Significance of DNA replication
- Is a fundamental process
- Of interest, as things do go wrong during it
- Viral replication can be useful to understand as it can be a potential target
Model of DNA replication
-Is found to be semiconservative (new products have 1 newly syn. strand and 1 old strand)
Replication of DNA in bacteria
- why done
- how it works for E. coli
-Bacteria smaller, easier to grow
- replication begins at Origin of replication; replication forks move bidirectionally around circular chromosome
- at replication fork, there is a complex of proteins -> are responsible for carrying out replication
Replication of Eukaryotic DNA
-enzyme responsible
- Similar concepts to prokaryotic
- Eukaryotes differ in that they have >1 origin of replication w/ forks moving away from this origin
- bidirectional replication holds true for all cellular systems
- DNA polymerase responsible for synthesising DNA
- DNA poly 1 first identified (is the one that synthesises DNA)
- adds to 3’ end of the primer
DNA polymerase
- what it needs, direction of synthesis
- How lagging strand is synthesised (and by what)
- Needs a primer to add bases to
- synthesises in a 5’ to 3’ direction
- 2nd strand is synthesised as a series of fragments (called Okazaki Fragments) that are subsequently drawn together
- DNA polymerase adds to RNA primers that are synthesised by primase (is an enzyme part of complex at replication fork)
- DNA polymerase 3 responsible for extending RNA primers (fills in the spaces)
RNA primers in DNA replication
- how they’re synthesized
- How they are removed - what happens
- Are produced by de novo synthesis -> is very error prone (NOT GOOD)
- are replaced with DNA
- After DNA poly 3 fills in gaps between primers, it dissociates
- DNA poly 1 binds to the bap between the DNA and RNA primer
- DNA poly 1 has a 5’ to 3’ exonuclease that allows it to degrade DNA or RNA in front of it - fills in behind it
- this action removes RNA and replaces it with DNA
- DNA poly 1 has a 5’ to 3’ exonuclease that allows it to degrade DNA or RNA in front of it - fills in behind it
- gap is sealed by DNA ligase
Processivity
- Is the number of bases added to a primer before the enzymes disassociates
- Differs between types of polymerases
- DNA poly 1 has processivity of 15-20 (about the length of RNA primer)
- DNA poly 3 holds DNA in place - is a very big subunit
- DNA poly 1 is a single monopeptide unit
Separating the DNA strands
- why need to separate
- how can be done
- need to separate the strands to replicate
- can be achieved by supercoiling -> usu allows the DNA to fit inside the cell
- super coiling with right twist = condense
- super coiling with left twist = separation of DNA strands
- can have molecules that are half condense and half relaxed (are enzymes that can reach the same effect)
Topoisomers -> what they are and what they do
Helicases -> also what they do
*Enzymes that introduce supercoils or relax supercoils in DNA
Helicases: enzymes that disrupt the H bonds that hold the two strands of the double helix together (responsible for ‘unzipping’ the DNA molecule)
DNAa protein
- what it does and effect it causes
- another factor that enables it do its job more easily
-what happens once job is done
- is a critical protein in DNA replication
- Binds to specific seq. at origin of replication
- DNA monomers start binding to each other and cause bending and twisting of DNA at that position
- Denaturation occurs at this site
- DNA monomers start binding to each other and cause bending and twisting of DNA at that position
- This site is also AT rich = more easily denatured (2 H bonds instead of 3)
- Once area denatured, other proteins come in (i.e. DNAb protein) and drives the process of DNA replication
DNAa protein, DNAb protein, DNAc protein
- > what they do and how they interact
- DNA polymerase 3 - what is it’s role?
- DNAg Primase
- DNAa protein binds to specific sequence (DnaA box - are 5 at origin) that unwinds the origin
- additional DnaA proteins bind to newly unwound single-stranded regions - DNAb protein drives the process of DNA replication (are helicases that unwind DNA)
- are 2 monomers of DNAb (one at each replication fork) - DNAc protein helps DNAb bind to DNA
- DNA polymerase 3 = major enzyme to replicate DNA (need monomers/complexes - one for each strand)
- DNAg primase = synthesises RNA primers
Replisome assembly -> overview
-DnaA’s job
- Assembly of replisome = orderly process that begins at precise sites on chromosome called Origins
- after unwinding begins after the binding of DnaA proteins, additional DnaA proteins bind to single stranded region
- 2 helicases now bind and slide in a 5’ to 3’ position to begin unzipping helix at replication fork
- Primase and DNA polymerase 3 holoenzyme recruited to replication fork by protein-protein interactions & DNA synthesis begins
*DnaA job = bring the replisome to the correct place in the chromosome for initiating replication
Components of Replisome (6)
-what is not included
- DNAb
- DNA polymerase 3
- DNAg primase
- DNA polymerase 1
- DNA ligase (seals gaps in strand)
- Single strand binding (SSB) protein
*DNAa is not included because it just aligns replisome to right spot
Role of SSB protein - why is it needed
- As DNA/RNA doesn’t like to be single stranded, it will H bond with itself
- In region of replication fork where there is s.s. DNA, it will form loops (cruciform loops)
- in replication, DNA polymerase tends to miss these structures are therefore hotspots for mutations - SSB binds to DNA & keeps it in extended form for replication and prevents intrastrand base pairing
- decreases mutations/lost DNA
The replisome - functions of parts;-
clamp proteins
helicase
DNAg primase
SSB proteins
*what happens to rest of DNA molecule in front of helicase??
