Mark Isalan Flashcards
General organisation of DNA in Eukaryotes?
Eukaryotic DNA is Heavily organized
DNA highly condensed to form chromosome –> organization driven by histone complexes which are positively charged so that they can interact with the negatively charged DNA
In the highly supercoiled form the DNA is not accessible to transcription factors and other enzymes
General organisation of DNA in Bacteria?
Even though the Bacterial genome is less organised than eukaryotic DNA. bacterial genome is not organised randomly
DNA is still has heavily organised supercoiled circular nucleoids –> Refers to DNA is associated with several proteins such as Histone-like nucleoid structuring (H-NS) protein (positively charged) as well as other proteins such as transcription factors.
General Overview of DNA synthesis?
DNA synthesis - Occurs in 5’ to 3’ direction
Remember that DNA has polarity and is anti-parallel
5’ end has a free phosphate
3’ -OH has a free -OH
Synthesis of more DNA requires DNA polymerase (works in a 5’ to 3’ direction) which uses ATP, Mg++ and nucleotide triphosphates
During synthesis of the 3’ -OH performs a nucleophilic attack on the incoming deoxynucleotide triphosphate in order to add the base pair to the growing chain
Product - Add Nucleotide + Disphosphate released
Is DNA a stable molecule?
No clear answer
- In one sense yes, as it can last for 100’s of years in permafrost –> providing readable DNA
- In another sense no, as DNA is always very active –> replication, transcription, unwinding –> resulting in many sources of damage and error.
What are mutagens?
Mutagen –> Refers to a physical (i.e. U.V.) or chemical (i.e. Free radical species) agent that changes the genetic material
How frequently is DNA damaged in one cell per day?
DNA is damaged approximately 10 000 times per cell per day –> all of which act as a source of potential diseases/disorders
Hence, this explains why the cell devotes a lot of energy to prevent DNA damage
Two main impacts of DNA damage?
- Block replication and/or transcription
- Cause alterations in the genetic code (mutation) –> impact the organism itself or offspring if germline cells are mutated.
What are the two main types of DNA damage?
- Chemical alteration to DNA –> which may be exogenous** (source of damage located outside the cell - environmental mutants such as UV radiation) or **endogenous (internal source of damage - internally generated damaging agents such as hydroxyl radicals – most common)
- Spontaneous damage to DNA –> DNA reacts with itself Includes deamination (losing an amine group) and depurination (losing a purine base) –> these changes tend to occur regardless of what you do, meaning they are inevitable.
Outline how UV may lead to the formation of pyrimidine dimers (exogenous - chemical change).
Example of exogenous agent causing DNA damage
- UV light induces formation of pyrimidine dimers (T-T (most common) C-T and C-C) –> 2 adjacent pyrimidines are joined by a cyclobutane(4C) ring structure (Double C=C converted to single and remaining electron forms C-C bond with adjacent pyrimidine)
- Consequence - The bases no longer function as normal Watson and Crick base pairing –> no longer serve its function as a base - no information
This explains why Solar UV irradiation is the cause of most human skin cancer.
Outline how DMS/EMS (Exogenous) causes DNA damage.
DMS/EMS both are alkylating carcinogens
Alkylation is the addition of methyl or ethyl groups to various positions on the DNA bases
For example,
Alkylation of the O6 position of guanine results in formation of O6-methylguanine –> this changes the W-C base pairing potential. Normal G forms three H-bonds but this no longer occurs when carbon 6 has become methylated.
Is normal cellular DNA methylation a source of DNA damage?
No! Different to the alkylation due to DMS/EMS
Normal methylation from the cell is normally not carcinogenic
Takes place on CPG islands and effects the major groove whereas carcinogens disturb the Watson and Crick base pairing.
Outline how benzo-(a)pyrene (exogenous agent) can damage DNA.
Many carcinogens (e.g.benzo-(a)pyrene) react with DNA bases, resulting in the addition of large bulky chemical groups to the DNA molecule
Result? –> messing up the normal W-C base pairing –> Base is misread by DNA polymerase (as thymine)
Are many carcinogens activated endogenously?
Yes!
Many carcinogens are activated endogenously, become free radicals, by reactions with cytochrome P450 enzymes
P450-important in clearance of compounds – ‘molecular dustbin’ –> may result in the formation of reactive intermediates.
What are the two main types of Spontaneous Damage that occur in DNA?
A) Deamination of adenine, cytosine and guanine –> E.g. Amine to carbonyl –> change from hydrogen bonding potential (donor –> acceptor)
Deamination examples - Cytosine to Uracil and Adenine to Hypoxanthine –> changes the W-C base pairing potential.
(B) Depurination –> removal of purine group –> resulting from cleavage of the bond between the purine bases and deoxyribose, leaving an apurinic (AP) site in DNA.
Depurination - losing an entire base –> leaves sugar behind with empty –OH group –>
Note - same thing can happen with pyramidines (depyrimidination)
What does changed W-C base pairing potential mean?
Basically, DNA damage leads to altered base pairing - chemical change to base results in difference base preference (Non-WC base pairing) - when it gets replicated the wrong complement base gets included.
Example
Adenine deamination changes the C-NH2 on carbon 6 to C=O, which changes the molecule from adenine to Hypoxanthine.
