Exam 2 Flashcards
histones
major DNA binding proteins that DNA wrap around
micrococcal endonuclease
- endonuclease from the bacterium Micrococcus
- treat eukaryotic DNA with this enzyme
- run product on an agarose gel
result: DNA banding pattern in multiples of 200bp
why? - nuclease cutting between regular complexes of 200np of DNA + protein
the nucleosome
- DNA wrapped around protein core (histones)
- the basic structure of chromatin
- consists of ~200 bps of DNA and an octamer of histone proteins
- is linked to other nucleosomes by Linker DNA
-endonuclease cleaves linker DNA and releases individual nucleosomes from chromatin
the DNA in the nucleosome
- DNA is wrapped around the outside surface of the protein octamer
- the length of DNA per nucleosome varies for tissues or species from 154 to 260 bp
- nucleosomal DNA is divided into
-the core DNA (145-147bp)
-linker DNA (7-115bp)
structure of the nucleosome
- the nucleosome is a cylinder
- DNA organized into ~one and two-thirds turns around the surface
- DNA enters and exits on the same side of the nucleosome
protein histones in the nucleosome
- small proteins rich in arginine and lysine residues
- charges»_space; it binds to the DNA
the histone octamer
- two copies each of H2A, H2B, H3, and H4
- core histones are HIGHLY evolutionarily conserved in eukaryotes
core histones
- H32 - H42 tetramer + two H2A-H2B dimers
- all histone N-terminal tails and H2A and H2B C-terminal tails extend out from the histone core
- tails are site for covalent modification
-important in chromatin function
Histone H1
- H1 is associated with linker DNA
- located at the point where DNA enters or exits the nucleosome
post-translational modification of proteins
- protein function can be modified by enzymatically adding small molecules to the protein
- changes protein shape
- changes protein reactivity (+ or -), etc.
- phosphorylation - adding PO4
- methylation - adding CH3
- acetylation - adding acetyl group
- ubiquitylation - adding ubiquitin
- Sumoylation - adding small protein SUMO
ALL of these modifications are reversible
nucleosomes are covalently modified
combinations of specific histone modifications define the function of local regions of chromatin
can multiple modifications sites in histones have more than one type of modification?
yes
- most have a single, specific type of modification, but some sites can have more than one type
functional effects of modifications: examples
acetylation of the lysine
- reduces the positive charge on the lysine
- causes decreased interaction with DNA
- acetylation of histones is associated with gene activation
methylation of lysine
- lysine retains the positive charge
- associated with gene inactivation
bromodomain
- proteins with the bromodomain in their structure can bind to histones that are acetylated
-allows transcription enzymes to bind - proteins have different domains that can recognize acetylated, phosphorylated, etc. modified amino acids
- this is how proteins recognize and interact with DNA
primary structure of chromatin
- a 10-nm fiber which consists of a string of nucleosomes
- “beads on a string”
secondary structure of chromatin
- formed by interactions between neighboring nucleosomes
- 10 nm strands may pack together closely to form densely packed higher levels of DNA folding
- would allow the DNA to be accessible for transcription
- easily reversible
higher order chromatin structures
secondary chromatin fibers
- folded into higher-order, 3D structures that comprise interphase or mitotic chromosomes
chromosome
- a discrete unit of the genome carrying many genes
- each chromosome consists of a very long molecule of duplex DNA
- plus approximately equal mass of proteins
bacterial chromosome
- bacterial chromosome is a single large circular DNA
where is bacterial chromosome located?
