chromosomes and DNA Flashcards
what are origins of replication?
specific DNA sequences that recruit initiator proteins, in E-Coli its called oriC (only has one, humans have over 100, 000)
Human origins of replication are not well understood, but are thought to be located near genes involved in initiating the cell cycle
briefly, what are the two steps that occur to initiate DNA replication, when do they occur and why are they separated?
G1 - formation of the pre-replicative complex (pre-RC)
GS - activation of the pre-replicative complex
The temporal separation of these 2 events ensures that:
each origin is used
Each chromosome is only replicated exactly once per cell cycle
explain how the pre-replicative complex forms in G1
Origin Recognition Complex (ORC) binds to Replicator sequence
Helicase-loading proteins Cdc6 and Cdt1 bind to ORC
They recruit Helicase Mcm2-7, which completes formation of pre-RC
***it remains INACTIVE due to low CDK levels
explain how the pre-replicative complex is activated in GS
CDK levels rise, activating the pre-replicative complex, telling the helicases to unwind the DNA. Two helicases create two bidirectional replication forks at the OR, going in opposite directions.
CDK remaining high throughout rest of cycle prevents more pre-replicative complexes forming
mutations in helicase - what is bloom syndrome?
Caused by mutations in gene for bloom DNA helicase, essential for genomic stability - removes roadblocks in the way of replication forks. Without it, replication forks collapse, DNA replication is no longer efficient - mutations - lead to cancer
Can’t go out in sunlight - UV interacts with DNA causing bulky adducts which cant be removed without this functioning bloom helicase
what is Werner syndrome?
Autosomal recessive mutation in Werner helicase, causes premature ageing
what is meant by processivity?
DNA polymerase needs high processivity - the ability to catalyse consecutive reactions without releasing the substrate
how is DNA polymerase’s processivity made to be high?
what protein increases processivity AND how is it loaded
increased by the sliding clamp protein: keeps DNA Pol in place and moves it along
Process -
Sliding clamp (ATP-dependent) binds at template-primer junctions, with a clamp loader
The clamp loader is displaced as it ‘loads’ DNA polymerase on instead, allowing the polymerase to bind to the sliding clamp
what is the function of single-stranded DNA binding proteins?
Exposing single strands as you unwind DNA results in hairpins as complementary bases on the same strand interact. SSBPs ensure DNA stays flat/linear and untangled
briefly, how does DNA synthesis work considering DNA polymerase can only add onto the 3’ end
DNA is antiparallel, so the strands run in opposite directions, and because new DNA has to be made 5’ - 3’, the two new strands will be formed in opposite directions, one following helicase which is nice and easy, one working backwards which is the annoying part
primase makes primers to provide a 3’ end for DNA pol to work on
leading strand only needs one primer at beginning to get started
he lagging strand works discontinuously in dribs and drabs, so you have to add a primer, make a bit of DNA, add another primer, make a bit of DNA etc… so on this strand you get DNA with gaps, called Okazaki fragments
The RNA primers are removed by ribonuclease H, and replaced with DNA by DNA polymerase. The nicks that remain after the primers are replaced get sealed by the DNA ligase (forms the phosphodiester bonds)
what are the different kinds of topoisomerases and what do they do?
unwinding DNA strands can create torsional tension
Topoisomerase 1 can nick ONE strand
Topoisomerase 2 can nick BOTH strands but requires ATP.
Topoisomerase (or DNA gyrase) is used to reduce strain on the DNA molecule and prevent positive supercoiling. It acts by making temporary nicks in the helix to release the tension, then sealing the nicks to avoid permanent damage
what is the main limitation of DNA polymerase?