Hypoxanthine behaves more like guanine therefore it preferentially binds to cytosine.
Thus, in the short term Hypoxanthine does not bond well with Thymine creating a bulge in the DNA.
In the long term, during DNA replication hypoxanthine will bind to cytosine –> resulting in a permanent mutation which may result in deleterious effects
What are common functional groups found in carcinogens?
Functional groups in carcinogens?
It can be a variety but all of them are reactive groups –> One common example would be aldehydes
Sometimes carcinogens aren’t even covalently reacting - e.g. intercalating agents sit in between the bases –> disrupting the DNA synthesis resulting in the wrong base being introduced –> e.g. ethidium bromide
What are the two general types of DNA repair mechanisms?
- DIRECT REVERSAL of chemical reaction responsible for DNA damage (in-situ) –> more common in bacteria
- EXCISION REPAIR –> Removal of damaged bases, replacement with newly synthesised DNA more common than direct repair in humans
Why? Because humans often lack some of the enzymes required for direct reversal
Outline the direct reversal of U.V. DNA damage in E. Coli?
- DNA damage via U.V. radiation
- Pyrimidine dimer created (cyclobutene rings)
- Photoreactivating enzyme uses photoreactivation (process driven by light as energy source) to break open cyclobutene ring to restore normal bases
How does the photoreactivating enzyme do this?
- Enzyme has a chromophore that can absorb light 300nm-500nm
- Transfers the energy to a non-covalently bound FADH-, which then transfer an excited electron to the pyrimidine dimer –> in turn splitting the ring.
- The now pyrimidine anion transfer its electron to FADH. (Radical) making the DNA good as new.
Note - Occurs in E. coli, yeasts, some plant and animal cells but NOT in humans
Outline how dealkylating enzymes can be used against the direct reversal of DNA damage.
Dealkylating enzymes –> removes alkylating group - enzyme has methyl acceptor group (–SH group)
- Enzyme comes in
- Finds bulge in DNA
- pulls of methyl group using –SH
- Base returns to its original form
- Enzyme needs to be regenerate using other proteins (reform –SH)
Specific Example
Alkylation - O6-methylguanine –> How can we fix?
- Can be repaired by enzyme: O6-methylguanine methyltransferase which is widespread in prokaryotes and eukaryotes
- Transfers the methyl group to its own cytosine residues -> reaction deactivates the protein which therefore cannot be strictly classified as an enzyme.
Types of Excision Repair?
- Base-excision repair –> Smallest type of excision - Base is removed leaving deoxyribose backbone intact so that another series of enzymes can add the correct base
2. Nucleotide-excision repair –> Nucleotide is removed leading to a gap in one strand (an oligonucleotide is usually removed) which is filled in by DNA polymerase
- Mismatch Repair –> Mechanisms for post DNA replication mismatches.
Outline how base excision repair is used to fix deamination of cytosine to uracil.
Base excision repair example - Convert Uracil back to Cytosine
- Uracil formed by deamination of cytosine, leads to a G:U mismatch –> results in DNA bulge
- Bulge is recognized by DNA glycosylase resulting in the bond between uracil and deoxyribose being cleaved by uracil DNA glycosylase –> leaves a sugar with no base attached in the DNA (an AP site)
- This site is recognized by AP endonuclease (cuts into intact DNA), which cleaves/nicks the DNA chain
- The remaining deoxyribose is removed by deoxyribose-phosphodiesterase –> leaves perfect gap for new nucleotide
- The resulting gap is filled by DNA polymerase and sealed by ligase - leads to incorporation of C opposite G.
Outline how nucleotide excision repair of thymine dimer (UV damage)
Nucleotide excision repair of TT dimer - Main mechanism in Humans
Note - This also occurs in many organisms or alternatively they use photo repair enzymes.
- Recognition of thymine dimers occurs by assembly of RPA, XPA and XPC-TFIIH at sites of damage - specificity is achieved mainly by the kinetic proofreading activity of TFIIH
- Helicase action by the XPD subunit of TFIIH generates a bubble around the dimer - creating the requisite DNA substrates for the structure-specific endonucleases XPF and XPG
- Cleave on both sides of the thymine dimer by 3’ and 5’ endonucleases creating nicks - XPF and XPG
- Oligonucleotide 24–32-nt in length (also known as the “canonical 30-mer”) dissociates from the duplex.
- The resulting gap is then filled by DNA polymerase and sealed by ligase (DNA pol I in E. coli/DNA pol β in human)
How does Nucleotide-excision repair differ between E. Coli and Eukaryotes?
-
E. Coli - Catalysed by 3 gene products – uvrA, B, C
a) UvrA recognises damaged DNA (Helix distortion),
b) UvrB and UvrC (endonucleases) cleave at 3’ and 5’ sides, excise 12-13 bases oligonucleotide
- UvrA, UvrB and UvrC form complex
c) uvrD (Helicase II) binds to the oligonucleotide segment in order to displace it –> allowing for DNA pol and DNA ligase binding.