nucleoid
- the DNA is bound to proteins
- the DNA is NOT enclosed by a membrane
the bacterial genome can be ___ or ____
relaxed or supercoiled
- supercoiled - coiling of the circular DNA so that it crosses over its own axis many times
eukaryotic chromatin
interphase chromatin
each chromosome is a long dsDNA
- heterochromatin - found in the edges of the nucleus and around the nucleolus
- euchromatin - less densely packed DNA, active genes
mitosis chromatin
- chromosomes are 5-10 times more condensed than in interphase
chromosome scaffold
- a proteinaceous structure in the shape of a sister chromatid pair, generated when chromosomes are depleted of histones
- eukaryotic DNA is attached to a protein scaffold
- in metaphase chromosomes, supercoiled DNA is attached to a protein scaffold
chromosomes can be stained to have banding patterns
- protease treatment and then staining
- stains the chromosomes to have a series of striations, called G-bands
- yields a characteristic banding for each chromosome
- each band can include many hundreds of genes
- allows us to study different regions of the chromosomes
polytene chromosomes
- some dipterans (like Drosophila melanogaster, the fruit fly) have huge chromosomes in interphase
-found in the salivary gland
cells - generated by successive replications of a chromosome without separation of the replicated chromosomes in mitosis
cytological map
- can label gene-specific probes to identify where specific genes are on the banding
puffs
- sites of gene expression/activity on polytene chromosomes expand to give “puffs”
- show that gene expression requires that the DNA must unwind
homologous chromosomes
pairs of chromosomes similar in size, shape and gene content
chromatid
one of the two DAN strands in a replicated chromosome
sister chromatids
chromatids from the same chromosomes
- join at the centromere
interphase
chromosomes replicate
prophase
chromosomes/sister chromatids condense and attach at the kinetochore to the mitotic spindle microtubules
metaphase
sister chromatids line up in center
anaphase
sister chromatids pulled to opposite opposite poles of the cell
telophase
sister chromatids in opposite poles and nuclear membrane reforms
cytokinesis/cell division
membrane pinches in middle to separate into two daughter cells
- cytoplasm is divided
cohesins
proteins that hold “glue” together sister chromatids
centromere
- a constricted region of a chromosome (the DNA) that:
- has unique DNA sequences and proteins not found anywhere else in the chromosome
- is where the sister chromatids attach to the mitotic spindle microtubules
- centromere region also contains the kinetochore
kinetochore
the proteins responsible for attaching to the spindle apparatus microtubules
the centromere
DNA is wrapped around:
- normal histone H3
- or a centromere-specific histone H3 variant, Cen-H3
- Cen-H3 allows binding of kinetochore proteins to form the kinetochore
- also has heterochromatin that is rich in satellite DNA sequences (repetitive DNA)
-function of the repetitive centromeric DNA is not known
replication overview
- supercoiled DNA must first be relaxed
- initiation
- elongation
- joining and/or termination
telomers
higher organisms with linear chromosomes
- have long series of short tandem repeated sequences called telomers
-may be 100-1000 repeats - human telomere repeats are
-TTAGGG-3’
why do we need telomers?
- DNA replication leaves a 3’ unreplicated end on one of the replicated DNA strands
telomeres are essential for survival
- DNA replication leaves one strand with unreplicated end
- next round of replication results in a shorter DNA
-due to shorter DNA template - and so on until genes near the ends are not replicated
telomeres are synthesized by telomerase
telomerase uses:
- the free 3’-OH of the telomeric strand
- its own RNA template of 3’-AAUCCC-5’
- a reverse transcriptase
- adds tandem repeats (5’-TTAGGG-3’ in humans) to the 3’ end at each chromosomal terminus
- extends the ends of the chromosomes to solve the so-called end replication problem
- telomerase uses RNA to complementary bind to end of telomer
- then the RNA polymerase of telomerase adds the complementary dNTPs to extend the end
how does telomerase solve the problem?
- repeat this step many times until it is long enough for the DNA polymerase to do another Okazaki fragment
- then DNA polymerase can fill in the gap
RESULT
* extends the end of the chromosome back to how long it should be
telomeres are essential for survival
telomerase is expressed:
- in actively dividing cells
-stem cells
-during development - not expressed in quiescent cells
-most other cells in the body - loss of telomeres results in senescence
-cell dies - cancer cells often have telomerase
telomer ends are “sticky”
- can be recognized by the DNA repair enzymes as a broken chromosome
- could be added to the end of another chromosome
- must have a way to prevent this!