DNA polymerase can only add nucleotides in the 5’ - 3’ direction because it needs the free -OH group on the 3’ end to bond with the phosphate group on a nucleotide’s 5’ end, so DNA polymerase only adds on at the 3’ end
mechanism of action is a nucleophilic attack
what are telomeres for?
removal of the primers (the initial one used for the leading strand, the last on used for the lagging strand?) leaves a short section of DNA at the end not copied, meaning important genetic info could be lost
telomeres = lots of a short repeating sequence at the ends of a chromosome, acts like a buffer as in some of this repeated sequence is lost, not important info. they define the end of the chromosome and maintain it’s integrity
can be replenished/lengthened by telomerase…
what is important about telomerase’s structure (include sequence)?
its a ribonucleoprotein - it is a protein with an intrinsic RNA component
the RNA component of the telomerase is what acts as a template for DNA synthesis
it’s sequence is AAUCCCAAU
meaning the DNA added on as telomere goes: TTAGGGTTA
explain how the telomerase shuffle works to lengthen telomeres
When the 9-base long RNA component of telomerase initially associates with the DNA, it does so at the 6-base long TTAGGG repeat, and therefore leaves a three-nucleotide (3’) overhang, which is then filled in
The 3 nucleotides added are ‘TTA’, which is the beginning of the recognition sequence that the telomerases intrinsic RNA component binds to
This allows the telomerase to move 6 bases forwards as it binds to the newly added TTA, leaving a 6 base overhang that can be filled in.
again the last three nucleotides are TTA, so the RNA component can move along another 6 bases and start at this new ‘TTA’ leaving another 6 base overhang to be filled in etc…
Does this hundreds to thousands of times to ensure there is no genetic information lost and chromosome integrity is maintained
what are a cell’s possible routes if DNA damage occurs (and it does not get repaired)?
DNA damage in cells that have proliferative capacity -
results in error during replication/failed repair attempts - mutations with selective advantage for clonal expansion - cancer
DNA damage in cells predominantly non-dividing -
blockage of transcription
if cell death doesn’t occur, there is reduced gene expression and functional decline of tissues/organs
results in aging (Werner’s syndrome)
cells are constantly under attack - what are some types of DNA damage and their causes?
Endogenous sources of DNA damage/lesions include hydrolysis, reactive oxygen species, by-products of metabolism
Exogenous sources of DNA damage include UV, X-rays, carcinogens, chemotherapeutics
what is deamination?
an endogenous source of DNA damage
A change of base due to an amino group being removed from a base by hydrolysis
Most common change is cytosine to uracil
During replication, the new strand made will then also have an incorrect base (it will pair with the mutated base in the template strand)
resulting in a mutated base pair
in deamination - what is a transition vs a transversion?
You mostly get transitions (a purine swapped for a purine/ pyrimidine for a pyrimidine) more so than translations (swapping a pyrimidine for a purine)
what is depurination?
(endogenous) N-glycosidic bond between base and backbone is hydrolysed resulting in an ‘abasic site’ - a base is missing
Replication occurs and the missing base obvi doesn’t get a pair.
This is a frameshift mutation, resulting in a missense protein - different amino acids are coded for from the mutation onwards (due to the three-base reading frame)
exogenous sources of DNA damage - what are pyrimidine dimers?
caused by UV light
When a covalent bond occurs between adjacent pyrimidines - Cs and Ts - forming a cyclobutane ring between the adjacent bases
This distorts the DNA making it difficult to replicate and can lead to mutations. Interstrand crosslinks can also form, as well as DNA and protein crosslinks. These also block replication and transcription
how are DS DNA breaks and SS ones caused? how are these kinds of DNA damage different?
DS = topoisomerase 2 inhibitors, ionising radiation etc…
SS = reactive oxygen species, chemotherapeutics etc…
these are both damage to the phosphate backbone rather than the bases
base excision repair - what is it used for, how does it work?
Repairs deamination and abasic sites
During replication, deamination can be recognised by uracil DNA glycosylase
This enzyme ‘flips’ the deaminated base out, like putting a sticky tab on a page that needs to be reviewed. The DNA repair pathway needs to come and fix it
Uracil DNA glycosylase will then ‘excise’/remove the base
AP endonuclease and phosphodiesterase come along and remove the section of sugar-phosphate backbone, leaving a SS break
DNA polymerase adds a new, correct nucleotide, DNA ligase seals the nick
nucleotide excision repair - what is it used for and how does it work?
Or the short patch repair pathway, for when more than one base is involved - pyrimidine dimers
- Damage is recognised, an excision nuclease cuts/excises the damage site a few bases upstream and downstream of the damage site - just takes out a little chunk
DNA helicase removes this cut out damaged section
The 12-ish nucleotide gap left behind is filled with DNA synthesised by polymerase, with DNA ligase sealing the nicks again
translesion synthesis - what is it used for and how does it work?