- Mutations of these genes leads to high sensitivity to UV - Eukaryotes - Catalysed by RAD gene products in yeast (7 different repair genes involved – highly conserved) –> analogous to humans
Mutations lead to xeroderma pigmentosum rare genetic disorder, affects 1:250,000 people, extreme sensitivity to UV light leading to skin cancers - all of which is due to the deficient ability to repair DNA by nucleotide-excision.
In Mismatch repair in E. coli, how does the E. Coli strand distinguish between parental and new synthesized DNA strand?
Main idea - Mismatch repair system detects and excises mismatched bases in newly replicated DNA
Important characteristic is that this system must distinguish parental strand from newly synthesised daughter strand because you want to change the base in the new strand (not change genetic code on the parental strand)
Different species use different methods to accomplish this…
In E. coli DNA is methylated by Dam methylase (Adds methyl group to Adenine in GATC) –> following replication the newly synthesised daughter strand will not be methylated resulting in hemi-methylated DNA –> so the cell can recognize any bulge on any unmethylated new DNA.
Outline the key players in DNA mismatch repair in E. Coli
Mismatch repair system in E. Coli is called MutHLS
Three enzymes:
- MutS recognizes bulges
- MutL is a helicase that walks along DNA (looping it), until it finds a methyl group so it can recognizes parental from daughter,
- MutH is a endonuclease that cleaves and nicks the new strand.
How does this table below highlight the importance of DNA methylation on mismatch repair?
Dam methylation is essential for mismatched repair in E. Coli –> conditions for repair shown in table
Table highlights how methylation of the strands directs repair
What about the last two rows?
Used a virus (PhiX174) to probe this system
- This virus lacks GATC sites so it doesn’t get methylated or cleaved by enzymes in the E. Coli cell
- But when we introduce GATC sites we achieved directed repair of the new strand during replication
Outline the different steps of the MutHLS Mismatch Repair Mechanism in E.Coli.
MutHLS Mismatch Repair Mechanism
- MutS – moves along the DNA strand until it recognizes a mismatch (bulge)
- Recruitment of MutL and MutH
a) MutL (translocase) binds to MutS –> Translocase with ATPase activity and forms DNA loops, walking along DNA looking for hemi methylated Dam sites close to mismatch to allow for strand discrimination
b) MutH (endonuclease) can then bind to MutL which can guide the cleavage on the opposite side of hemi-methylation (allows for discrimination between New and Old)
Note - Cut site can be away from the mismatch (12-13 nucleotides) either upstream or downstream –> doesn’t matter as the entire region is removed (stochastic) –> the presence of the MutHLS complex indicates the presence of a mismatch
Once a nick has been made by the MutHLS complex, how is the DNA region containing the mismatch removed?
Once the cut has been made exonucleases are used to excise the mismatched region –> the kind of exonuclease will depend on the nick is made upstream or downstream of the bulge.
Either end up with an exonuclease eating away at the DNA..
- 5’ to 3’ direction - Nick is made upsteam –> Exo 7 or Recj
- 3’ to 5’ - Nick is made downstream –> Exo 1
The gap that is created filled by DNA Pol III and sealed by ligase.
Note - Exonuclease is used as we are chewing away from an end of DNA (nick)
In Mismatch repair in mammalian cells, how are the parental and daughter strands distinguished from eachother?
Instead of CH3 (methylation) groups you are looking for strand breaks
The newly replicated strand is distinguished from the parental strand because it contains strand breaks since eukaryotic DNA contains many replicons it will have many strand breaks due to…
- Leading stand –> no Okazaki fragments but there are multiple replication initiation points –> creating nicks that can be recognised at each replicon.
- Lagging strand –> presence of Okazaki fragments means that the daughter strand will have many breaks
WHat is the equivalent of the MutHLS system in Eukaryotes?
Eukaryotes –> MSH complex responsible for mismatch repair
MutS
MutL
Helicase
Exonuclease
In humans, what can mutations in hMsh2 and hMlh1 genes cause?
Mutations in hMsh2 and hMlh1 genes are a cause of inherited non-polyposis colorectal cancer
Affects 1:200 - Causes ~15% of UK colorectal cancers.
What are the two ways that a double stranded break in DNA can be repaired?
When double stranded breaks occur we can have one of two things happen:
- Non-Homologous end joining - NHEJ
- Homologous Recombination - H.R.
Note – Normally Mismatch repair is not used as this happens after replication whereas H.R. happens during.
How can double stranded breaks be created?
Double strand breaks can occur due to DNA damage
There are many factors that can cause a double stranded break (Physical/Chemical factors) - Frequently occur during DNA replication.
Note - Apart from Physical/chemical factors we also have nucleases that come in and cut DNA
What are the two common ways that double stranded breaks occur during DNA replication?
- DNA damage leading to a nick in the DNA backbone –> during DNA replication DNA polymerase action is forced to stop –> thus creating a gap or dsbreak in the newly synthesized daughter DNA
Image context - Remember that there is going to be another replication bubble which will replicate towards that gap
- DNA damage causes DNA lesions –> Results in blocking DNA polymerase during synthesis which will stall at the lesion –> We are left with a single stranded region that is more prone to breaking –> increasing the likelihood of a full dsDNA break
Outline what NHEJ is?
DNA double stranded break –> What know?
