Exam 2 Flashcards
Requirements for DNA synthesis
helicase, polymerase, primer, base pairs (dNTPs), ligase, template strand, and primase
DNA polymerase
Fingers
The fingers are like the grabbers of DNA polymerase.
They help grab onto the building blocks (nucleotides) and the DNA template.
Think of them as the hands that hold onto the parts needed for building the new DNA strand.
Thumb
he thumb is like the stabilizer of DNA polymerase.
It helps hold the DNA polymerase onto the DNA strand.
Picture it as the part of the hand that keeps the DNA copying machine steady while it’s working.
palm
The palm is like the workspace of DNA polymerase.
It’s where the actual building of the new DNA strand happens.
Imagine it as the table where the DNA copying machine puts together the new DNA strand.
template
The template is like the guide for DNA polymerase.
It’s the old DNA strand that serves as a template for building the new strand.
Think of it as the blueprint that DNA polymerase follows to make the new DNA strand.
primer
The primer is like the starter for DNA polymerase.
It’s a short piece of RNA or DNA that provides a starting point for building the new DNA strand.
Picture it as the first piece of a puzzle that DNA polymerase uses to begin copying the DNA.
The palm catalysis
Site of catalysis
(finish)
Palm 3 prime exonuclease proofreading
error rate:
E,coli poly with no exonuclease domain
with error: in 10,000 nt (10^5)
exonuclease domain: about 1 error in 1,000,000 nt with this (100 fold better fidelity)
Fingers
O-helix: Interacts with incoming nt and keep clamped down to help phosphier bonds ( keeps them still and in place for bonding)
Forms ionic interactions with negative phosphates ( secure)
R group: Base stacking interactions stabling ( bricks adding extra stability)
o helix
clamps down incoming dNTPs (building blocks of DNA)
Tyrosine forms stabilizing base stacking with nitrogenous base (like the glue and ATCG are the blocks glued together)
Lys and arg (magnents) interact with positive phosphate
help enzyme hold onto DNA strand tightly while working. (grabbing onto a steering wheel)
Thumb
maintains interactions with the primer (starter) and the template (old strand) strand (holding something in place while fixing it , new DNA made)
promotes processivity (adds new DNA quickly and not stop)
high processivity= high syth speed (nt/sec) (many DNA at once)
processivity
Rate of nt (building blocks) added per polymerase binding
DNA synthesis – One cycle of DNA polymerase
- Incoming nt forms H-bonds base pairing w/ next available template base (finding the right puzzle piece to fit the space)
- The fingers closed around the new nucleotide (grabs it so it doesn’t leave)
3.Base moves into alignment with critical catalytic metal ions (helpers to make sure everything is right)
- Formation of new base pair phosphodiester bond (new building block to growing strand like snapping puzzle pieces together)
- Reopening of fingers and movement of the primer template junction up one nt (thumb “usually” holds polymerase on the strand)
( like taking a step forward in a line. The thumb helps keep it attached to the DNA strand, like a stabilizer)
Replication initation
ORI:
-site of begining of DNA rep
- AT rich (easier to break many nt at the same time to form the replication bubble)
Replicon: seq of DNA replicated from a single ORI
all the DNA that’s replicated from a single starting point (the ORI)
Think of it as a segment of a book that you’re photocopying—it’s all the pages you copy from a single starting page.
Replicator: All of the proteins required to replicate the replicon
(group of people with photocopy machines and supplies needed to copy the book segment (replicon) )
Initiator: Protein complex required for replication initiation (part of replicator)
Imagine it as the coach who says “Go!” to start the race (replication) at the starting line (ORI).
Rep init in euks
Histones: Shifted away from DNA sequence to be replicated and then shifted back
(DNA’s bodyguards.
wrap around , protecting it and keeping it organized)
When it’s time to copy the DNA, histones temporarily move out of the way so the copying machinery can access the DNA sequence.
Afterwards, they shift back in place, like guards returning to their posts after a break.
Pre-rep complex: All proteins bound to the ORI prior to replication initiation
(pre-rep like a team getting ready for a big job,
all proteins that gather at (ORI) before DNA replication begins. the crew assembling all their tools before starting work.)
ORC: complex of initiator proteins that bind to the ORI (recruits Cdc6 and Cdt1) ( as a team of explorers planting a flag at the starting point to signal where the race (replication) will begin)
Cdc6 and Cdt1: Recruit the helicase MCM2-7 (one on each end of complex)\
Imagine them as managers hiring workers to set up the equipment needed for the job.
Separation of double helix
DNA helicase (molecular zipper):
- breaks H-bonds between nt (unzipping or opening up the double helix)
- endothermic reactions so hydrolyzes ATP to break bonds (needs energy, so it uses ATP)
- Hexametric ring complex that encircles one single strand of DNA ( hula hoop around a dancer’s waist)
SSBs (DNA’s bodyguards):
single stranded binding protein
very basic and polar (parts that are attracted to the DNA’s negative charge )
bind very quickly to ssDNA (making sure nothing harms it.)
Topoisomerase(untangler):
- relieves positive supercoiling caused by replication (cut and reattaches)
-causes negative supercoiling
- DNA gyrase type 2 topisomerase breaks both strands
Priming for DNA synth
Limitations of DNA Poly
- needs a primer to start syth (Without the blueprint, the builder doesn’t know where to start)
primase
- RNA polymerase that makes short 5’ RNA priers 5-10 nt on a ssDNA substrate (drawing the first lines on a canvas before an artist starts painting.)