Allows damaged section to be skipped over
Sliding clamp reaches some DNA damage, the DNA polymerase dissociates from the clamp and is replaced with translesion polymerase
This polymerase isn’t as accurate as the normal, so skips damaged bases
Good in that it allows replication to continue, but is quite mutagenic because its not repairing the DNA
name the two methods used to repair double stranded breaks, when they occur, and the major difference
Non-homologous end joining -
Occurs in G1 of the cell cycle
Simply rejoins the breaks and is therefore error-prone, genetic information is often lost
Homologous recombination -
Only occurs in S-phase and G2 because it uses the newly synthesised DNA as a template
Complex, but results in error-free repair
how does non-homologous end joining work?
Recognition of DNA Ends: NHEJ is initiated when a cell detects double-strand breaks (DSBs) in the DNA. MRN complex resects the DNA slightly to give a 3’ overhang
Binding of Repair Proteins: Proteins Ku70/Ku80 and DNA PK, quickly bind to the each of the broken DNA ends, forming a complex known as the Ku heterodimer, pulling the two ends together
End Processing: endonucleases then come along and remove the 3’ overhang
Ligation: DNA ligase joins the ends together
how does homologous recombination work (six steps)?
- At the double stranded break, MRN complex resects the two strands to produce 3’ overhangs (because this is needed for DNA polymerase to later be able to add on)
- Proteins like Rad51 and RPA coat the 3’ overhang, causing recruitment of more proteins such as BRCA1 and 2 to help with…
- Strand invasion, where the damaged DNA finds its sister chromatid at the section in need of repair, and ‘invades’ so that it can get a look at a correct version of the complementary strand and use it as a template. Note, this first ‘crossover’ where the damaged strand invades is called a holliday junction
- DNA polymerase will use the undamaged template to synthesise the missing section of DNA and the once broken strand rejoins its original strand note, now that there are two crosses over, 1. Where the strand first invaded and now 2. Where it is leaving to rejoin it’s original strand, this is called a double holliday junction
- The newly synthesised DNA now acts as a template itself for the other break - in the other DNA strand (as this is for DS breaks)
- DNA ligase seals the nicks
what are the three checkpoints in the cell cycle?
G1 checkpoint - damage here is repaired by non-homologous end joining
Early S phase checkpoint - also likely repaired by non-homologous end joining as no DNA replication has actually occurred
End of G2 checkpoint/entry into mitosis - repair would occur by homologous recombination as DNA has been synthesised to be used as a template
what is the process/cascade that is involved in the cell cycle check points?
H2AX detects where DNA damage is
ATM and ATR kinases are then activated
Check 1 and Check2 are then activated
P53 is then phosphorylated/activated and made stable
P53 binds to regulatory region of p21
This deactivates S-CDK, preventing S phase from occurring, and so the cell cycle stops for DNA repair or, if not, cell death
give two examples of human diseases cause by DNA repair defects?
Xeroderma pigmentosum - defects in NER (nucleotide excision repair), cause 2000 fold increase in likelihood of skin cancer
Breast cancer - BRCA1 and 2 gene mutations occur in 80-90% of inherited breast cancers. These genes are involved in homologous recombination
how can cancer be treated with synthetic lethality?
this idea is used to harm cancer cells without harming healthy cells as much
the idea is that cancer cells have, already, a specific gene mutation that knocks out a certain pathway e.g. for a particular kind of DNA repair
you then knockout another pathway, e.g. a different DNA repair method, so the cancer cell cannot survive with both pathways not no functioning
proved effective for cancer cells with mutations causing non-functioning of BRCA2 (forced to rely on another pathway to survive, so knock this pathway out etc…)
name four methods used to study DNA damage
Expose cell lines to sources of DNA damage and compare
Using immunofluorescence to look for proteins involved in DNA repair (lots of fluorescence = lots of damage must have occurred)
Comet assay - Cells treated with drug, if DNA damage occurs it leaks out of the cell in electrophoresis
Western blots to look art expression of different proteins involved
what are chromosomes made of?