telomers form circular loops at the end of chromosomes
the protein TRF2
- allows the 3’ telomer unit to invade into its homolog in an upstream region of the telomere
- forms the t-loop
- t-loop prevents DNA repair enzymes from recognizing the 3’ end as a DNA break
telomeric binding proteins
(TRF1, TRF2, Rap1, TIN2, TPP1, and POT1)
- form the Shelterin complex
- function to protect the telomers from DNA damage repair
- can result in chromosome ends sticking to other chromosomes
2. also function to control telomer length by inhibiting telomerase - the more shelterins bind, the less telomerase can bind to add more DNA
replicon
- a unit of the genome in which DNA is replicated
- bacteria usually only have one replicon
- eukaryotes can have many replicons
- each replicon contain an origin for initiation of replication
origin- a sequence of DNA at which replication is initiated
terminus - a segment of DNA at which replication ends
Meselson and Stahl experiment 1958
- grow organism on “heavy” 15N to label DNA
- then grow on medium with “light” 14N
- allow DNA replication to occur
- isolate DNA and ultracentrifuge in dense medium
what meselson and stahl found
parental DNA
- all heavy DNA
1st generation
- all medium hybrid DNA
2nd generation
- mixture of light and medium DNA
suggests DNA is replicated semiconservatively
- resulting replicated DNA strand is one parental and one new
semiconservative replication
- replication accomplished by separation of the strands of a parental duplex
- with each strand then acting as a template for synthesis of a complementary strand
replication bubble
in electron microscopy:
- a replicated region appears as a bubble within nonreplicated DNA
- a replication fork is initiated at the origin and then moves sequentially along DNA
when is replication unidirectional?
when a single replication fork is created at an origin
when is replication bidirectional?
when an origin creates two replication forks that move in opposite directions
bacterial DNA is usually a singular circular replicon
- bacterial replicons are usually circles
- they replicate bidirectionally from a single origin
- the origin of E.coli is oriC
-245 bp in length
the eukaryotic cell cycle
cells cycle between:
- mitotic (M) phase = when cells actually divide
-
interphase = the non-dividing phase
-the chromosomes are generally uncoiled in euchromatin and heterochromatin
cells spend most of their time in interphase
chromosome replication occurs:
only during interphase
- not during mitosis
DNA synthesis occurs in the S phase of interphase
the gap phases
interphase has two gap phases
- no DNA synthesis occurs
- G1 and G2
G1 phase
cell growth
- everything in the cell is doubled except DNA
- cells prepare for DNA synthesis
- make all enzymes for DNA synthesis
G2 phase
- after DNA synthesis
- preparing for mitosis
organelles replicate and additional cytoplasm is made in G1 and G2
G1 checkpoint
progression from G1 into S phase is tightly controlled
checkpoint
a control mechanism that prevents the cell from progressing to the next stage unless specific goals and requirements have been met
- no damage to the DNA
- must be a certain amount of cell growth
each eukaryotic chromosome contains many replicons
- too much DNA for just one origin/replicon
- not just DNA, but DNA + histones
- eukaryotic replicons are 40 to 100 kb in length
- multiple origins of replications that ultimately merge during replication
individual replicons are activated at characteristic times during what phase?
S phase
- early replicating
- late replicating
- see ~100 to ~300 replication foci when stained
- eukaryotic replication takes over 6 hours to complete
-S phase
origin recognition complex (ORC)
~400 kDa protein complex
- highly evolutionarily conserved in eukaryotes
- binds to eukaryotic origin
- is cis-acting
causes DNA replication initiation on the strand of DNA to which it binds
how is replication initiated? - experiment
- inject nucleus into a Xenopus (frog) egg
- DNA replicates
- but it cannot replicate again
-occurs only once - but if you permeabilize the nuclear membrane before injection
-DNA will be replicated more than once
permeabilizing the nuclear membrane
allows new protein made in the cytoplasm to enter the nucleus and initiate replication again
if you permeabilize before injection:
- suggests that a protein is found in the nucleus that allows initiation of replication
- once replication occurs, the protein is used up or inactivated
licensing factor
controls eukaryotic re-replication
- is necessary for initiation of replication at each origin
- present in the nucleus prior to replication, but is removed, inactivated, or destroyed by replication
licensing factor cont.