One method of repairing the double stranded break –> Non-homologous end joining (Simplest Outcome)
NHEJ - When a series of proteins bind and stabilize the DNA ends and bring them together to allow them to rejoin and re-ligated.
More and more evidence is showing that bacteria are able to carry out such processes.
Explain the step by step process by which NHEJ occurs.
- DNA double stranded break
- Ku (Ku70/K80 heterodimer) is a protein that binds to DNA double-strand break ends - high affinity for DNA ends - sequence independent since it binds to the sugar backbone - ring shaped structure that can accomodate DNA
- Note that Ku is evolutionarily conserved from bacteria to humans - Ku70/K80 heterodimer binding leads to the recruitment of DNA-PKcs - DNA-dependent protein kinase, catalytic subunit –> Activation of kinase activity –> Shown to lead to the formation of a synaptic complex that holds the two ends of the broken DNA molecule together
- Depending on the nature of the break, different DNA end processing enzymes may be required, including those that resect DNA ends, fill in gaps, remove blocking end groups, and make the ends ligatable.
e. g. Artemis, PNKP, APLF, Polymerases μ and λ, etc. - The final step in the repair of a DSB is ligation of the broken ends by DNA Ligase IV
- Dissolution of the NHEJ complex
(Anthony J. Davis and David J. Chen, 2013)
5.
- S
Is NHEJ an error prone process?
Yes, it is an error prone process
Quite often we end up with flaps that associate incorrectly due to microhomologies (as shown) or the ends fold over forming hairpin or even sometimes reverse Hoogsteen base pairing.
e.g. GGG/G forming triplex and quadruplex structures –> source of slippage when DNA polymerases passes - resulting in base deletion (extra flaps are resolved/removed) or addition
One would have to predict knockouts by examining on a case by case basis.
Pros and Cons of NHEJ high error rate?
Good - or evolution - Increase diversity
Bad - Mutations may reduce the fitness of the organism
What cells in the body exploit the high error rate of NHEJ?
Immune system - antibody production
Some cells exploit this high mutation rate –> cells involved in antibody production use (Known as VDJ recombination) –> increase the rate of mutation in antibody genes - creates new mutants - makes us more resilient to invaders
Note - Modern genome editing technology (e.g. CRISPR/Cas9) to make targeted mutations and knockouts NHEJ is used after CRISPR makes a break in the DNA.
Outline how homologous recombination can be used to repair ds DNA breaks?
Homologous Recombination (HR) –> refers to the exchange of DNA with homologous DNA - can be used to repair double stranded DNA breaks.
Classic way of looking at it is homologous recombination between chromosome in meiosis
BUT!
It can also take place in the cell between homologous DNA - remember we have two copes!
Idea - we are missing a piece of DNA –> Where can we get the correct DNA? Homologous DNA acts as a template
Outline the two main ways in which HR is used to fix dsDNA breaks.
H.R. essentially lines up homologous DNA (readily found at the replication fork and swaps it between strands - relatively error free.
Variations of homologous recombination can occur to maintain the replication fork but two examples are…
- Fork regression –> lesion blocks polymerase –> end up getting the single strands coming off and priming on each other –> creating extra copies of homologous DNA
-
Strand invasion –> Blue/Red strands are complementary to opposite strands so they can act as primers –> meaning that one strand can rip off and act as a primer for the other strand
- So homologous strand invades and acts as a template for missing region
Outline the general use of HR?
Basically, H.R provides a general mechanism for repair where intramolecular template information has been lost
Used in a variety of situations…
- Double strand breaks –> most common
- Lesions bypassed during replication - Repaired
- In the Long term - source of evolution –> due to imperfect recombination leading to parts of the genome being duplicated/deleted.
Outline the general process of Homologus recombination
Blue - Parental / Purple - Daughter
- Chemical lesion in one parental strand –> results in missing information during replication as lesion is not copied –> gap has been created which means that informarion has to be retrieved from homologous DNA.
- Break open the homologous strand
- Reform phosphodiester bond so that the genetic information is swapped (Homologous recombination)
- Anys gaps are then filled in with DNA polymerase and Ligase.
Outline the different possible HR combinations that are possible.
H.R. can occur in a variety of ways but involves the same basic processes –> as long as there is complementarity (5’to 3’ or 3’to 5’)
- Single crossover –> large segments swapped
- Double Crossover –> everything between the crossover is swapped.
- Intramolecular (within the same strand)
a) Direct repeats - Deleting everything between the two repeats
b) Inverted repeats - Invert the red region –> red region changes orientation relative to surrounding DNA
Outline the different steps in the E. coli RecBCD pathway.
- Ds Break
- Eaten away by exonuclease RecBCD (5’ to 3’) until you reach chi site –> this leaves 3’ overhang
- RecA binds and stabilizes 3’ overhang
- RecA invades homologous DNA in genome –> strand invasion allows it to base pair with the homologous DNA - displacing the other strand forming a D loop.
- Once homology has been found - you have to nick and cut homologous strand using enzyme (allow for strand exchange)
- Fill in and ligate the nicks that have been formed - swapping the strands
- End up with 2 homologous strand that are paired up with a cross over between them which is known as a holliday junction (unstable structure that can migrate)
- Holliday junction can migrate (Branch migration driven by molecular motors) - when this happens the DNA is swapped –> swapping genetic info –> fundamental mechanism of HR
- Once missing DNA is exchanged - Holliday junction cut and re-ligated –> Known as resolution (random process – endonucleases that come in and cut the crossover region)
Further elborate how RecA binds and facilitates strand invasion for HR?