- Clamp proteins: hold DNA polymerase in place on DNA
- helicase unwinds strands
- DNAg primase synthesises small RNA primers
- SSB proteins binds to s.s. DNA
*rest of DNA molecule gets knotted up infront of helicase -> is another topoisomerase that unknots the DNA before the helicase gets to it
Movement of replisome
-term for end of ‘cycle’
- Replisome moves around the circular chromosome until reaches point opposite the origin = TERMINUS
- at this point, the linked circles are separated by a topoisomerase
- the separated circles then migrate to opposite poles of the cell
Replication in Eukaryotes -> how it is different
How to get rid of RNA primers and replace it with DNA? (Overview)
- Eukaryotes have linear molecules
- multiple ORI
- If RNA left, will get degraded in cell
- to get rid of RNA primer, can use 3’ end of chromosome (are repeats of a short sequence motif = telomeres)
- may be 10^5 to 10^7 telomeres added to the chromosome
- in human somatic cells, they are not replaced (gets shorter after every round of division (other species, telomerase replaces these repeats)
- may be 10^5 to 10^7 telomeres added to the chromosome
What structures allow RNA primers to be replaced with DNA
- what these structures provide
- what telomeres do -> an absence of telomeres
- Hairpin structures allow RNA primers to be replaced w/ DNA
- telomeres at each end of chromosome (they also protect chromosome from degradation by nuclases)
- Hairpin loop provides 3’ end where DNA polymerase can bind and replace RNA primer
-Progeria: Where people age very quickly due to having very short telomeres
Another way to replicate the ends of the chromosome
-and what organism usually uses this
-Use protein primers
-DNA polymerase is able to bind to cytosine and initiate synthesis of the complementary strand
(Cytosine is attached to a protein that binds to the template)
*these are mainly used in viruses
Why is DNA replication linked to cell division?
- Replication of DNA followed by cell division
- each daughter cell needs a copy of the chromosome
- variation in chromosome number is not acceptable
- process of cell division must be linked to the process of DNA replication
Cell cycle (4 phases)
G1 -> S -> G2 -> M
G1: interphase (cell growth) - need enough cell components for cell division to occur and support 2 daughter cells
S phase: DNA replication
G2: Recovery from DNA replication - is where the cell checks the newly replicated DNA for mistakes (as replication occurs v. quickly & genomes can be huge)
-if too many mistakes, cell undergoes apoptosis
M phase: where mitosis occurs
Result of having same cell cycle operating in each cell
-Each daughter cell has a copy of the genome
Cell cycle in bacteria (Overview)
- bacteria are roughly similar, but less complex (don’t have spindle fibres, intracellular membrane systems etc)
- therefore divide by binary fission
- they grow, replicate dna and then segment DNA for 2 cells (chromosomes attach to membrane of cell - when new membrane grows at centre of cell, it pushes cell’s genome apart)
Regulation of the cell cycle
- how it is usually balanced
- what unregulation leads to
- what we have learnt from this
- Eukaryotic cells do not divide continuously - process is regulated so that organs are the correct size
- cell division & apoptosis is in constant state of equilibrium
- Unregulated cell division = cancer
- studying cancer cells have led to the identification of many cell cycle regulatory proteins
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DNA & RNA11
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Genome Organisation13
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Laboratory Techniques & Theory25
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Proteins8
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Polysaccarides & Lipids8
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Viruses6
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DNA sequence databases6
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Gene Expression - Transcription19
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RNA Processing10
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RNA Translation13
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Gene Regulation35
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Development11
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Signal Transduction16
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Apoptosis5
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DNA Replication29
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Mutation and DNA Repair30
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Recombination33