- active licensing factor is present in the nucleus
- replication inactivates it
- new licensing factor is made in the cytoplasm
- but it cannot get into the nucleus through the nuclear membrane
- breakdown of the nuclear membrane in mitosis allows new licensing factor to associate with the nuclear material
what is this licensing factor?
- cyclins and cyclin-dependent kinases (Cdk)
- they are Helicase Activating Proteins
- Cdk enzymes must bind cyclins to be active
-becomes the “ON-OFF” switch
helicase
the enzyme that separates the strands of DNA for replication - a crucial initial step for replication
topoisomerase
- relieves stress coiling
- relaxes the supercoil
- moves in front of the replication fork
Type I topoisomerase
ssDNA nick to allow cut strand to rotate around the uncut strans
Type II topoisomerase
both strands of DNA are cut and the intact strand is passed through the cut to relieve the tension
initiation
- origin recognition complex binds to the origin
- DNA strands must be separated and stabilized
-creates replication bubble - DNA synthesis is initiated at the replication forks
elongation
replisome
multiprotein complex that assembles at the replication fork to undertake synthesis of DNA
- it contains DNA polymerase and other enzymes
- is assembled de novo (at that time and place) into the replisome complex
- replisome moves along the DNA as the DNA is unwound
- daughter strands of DNA are synthesized
joining and/or termination
- joining of replication forks or termination of DNA synthesis
- separation of the duplicated chromosomes
two types of DNA polymerases
- DNA-dependent DNA polymerase (or DNA polymerase)
- DNA Repair Polymerase
DNA-dependent DNA polymerase (or DNA polymerase)
- also known as DNA replicase
- is only one subunit of a large protein assembly called the holoenzyme
- responsible for semiconservative replication
DNA repair polymerase
- responsible for excising damaged DNA bases
- then synthesizing new DNA to replace the excised DNA
all DNA polymerases:
- synthesize DNA antiparallel from 5’ to 3’
- require a template that is 3’ to 5’
-nucleotide choice is determined by complementary base pairing with the template - require a free 3’-OH to add nucleotides to
- polymerase makes the phosphodiester bond
bacterial DNA polymerases
- bacterial (& eukaryotic) cells have several different DNA polymerase enzymes
- DNA polymerase III - is the bacterial DNA polymerase
- does semiconservative replication
repair DNA polymerases
DNA polymerase I
- repair of damaged DNA
DNA polymerase II
- required to restart a replication fork when the process is stopped by DNA damage
DNA polymerase IV and V
- allow replication to bypass some types of DNA damage
- error-prone polymerases
DNA polymerase I
- only polymerase that can do Nick Translation
- DNA polymerase I has a 5’-3’ exonuclease activity
- can remove nucleotides in front of the polymerase
- it can also recognize a single strand nick in the DNA
- inserts a 5’-3’ exonuclease removes nucleotides ahead of the polymerase as it makes new DNA
Klenow Fragment
- DNA polymerase I cleaved with an enzyme
yields 2 parts:
1. larger fragment = polymerase + proofreading enzyme
- used in the laboratory to synthesize DNA
- smaller fragment is the 5’-3’ exonuclease
fidelity of DNA replication
- how well does the polymerase copy the DNA?