highly coiled chromatin
Chromatin = beads (nucleosomes) on string. DNA helix is wrapped around the nucleosome ‘beads’
Nucleosomes = made up of 8 core histones, H2A, H2B, H3 AND H4 (there’s two of each). The N-terminal tails of the 8 histone subunits project out and a free to interact with other proteins, allowing for regulation of chromatin structure and function
why is H1 - the linker histone - different from the others? what does it do?
structurally how is it good at it’s job?
straps DNA onto the histone octamer (so not actually in the nucleosome)
Keeps chromatin structure of transcriptionally silent/ not needed regions tightly wrapped away
Rich in lysine and arginine = basic = binds to DNA readily, but doesn’t need to be sequence specific - binds to the -ve phosphate backbone
how are chromosomes viewed?
Chromosomes are easiest to distinguish at metaphase, displayed in a karyotype
In interphase different stains can make it possible to look at different chromosomes (looks like a big blob) but location is preserved in specific ‘sub-nuclear territories’
how is DNA kept accessible despite the nucleosomes?
DNA remodelling enzymes remove nucleosomes in order to open up DNA sequences needed for transcription or when replication is occurring
what are fractal globules?
a level of organisation of chromatin within a chromosome, in a way that allows chromatin to be condensed and decondensed without getting knotted. described as a pattern within a pattern
where is transcriptionally inactive DNA stored?
at the nuclear periphery
centromeres - what are they?
how does it interact with the kinetochore?
regions of alpha-satellite repeated DNA sequences where the chromosomes are connected
Kinetochore recognises the repeated sequence and binds to it. Kinetochore has an inner and outer ‘plate’
Inner plate binds to the repeated sequence the outer plate binds to the microtubules of the mitotic spindle
Ensures faithful segregation of sister chromatids
structure of the genome - %s?
is this the same in more complex organisms
Only 1% is coding
20% is introns
50% is transposons
Rest of genome is non-repetitive DNA neither introns or codons
More complex organisms = more protein coding genes AND more non-protein coding DNA, for regulating transcription and organising access to protein coding genes
what is a DNA transposon?
DNA that hops from one region to another using an RNA intermediate
These regions of DNA will also encode transposase, an enzyme that can cut out the transposon, and move it to anywhere in the genome. Its original position rejoins, then it joins elsewhere. Can be quite a problem if it joins in the middle of an important gene or fails to join properly
what is a retroviral-like retrotransposon?
not the same as a DNA transposon, it is a bit like a retrovirus (its not a pathogen it’s endogenous in think) but doesn’t have a functional envelope - i.e. it doesn’t burst out of the cell
how it works -
The transposon is an mRNA molecule in an envelope.
This mRNA molecule is released and reverse transcribed to DNA.
a cDNA strand is then made from it to give you a dsDNA molecule.
Integrase then adds this DNA into the target DNA (the new region of ‘host’ DNA the transposon copy is being placed).
When transcribed by the ‘host’, another virus-like particle is formed with an envelope and everything and cycle repeats
what are non-retroviral polyA retrotransposons? include how they are different from retroviral-like retrotransposons
These do not have to use a virus-like capsid and envelope to transcribe the RNA back into DNA
An RNA transcript of the retrotransposon is made by RNA polymerase II. This transcript includes the entire retrotransposon sequence, as well as the poly(A) tail.
Processing: The RNA transcript is processed to remove introns and to form a mature RNA intermediate
you get reverse transcription, cDNA, integration just like ‘retroviral-like retrotransposons’
key difference -
Circularization: Once the cDNA copy is integrated into the host cell’s genome, it can be circularised by a process called end-to-end joining. This circular DNA can then be transcribed to produce new RNA transcripts of the retrotransposon, starting the replication process over again
what is the purpose of transposons and retrotransposons?
Transposons don’t help really, so they are now mostly defective ancient relics.
Despite their lack of function they have multiplied in the human genome across the years when compared to other mammals, particularly smaller mammals like mice???
almost 50% of the human genome is effective transposons
they do contribute to genetic diversity and could potentially provide selective advantages (tho more likely cause problems)
