 reaches 5’ end of the RNAprimer Pol3 is swapped for Pol 1
- Pol1 removes the RNA primer and resynthesizes a short tract DNA
Elongation of DNA during replication in prokaryotes
-Pol 3 completes one Okazaki fragment then moves to initiate DNA synthesis of a new one
- in the mean time Pol 1 exchanges places with Pol 3 to remove RNA primers
- DNA must form a loop so both strands can replicate simultaneously
All proteins involved in prokaryotic DNA replication
- topoisomerase (gyrase)
- helicase
- single-stranded binding protein
- DNA primase
- DNA polymerase III (and beta clamp)
- DNA polymerase I
- DNA ligase
What makes eukaryotic chromosome replication unique?
- shorter RNA primers and Okazaki fragments
- replication only occurs in the S phase
-multiple polymerases (at least 15) - bidirectional replication from multiple origins on each chrom.
- nucleosomes that need to be removed from parental DNA and re-assembled onto new DNA
-telomeres: shorten at each round
telomeres
protect the ends of chromosomes from degradation
what is the problem with telomeres and eukaryotic DNA replication
- the chromosome end will be degraded causing chromosome shortening every round
- we can’t put a primer right at the end of DNA since telomere is there and primate needs to sit on chromosome
how does replication happen on linear DNA with telomeres
- the terminal primer is positioned 70-100 nucleotides from the end of the chromosome which leaves a gap that is not replicated