- substitutions = inserting the wrong base
- frameshifts = inserting an extra nucleotide or deleting a nucleotide
- DNA polymerases have a proofreading ability
-due to a 3’ to 5’ exonuclease- is used to excise incorrectly paired bases
-detects A-C or A-A mispairing
- is used to excise incorrectly paired bases
DNA synthesis
- dNTP binds to template base by complementary binding
- polymerase catalyzes joining of phosphate to 3’OH of primer
- using energy of pyrophosphate release
DNA polymerases have proofreading ability
- if wrong base is inserted and added to the chain
-causes a warp in the chain that slows the polymerase - allows the 3’-5’ exonuclease to back up and remove that wrong base
- polymerase then has the chance to add the right base
- the fidelity of replication is improved from proofreading by a factor of ~100
so DNA polymerase I has:
- polymerase to synthesize DNA 5’-3’
- 3’ to 5’ exonuclease for proofreading
- 5’-3’ exonuclease that can allow Nick Translation
-removes bases in front of the polymerase
(note: all bacterial DNA polymerases have the 3’-5’ proofreading exonuclease)
DNA polymerases have a common structure
the right hand
- DNA lies across the “palm”
-catalytic site - a groove is created by the “fingers” and “thumb”
- DNA slides through the groove as synthesis continues
DNA synthesis differs on each DNA template
leading strand synthesis
- DNA synthesis goes 5’-3’
- DNA pol advances continuously when it synthesizes the leading strand
problem is the “lagging strand”
- DNA synthesis MUST go 5’-3’
- as DNA synthesis on the lagging strand beings
-the leading strand is still moving ahead
-this leaves a gap on the lagging strand
-so the pol must bind again near the fork and start a new strand
lagging strand DNA synthesis is discontinuous
- lagging strand is synthesized in many fragments
- called Okazaki fragments
- these Okazaki fragments must then be connected together
helicase
separates (or melts) the DNA duplex
- E.coli has 12 different helicases
- most are multimeric
- most common is a Hexamer
helicase action
- helicase encircles a ssDNA strand
- alternates between a dsDNA binding conformation and a ssDNA binding conformation
- this separates the DNA strands
- requires the energy of ATP hydrolysis
unwinding of DNA exposes ssDNA
must:
- protect the ssDNA
- prevent it from re-annealing with the other strand
-
leading strand DNA is synthesized quickly
-no real need to protect the ssDNA -
lagging strand DNA must wait until there is sufficient space to begin an Okazaki fragment synthesis
-must protect this ssDNA
single-stranded binding proteins (SSB)
- SSBs bind to the lagging strand ssDNA after unwinding by helicase
- SSB binding is cooperative
-binding of one SSB enhances the binding of subsequent SSBs
DNA synthesis requires a primer
- all DNA pol require a 3’-OH priming end to initiate DNA synthesis
RNA primer is provided by primase
- small RNA polymerase
-makes RNA 5’-3’
-does not require a free 3’-OH - makes an RNA primer for DNA synthesis
- associates with the oriC complex in bacteria
-which binds to the origin - recognizes the origin and synthesizes ~10 bases of RNA complementary to the DNA at the origin
priming DNA synthesis at the origin
requires:
- binding of the helicase to unwind the DNA
- binding of SSBs to protect and prevent re-annealing
- primase binds and makes a complementary 10 bases of RNA
coordinating synthesis of the lagging and leading strands
- leading strand is synthesized continuously
- lagging strand is synthesized discontinuously
- polymerase must continually detach and move back to the replication fork
-
E.coli uses the same polymerase for both (circular chromosome)
- eukaryotes use different enzymes for leading and lagging strands
DNA polymerase III Holoenzyme (replisome) subunits
-
2-DNA polymerase III catalytic cores
-a catalytic subunit (alpha)
-and a 5’-3’ proofreading subunit -
2 clamps
-ensures processivity = that the polymerase stays with the DNA -
a clamp-loader/dimerization complex
-loads the DNA into the clamps
-brings the 2 polymerase complexes together
DNA polymerase holoenzyme assembly
- a clamp loader complex assists in loading the circular clamp around the DNA strand
- catalytic core polymerase associates with each template strand
- the dimerization subunits of the clamp loader helps in dimerization of the 2 core polymerases
- the clamp loader stays associated with the polymerase on the lagging strand
- it will be needed to reload the polymerase after each Okazaki fragment
the “sliding” clamp
- the clamp forms a ring around the DNA
- holds the polymerase strongly to the DNA
- makes the polymerase
highly processive =
stays with the DNA
- makes the polymerase
- the clamp associated with the polymerase on the lagging strand dissociates at the end of each Okazaki fragment and reassembles for the next fragment