Homology search and strand invasion - RecA protein structure carriers this out
- It can bind to single stranded DNA (3’ overhang)
- Not 100% understood how it mediates strand invasion but we know that the first step is triplet formation.
- For triplet formation - Homologous DNA needs to be unwound –> short stretches doesn’t require much energy but longer stretches do require ATP hydrolysis.
- RecA coats DNA but allows enough space so that the DNA bind to it complementary homologous DNA
- Once homology is found, homologous DNA is nicked and ligated –> forming holiday junction –> allows for branch migration and geentic information exchange
Note - Helical nature of RecA filament means that it can form a triplex structure with a homologous DNA duplex
Is the RecBCD pathway used for single and double crossover?
Yes, it can be used for either single or double crossover
Note - double crossover is possible but hasn’t been studied as much
Explain what a Holliday junction is?
Holliday junction
During strand invasion, the invaded duplex is nicked which creates 2 x 3’ –OH strand which can then recombine with the opposite 5’ to 3’ homologous strand
This junction between strand (crossover) is known as the Holliday junction
Junction is a dynamic structure - moves along DNA unwinding in one direction and reforming in the other.
In the middle the DNA is not base-paired as they are being exchanged
What proteins are present to stabilize the Holliday junction?
The Holliday junction stabilized by recombination proteins (RuvA/B) which are also essential to drive branch migration which promotes exchange of strands.
Outline the what exactly RuvA and RuvB are and their specific roles.
We know that both RuvA and RuvB stabilize the junction but more specifically…
- RuvA - flat structure that binds the 4-way Holliday junction –> A hydrophobic ‘pin’ in the middle helps to separate the strands as it is a lubricating hydrophobic pin in the middle as the DNA backbone is hydrophilic (charge) - Energy dependent process (ATP)
- The RuvB ‘motors’ bind either side of the RuvA complex and uses ATP to translocate DNA, opens it up and thus allows genetic information to be swapped.
How much does the Holliday junction migrate?
How much migration? –> random/stochastic process – creates genetic variation
What happens after branch migration? How do we resolve the structure?
Resolution of the holliday junction refers to the removal of holliday junciton
In order to return to original state –> There are two options..
Cut Horizontally or Vertically –> leads to two outcomes..
Horizontal cut –> also known as gene conversion (one gene jumps from one to another) - patches
Vertical cut –> Also known as crossover - information from one chromosome is swapped with the other chromosome – splice
Check
What is the role of RuvC?
RuvC is the endonuclease in Bacteria - other organism have different endonucleases.
RuvC is responsible for the cleavage of the holliday junction
Position of RuvC binding and cleavage delineates/decides the outcome –> It is not known how this is coordinated between the patch and splice variations –> we think it’s a random process but there is a difference between different cell types.
What does a heteroduplex refer to?
After cuttting the holliday junction we get Heteroduplexes (DNA that has inherited DNA from other DNA regions)
Is the RecBCD (HR - Strand invasion) prone to errors?
Process is prone to getting bulge/mismatches –> mismatch repair is used to repair any errors
Generally speaking, what important processes is H.R involved in?
- Contributes to much of the variation in offspring –> meiosis - gene shuffling –> Scrambles the genes of maternal and paternal chromosomes leading to non-parental combinations
- Forms physical links between homologous chromosomes to allow chromosome alignment during meiotic prophase
- Evolution – horizontal gene transfer –> i.e. bacteria taking in foreign DNA
- Important in DNA repair –> Alternative way to bypass lesion
- Exploited in biotechnology: genome editing with CRISPR/Cas
Note - H.R. is conserved in almost every single cell –> exceptions very minimal organisms.
Does the meiotic recombination in yeast follow a similar recombination mechanism as seen in E. Coli (RecBCD)?
Yes!
Note that this time we get a double stranded break allowing for two overhangs to invade the homologous DNA
Forms a Double crossover during Meiosis since there are two branch points
Used to create offspring with different DNA combinations
Do H.R. and mismatch repair occur in isolation?
No! All these mechanisms of DNA repair may occur simultaneously
However, whether or not they do occur we can end up with different outcomes as shown in the attached image
Hypothetical…
Following recombination there is a possibility of a mismatch, two situations…
- Mismatch repair is repaired –> DNA sequence is restored to parental sequence - Homozygous ‘A’
- Not repaired (happens by random) –> Changes will persist and following recombination will be inherited by one of the daughter progeny –> Heterozygous
What is site specific recombination? How can it occur?
Specific recombination mediated by viral elements - Similar mechanism to normal H.R. –> uses enzymes called recombinases
Recombinases recognize sequence specific targets that are homologous and allows for recombination.
There are two main outcomes…
- Inverted repeat changes orientation
- Insertion or deletion which either adds/removes DNA.
Result of reaction is dependent on the orientation of the repeat sites
Difference between site specific and general HR?