DNA synthesis
see ch. 11 notes
status at this point
- leading strand continuing to replicate DNA
- lagging strand
-newly synthesized Okazaki fragment terminate just before the previous Okazaki fragment
-must remove the RNA primer
-synthesize DNA to replace the RNA primer
-seal the last phosphodiester bond between the Okazaki fragment
removing the RNA primer (in bacteria)
- DNA polymerase III dissociates leaving a gap and the RNA primer
- DNA pol I uses the “nick” to attach and uses its 5’-3’ exonuclease to remove the RNA primer and replace with DNA
ligation
- DNA pol I is released leaving a “nick” between the Okazaki fragments
- DNA ligase seals the remaining phosphodiester bond at the nick
- DNA ligase uses energy from ATP to catalyze the reaction
DNA replication in eukaryotic cels
eukaryotes have 3 polymerases for DNA synthesis
-
DNA polymerase alpha/primase initiates synthesis of new strands
-has DNA pol activity
-and RNA pol activity for making its own primer - DNA polymerase ε synthesizes the leading strand
- DNA polymerase δ synthesizes the lagging strand
- all 3 are linked in the replisome
eukaryotic DNA replisome is similar
- helicase (MCM) binds the DNA
- pol α/primase detaches (but remains with replisome)
- clamo loader (RFC) mediates binding of the clamp (PCNA) to the ssDNAs
- clamp and pol δ release to make another Okazaki fragment
- but in subsequent okazaki fragments, pol δ continues and into the RNA primer
primer removal and ligation
- polδ displaces the RNA primer
- leaving a flap of RNA
- FEN1 nuclease removes the RNA primer flap
- polδ then fills the gap
- DNA ligase I seals the remaining nick
what if the polymerase encounters an error or nick in the DNA?
DNA synthesis stops and replication fork may collapse
3 options:
- cell death (not really acceptable)
- skip over the lesion = lesion bypass
- repair by recombination
lesion bypass requires polymerase replacement
- the replication fork stalls
- the replication complex must be replaced by error-prone polymerases
-DNA pol IV or V (for bacteria)
-can copy through the error and incorporating the error - error-prone polymerases removed
- restart DNA synthesis with primosome
-primase and polymerase complex that restarts synthesis - then primosome is replaced by the replisome
recombination to repair the lesion
- excise damage
- information from the undamaged other strand is used to repair the damaged sequence
- DNA synthesis is restarted
- DNA repair is used to fix the gap later
termination of replication
bacteria:
- the two replication forks meet halfway around the circle
- ter sites that cause termination if the replication forks go too far
termination of replication
eukaryotes:
- telomer region
- leading strand completes replication
- lagging strand has unreplicated end
simple repairs
includes:
1. direct reversal of DNA damage
2. base excision repair
- remove the base and replace with appropriate base
3. nucleotide excision repair
- remove and replace the whole nucleotide
4. mismatch repair
- requires determination of “new” and “old” DNA strands
recombinant repairs
- repair mechanism that retrieve an undamaged sequence from the other replicated DNA strand
- nonhomologous end joining
- translesion/error-prone repair
replication errors
- missed by the 3’-5’ proofreading exonuclease
- results in a major distortion of the DNA strands
- repaired by the mismatch repair system
-if not, becomes a permanent change in the DNA
spontaneous base deamination mutation
e.g. spontaneous deamination of cytosine to uracil
-can also happen to other bases
- creates a mismatched U-G pair
- results in a minor structural distortion of the DNA
- uracil is preferentially removed by base excision repair
loss or removal of a base
depurination
- spontaneous loss of a base by hydrolysis
- blocks DNA replication and transcription
- corrected by nucleotide excision repair system
mutagens
- substances that increase the rate of mutation by inducing changes in the DNA sequence
- either directly or indirectly
carcinogen
a substance that promotes the formation of cancer
UV light
- UV light at ~260nm is absorbed by the bases
- can cause a photochemical fusion of two pyrimidine bases side-by-side
-
thymine-thymine dimer
-covalent bonds - blocks replication and transcription
- corrected by nucleotide excision repair
alkylating agents
- includes mutagens like nitrosamines
- found in tobacco products or produced from some food preservatives like nitrates
e.g. methylation of guanine
- creates a bulky base that distorts the DNA
- inhibits replication and transcription
methyltransferase
used for direct reversal
- transfers the methyl group to the methyltransferase
reactive oxygen species
- oxidizing agents generated by ionizing radiation or chemical agents
- generates free radicals
- superoxides, hydroxyl radicals, hydrogen peroxide, etc.