- Site specific Homologous DNA region that is targeted is very short i.e. 10/20 B.P –> Enzymes only recognize these specific homolgous sequences
- H.R. targets larger regions –> Basically you don’t need specifc homologous regions –> Enzymes function more generally
What are integrases?
Integrases are an important class of transposition enzymes - Which allow for Site specific recombination
Outline how a Lambda phage integrase (Int) leads to the integration of a plasmid into the E. Coli Genome.
- Site specific Att sites (homologous regions) POP’ (phage) and BOB’ (Bacteria) line up with Lambda phage integrase
Note the region that is Homologous is ‘O’ - present in the middle of the Att sites.
- Lambda phage integrase allows for the crossover the of DNA
- Leads to the insertion of the Phage plamsid
Apart from the integrase itself - The process also requires some enzymes from the host cell to complete the process
Does Lambda phage integrase require energy to perform site-specific recombination?
No!
No ATP hydrolysis needed as energy is transduced using tyrosine in the enzyme active site –> traps energy in a high energy phosphate intermediate –> drives final crossover
Outline the more precise mechanism behind Integrase Enzyme used for site specific recombination.
Are there many different kinds of integrases and recombinases?
Yes!
Each recognizing specific homologous sequences
Example - Cre recombinase from yeast
What are Transposons?
Transposable genetic elements –> Transposons (‘jumping genes’) are mobile genetic elements that move randomly around the genome
Sequence homology is not required - Known as ‘selfish DNA’
Different types:
- Simple transposons contain only the genes required for their transposition (transposases) –> like mini viral like elements
- Complex transposons carry other genetic information e.g. antibiotic resistance
Note - Transposases similar to recombinases - cuts and swaps DNA around.
General overview of the Bacterial genome structure?
- Bacterial genomes are parsimonious (efficient)
- Organized into operons
- No gaps - No introns
- Regulation/co-regulation - highly organized
Why?
Explained by increased rate of evolution –> due to their short life cycle (20 min) - allows for genome to evolve quicker –> optimizing it
What does a karyogram of a human genome show us?
Karyogram shows chromosomes - all 23 pairs (Mother + father) - diploid
- Ordered in size
- Shows banding - known as G-banding –> Tryptic digest then Giemsa stain
a) Dark bands correspond with AT rich regions
b) Light bands correspond with GC rich regions
Banding pattern can be used to…
- identify unique patterns of bands on each chromosome, which can help…
- identify chromosomes
- diagnose chromosomal rearrangements
What do sister chromatids and centromere refer to?
During prophase… replicated DNA comes together to form chromosome…
Which is formed of two sister chromatids held together at a constriction point called the centromere.
What do B chromosomes, Holocentric chromosomes and Extrachromosomal DNA refer to?
Other physical structures
- B chromosomes are extra (supernumerary) chromosomes to the standard complement that occur in many organisms - not essential for life
Originate in a number of ways including derivation from autosomes and sex chromosomes in intra- and interspecies crosses - Example in deer.
- Holocentric chromosomes – the entire chromosome acts as a centromere. Best known example C. elegans
- Extrachromosomal DNA Plasmids Organelle DNA in mitochondria/chloroplasts important for function
Are there trends that can be seen on the chromosomal level in terms of gene distribution? What specific terminlogy is used to help describe the layout of genes on chromosomes
Generally speaking, the gene distribution is uneven and differ between eukaryotes
But!
Generally speaking, we observe a lower density noticeable around centromeres (less dense)
Terminlogy
- Gene-rich regions – High density
- Gene Deserts – Low density
- Multi Gene Families – Genes that are related occurring in more than one place (10 different places)
- Gene Superfamilies – 100/1000s of copies of a particular gene spread across the chromosome i.e. zinc fingers
What proportion of the DNA in the human genome is associated are protein coding genes/associated with genes vs. not associated with genes (intergenic)?
Genes or gene-related sequences –> Less than half (38%) of the genome has DNA associated with genes
Integenic (Between genes - i.e. not genes associated) –> 62 % of the human genome is made of intergenic region which is classified into two categories:
- Unique
- Repeats
What are characterisitics of Repeated Intergenic Regions?
- Has a different GC content than the rest of the genome (lower)
- Present in all eukaryotes
- Normally categorized by length
- Can take up roughly 10% up to 50% of genome
- No coding function
- Can be separated by CsCl centrifugation due to different physical properties
- Potentual roles - Some may play a structural role:
a) Centromere
b) Telomere - regulating mortality of cell In Vertebrates TTAGGG ~ 2500 times in humans
Why does Satellite DNA form?
- DNA replication
Both Microsatellites & Minisatellites are unstable - mutations in DNA repair systems can permit expansion which is a common occurrence in cancer cells.
Why is it unstable –> due to the way that DNA is replicated
During DNA polymerase action we can get mistakes –> priming further back add copies whereas priming further forward removes copies
- DNA recombination
Unequal Crossing Over (prophase I of meiosis) –> homologous DNA incorrect prime on eachother
Take away message - You get different number of repeats due to processes such H.R. and replication errors.
Outline the process of DNA transposition.
First discovered in Maize by Barbara McClintock
Different to other genome wide repeats - No RNA intermediate
Outline the process of LINES are created.
Outline the process by which SINES are created
How does gene duplication result in the a genome acquiring a new gene with a new function - what normally happens?
How gene duplication can lead to a new gene…
- Ancestral gene has promiscuous activity (i.e. main reaction + side reaction which has no biological function)
- Overtime the side reaction which had no initial function gains a function (bifunctional)
- We then get gene duplication and divergence - each gene carry out one of the functions from the bifunctional gene –> Results in increased fitness
Outline how recombination - unequal crossing over leads to DNA duplication
Remember there are two types of recombination
- Between Homologous chromosomes in meiosis (they don’t pair up during mitosis - no formation of bivalent pairs)
or. .. - Between sister chromatids in mitosis & meiosis
In both cases…
Unequal crossing occurs when similar sequences that are not in the same locus as each other on each chromosome undergo recombination.
e.g. repeat sequences that don’t align up properly which is then followed by recombination - resulting in duplication and deletion.
Outline how DNA Amplification during replication leads to DNA duplication?
Error that occurs in the replication Bubble during DNA replication
DNA recombination with the misaligned repeat regions leading to duplication and deletion - daughter cell will inherit 2 copies or no copies
Outline how Replication slippage leads to DNA duplication.
Replication slippage - Smallest type of error creating microsatellites
- Random process - replication is a dynamic process/enzymes are not always present at the stand (they detach)
1. Base pairing breaks/melts due to ambient temperature (bound to happen as its in a dynamic equilibrium)
2. DNA loops out but primes/attaches at the wrong base
3. Polymerase reattaches and starts adding bases - one strand will receive a insertion but the other a deletion - evident after another round of replication.
Probability of slippage is also dependent on bases present - AT less hydrogen bonds than GC.
Under what circumstances can we say that successful gene duplication occurs?
Under the following circumstances…
- 1 copy at least retains original sequence
- Gene gets duplicated and doesn’t change - increasing Synthesis rate
- 1 copy remains selectively neutral - It may accumulate mutations (~0.1% per million years) to form gene relics (‘Pseudogenes’) or become non-functional.
- 1 copy acquires new function or a sub-function ‘Neofunctionalisation’ e.g. new gene undergoes new/different selective pressures resulting in adaptation to provide a fitness advantage.
What are pseudogenes?
Pseudogenes - Characteristics:
- Not rare
- Copies of functional genes with altered/missing regions - often contain frameshift/nonsense mutations
- May serve regulatory roles - antisense RNA strands (RNA-RNA control).
- Increase genome size (cost/benefit)…
Normally speaking, evolution selects for certain things as the benefits outweighs the cost, but it is possible that things (e.g. pseudogenes) are just a by-product of evolution. Nevertheless, the fact that many organism have them would argue disagree with that.
What are the two types of pseudogenes?
Types of Pseudogenes
- ‘Non-processed’ pseudogenes - genomic region (DNA) is duplicated to form pseudogene –> pseudogene is non-functional due to incomplete duplication or inactivating mutations - not selected for on a evolutionary basis. Furthermore, pseudogene is missing regulatory region.
- Processed’ pseudogenes - DNA is transcribed to form RNA. RNA gets reverse transcribed to form cDNA which gets inserted into the same or different chromosome.
Evidence:
- Lack introns/promotors
- Contain poly(A) tail/flanking repeats
Why does this occur? Random stochastic process - we get random priming allowing reverse transcriptase bind form cDNA.
Can Pseudogenes can be reactivated to give a new protein?
Yes - retrogenes
Retrotransposition - This normally happens if a retrogene gets reintegrated by chance near a regulatory region –> over time the gene tends to evolve and become more and more functional (correct reading frame, etc.)
Retrogenes defining feature - missing introns
Example: Testis specific pyruvate dehydrogenase
Note –> when a pseudogene gets reintegrated it can either pick up a promotor in forward or reverse direction - Make RNA (sense) to produce gene of interest or reverse RNA transcript (antisense) which can serve a regulatory function - alter parent gene splicing or degrade target.
What are the two arrangements of multigene families in the genome?
- Tandemly located (back to back): α-globin genes (chromosome 16) & β-globin (chromosome 11)
- Dispersed - e.g. 3 aldolase genes on different chromosomes.
Outline the importance of the gene duplication in the globin superfamily.
What type of clustering does the globin superfamily show?
Globin Cluster structure –> Globin family shows both Co-location of genes in the clusters as well as dispersal of clusters
Similar organization in all vertebrates but lengths vary and different distribution of pseudogenes.
Changes between embryonic and adult genes?
What organisms have a high degree of polyploidy?
It is thought that ≤ 80% of flowering plants species originated via recent polyploidy –> examples include: Oats, cotton, potatoes, bananas, coffee, sugar cane, peanut, apples
Useful as it Generates new properties - Larger genome results in large cell size - not really understood why
But…
It turns out that polyploidy can also occur in vertebrates –> More common in invertebrates, fish & amphibians but rare in mammals.
What is the mechanism for Polyploidization?
Two main scenarios
- ‘Autopolyploidy’ (‘Auto’ = same) - Due to mistake (meiosis error) in forming gametes the cells aren’t fully reduced (production of diploid gametes) - upon fertilization –> result in new ploidy
If its viable can lead to speciation - 1-40% frequency of formation.