- oxidation of guanine
- causes base pairing with adenine
- correct by mismatch repair
ionizing radiation
- gamma radiation or X-rays
- directly ionizes the deoxyribose
(indirectly causes reactive oxygen species) - causes double strand DNA breaks
- kills rapidly proliferating cells (cancer treatment)
- corrected by recombinant repair
direct reversal of DNA damage
- alkylating agents resulting in methylation of guanine to methyl-guanine
-
methyltransferase
- transfers the methyl group to the methyltransferase
base excision repair
- for spontaneous base deamination
- cytosine deamination to uracil
- also alkylated bases
- causes the removal of an individual damaged base
- glycosylases cleave the bond between the deoxyribose and the base
- results in a removal of a base
DNA pol β
- eukaryotic DNA pol specific for base excision repair
- replaces the nucleotide
the nucleotide excision repair system in bacteria
- removes mispaired or damaged bases then synthesizes a new stretch of DNA to replace them
- used for almost all excision repair in E.coli
E. coli Uvr system
- UvrAB dimer recognizes the damage
- UvrA is released
- UvrC joins UvrB
- UvrBC cuts DNA strand above and below the damage
- UvrD (helicase) then unwinds DNA
- DNA pol I then binds
- removes the damaged area as it synthesizes new DNA
- DNA ligase seals the nick
excision repair system - eukaryotic
Xeroderma pigmentosum (XP)
- a human disease caused by mutations in any one of several nucleotide excision repair genes
- hypersensitivity to sunlight and UV light
- results in skin diseases and cancers
- disease was helpful in understanding the excision repair system
two major pathways in eukaryotes
- global genome repair
- transcription-coupled repair
global genome repair
XPC protein detects the damage and initiates repair
transcription-coupled repair
- damage is recognized by the RNA polymerase during transcription
- RNA pol stalls at the damage
- the RNA pol is released
both then use similar pathways
- TFIIH transcription factor complex binds
- helicase in complex unwinds the DNA
- complex endonucleases cut DNA above and below damage
- DNA pol δ/ε
-eukaryotic replisome polymerases - excises and replaces the gap
- DNA ligase seals the nick
mismatch repair - bacteria
- purpose is to repair mismatches immediately after DNA replication
- relies on MutS complex
- dimer that embraces the DNA
- moves along the DNS looking for distorted DNA due to mismatched bases
but how does MutS know which strand has the wrong base?
- after DNA synthesis in bacteria
- Dam Methylase methylates DNA at -GATC- sequences all along the DNA
- but shortly after synthesis, new DNA is not methylated
- so unmethylated DNA would have the mistake
-the original template DNA would be methylated
mismatch repair mechanism
see ch. 14 notes
mismatch repair in eukaryotes
- similar to E. coli
- eukaryotic DNA is methylated BUT mismatch repair DOES NOT use methylation to identify the new strand
how do eukaryotes recognize the correct DNA strand?
- mismatch repair is coupled to the DNA synthesis machinery
- uses nicked Okazaki fragments to ID new strands
- also associated with clamp to determine the new strand
- then uses a similar mechanism to remove and replace the mismatch nucleotide
genetics
patterns of inheritance
- single genetic locus or just a few loci
genome
an organisms complete set of DNA, including all of its genes
- also includes mitochondrial and chloroplast genomes
- extrachromosomal DNA and other forms of inheritance
genomics
genome-wide studies via DNA or RNA sequencing + bioinformatics
classical DNA sequencing: Sanger method
main principle: chain termination method based on terminating DNA synthesis one nucleotide at a time
components:
1. DNA primers (only one per sequencing rxn)
2. DNA pol
3. nucleotides (dNTPs)
4. labeled modified nucleotides dideoxynucleotides (ddNTPs)
dideoxynucleotides (ddNTPs)
chain-terminating nucleotides, lacking a 3’-OH group required for the formation of a phosphodiester bond during DNA elongation.
incorporation of a dideoxynucleotide into the elongating DNA strand therefore terminates the extension, resulting in DNA fragments of varying length
classical sanger method
see notes
dye terminator sequencing
- an easier way
- sanger method based
- non-radioactive
- more automated