Theoretically, Endoreplication can also be a cause of polyploidy in autopolyploidy - replication of the genome without going through mitosis
- ‘Allopolyploidy’ (‘Allo’ = other) –> polyploid offspring is derived from two distinct parental species
If a 2n gametes fertilizes with an n gamete, can it reproduce with the parents?
No!
If due to Autopolyploidy –> you end up with 2n combining with n creating 3n offspring –> this offspring can not reproduce with the parents
Why? Unequal number of chromosomes during cellular divison
Why is autoploidy uncommon in animals with 2 distinct sexes?
Elaborate on the idea of allopolyploidy? What are the two models proposed for how allopolyploidy occurs (conceptually)?
What is endoreplication?
Why are triploids in allopolyploids no good?
They are tolerated in first generation but offspring are infertile
Example - 2n + n –> 3n - mature adult plants
When 3n tries to reproduce and produce offspring via meiosis - only 2 chromosomes (out of three) can form bivalents –> chromosomes don’t come together correctly and we end up with unbalanced gametes.
Outline how the concept of polyploidy is used to create fertile Triticale plants.
Example of Alloploidy - Triploid Wheat-Rye hybrid - two species
Wheat (triploid) produces a high yield and Rye (diploid) has high disease tolerance - useful to combine to produce Triticale
Problem! F1 generation has 21 chromosomes - lead to meiotic problems (improper bivalent formation) - infertile
Solution? Add chemical colchicine - cancer drug to F1 generation –> interferes with chromosomes - leads to doubling of F1 gametes
Hence upon fertilization of doubled gametes - 21 (gametes) to 42 –> 42 is even so all chromosomes can pair up we get allotetraploid
After Whole Genome duplication occurs, how does the cell attempt to reach balance?
After WGD there are Drastic change in gene expression –> Hence, the cell attempts to achieve chromosome expression balance - meaning that it restores diploid like behavior.
This is achieved in several different ways:
- Genetic control - transcription factors are regulated via feedbacks loops to ensure correct expression
- Epigenetic - DNA methylation/chromatin reorganization changes gene expression accordingly.
Consequence of doubling the number of genes –> allows organism to carry new mutations –> as you already have the wild type - new functions/ sub-functions
Why is it hard to identify ancestral polyploidisation?
Genome duplication takes place –> Over time we lose particular genes copies –> Another Genome duplication takes place –> Over time we lose particular gene copies
Hence, the number of gene copies is not constant - changes –> hence, making it hard to identify WGD
Different ways to detect WGD?
How to discern auto and allo-ploidy?
Is there evidence for WGD of Saccharomyces cerevisiae?
What is the Hox gene family? What does it do?
Example of WGD –> Hox gene family - homeobox genes involved in animal development –> they occur in clusters.
Hox genes - DNA binding proteins/code for transcription factors –> turn genes on/off in order regulate development
Hox gene T.Fs utilize the Homeodomain
Hox gene family –> provides a very quick way for evolution to change the body plan of an organism
Any mutations in the cluster - results in major changes in the body plan
Generally speaking, how do Hox clusters influence the development of the embryo’s body plan?
How does the Hox gene family provide evidence for WGD?
Genome duplication and sequence convergence (evolution) in genes and regulatory regions has resulted in morphological complexity thus allowing complex body plans to be formed –> Duplication & manipulation of Hox genes allow for variety the spice of life bbbyyyy
Example: Insects have 1 Hox cluster whereas vertebrates have 4 - indicating that WGD has allowed for the increased copies of these clusters to exist and to be fine tuned through evolution.
What is the 2R hypothesis?
Outline what is going on in the attached picture?
Hox clusters
Genome Duplication Benefits?
What are the different ways evolution is using to increase genome complexity?
Difference between Exon Shuffling and alternative splicing?
Note - They are NOT the same thing!
What is the exon shuffling theory?
Note - Exon duplication and Shuffling are different concepts
But…
They come hand in hand
How do the rates of exon duplication + exon shuffling compare to gene duplication?
How can Illegitimate non-homologous recombination lead to exon shuffling?
Illegitimate non-homologous recombination is one-way exon shuffling takes place –> only requires a short primer/overlap/homology.
Structural domains of different proteins are joined together - due to the low levels of homology for this process can occur quite easily even though we are dealing with different genes.
Topoisomerases - nicks DNA and ligates non-homologous region –> leading to the shuffling of exons
How can LINES (Long Interspersed Nuclear Elements) jumping around the genome lead to exon shuffling?
How can DNA Transposons result in exon shuffling?
What is something that you have to consider during exon shuffling (or any insertion/deletion in the genome)?
What is the splice frame rule?
Is there evidence for exon shuffling in genomes?
Is there any correlation between organism complexity and exon class? Why does this arise?
YES bby
Exon shuffling example?
Why does evolution favour exon shuffling? What is particularly beneficial?
Futhermore…
Shuffling promotes self-interaction capacity (very common) - Allows formation of homodimeric proteins –> These protein domains self-interact
These domains tend to be stuck in exon shuffling compatible structures –> leads to formation of families and superfamilies.
Main differences and similarities between exon shuffling and gene duplication?