Leading strand
- requires 1 initial primer (Imagine it like driving on a clear road without any traffic jams)
Lagging strand
- new primer for every Oskaki fragment (driving on a road with traffic lights where you have to stop and start multiple times.)
Why primase does not form primers randomly in rep bubble?
Primase - Only comes in to work to build Okazaki frags
- binds to helicase and its activity increases 1000 fold over unbound (productivity skyrockets so it runs smooth)
Step 4 Other players
DNA Sliding Clamp:
Beta clamp (hula hoop that encircles the DNA strand, keeping DNA polymerase in place like a guide.)
Thumb not good enough
- 2nd protein complex ring that encircles (better grip)
Clamp loader (y complex):
Uses ATP to load B-clamp onto DNA (worker using a tool (ATP) to place the hula hoop (sliding clamp) onto the DNA.)
t Protein:
-Flexible arm protein that binds the y complex and DNAP (bridge that connects two separate parts of the replication machinery.)
DNA Pol 3 holoenzyme:
many proteins in acomplex where all functions help a main protein (group of helpers surrounding DNA polymerase, providing support and assistance to ensure efficient replication.)
DNA polymerase in E.coli
Pol 1
- DNA repair and primer removal poly, low processivity (not good at long DNA)
3’ - 5’ and 5’ -3’
Pol 2
Everything in poly 1 minus primer removal ( like backup)
Pol 3
Main rep poly (processivity – 50 nuc per binding)
Exonuclease activity: 3’-5’ (proofreading)
Pol 4 and 5
- Transversion poly (awesome!)
DNA poly in Euks
Pol a:
- Binds DNA and acts as a primase to Syth the RNA primer ad changes confirmation and continues synth DNA
(worker who first draws the outline of a picture (making RNA primers) and then fills in the details (synthesizing DNA) to complete the job)
Pol s:
- main rep ply in Euk on the lagging strand
(lead worker handling most of the construction work on a complex project.)
Pol e:
- main rep ply in Euk on the leading strand
(lead worker handling the bulk of the construction work on the smoother, more straightforward part of the project.)
Steps 5; Finishing up
RNase H:
- degrades the RNA primer 5’ exonucleus degrades lost RNA nt (chops off RNA nucleotides one by one from the 5’ end, acting like a pair of scissors trimming away the excess RNA.)
DNA polymerase:
Syth across gap until leaves a “nick” (works across the gap until it reaches the end, leaving behind a small unconnected spot called a “nick” in the DNA strand.)
DNA ligase:
Cant add nt but repairs gap (glue that sticks the pieces of a broken road back together, sealing the nick left behind by DNA polymerase.)
Nucleosomes:
Moved back in place (shelves being temporarily shifted aside during construction and then put back in place once the work is done.)
Finish rep for linear
End of rep problem:
Poly needs RNA primer, cant syth last set of nt bound to the most 5’ primer (lagging and leading)
Telomeres:
Potential advantage (gives Euk security that cells cant forever divide)
Cancer expresses telomerase
Semiconservative
Each progeny gets some new and some old DNA
Bidirectional
Replicate in both directions
Semidiscontinous
Leading strand: Continues
Lagging strand: Discont.
Semi for both
Priming for DNA synth
All DNA Polys seem to need a primer
When can mutations occur
During and NOT during replications
3 potential repercussions of mutations
Delanteous: 2nd most common
Advantageous
Neutral: most common
Point mutations
Mutation of single base pair/nt
Translation:
Pyr to Pyr (Pur to Pur)
Transversions:
Pur to Pyr (Pyr to Pur)
Consequenes in a coding reigon
Silent:
No change in amino seq
Missense:
Change in amino seq
Nonsense:
Stop codon (early end)
Neutral:
No change in Protein function
The Problem and what we see in the cell
Replication Error Rate: – 1 in 10^7 nt
See: – 1 error in 10^10 (needed for survival)
Why: If we don’t have this (fidelity) cells eventually die from too many mutations
Others: Lots from enviorment
Mutant
Mutant genes and organisms
Mutagen
Anything that can cause a mutation
Mutagenisis
Process of causing mutation
Site-Specific mutagenesis
Intentional mutation of a specific DNA (in a lab)
Classification of Mutations
Base addition/deletion (frameshift): can be any # of nucleotides. Add or delete multiples of 1 or 2 nt (very severe changes amino acids)
Huntington’s: CAG repeats (not frameshift but severe)
Conditional Mutations: exhibit the mutant phenotype under specific conditions both at molecular and physiological
Mismatch repair following DNA replication
The problem: You WILL get errors in DNA replication
The causes: Mis-incorporation of bases (“wrong” ones added)
Base tautomerism (a diff process where wrong nt added)
Mismatch by tautomeric forms of bases
Base pairing of imino and keto forms
Adenine (imino) binds to cytosine major form (amino form)
cytosine (imino) binds to Adenine major form (amino form)
Thymine (enol) binds to guanine (keto)
guanine (enol) binds to Thymine (keto)
E.coli mismatch repair
effectiveness: Increases accuracy of DNA synthesis by 2-3 orders of magnitude
Time is a factor: cell has one round to fix errors before undetectable
3 enzymes in E.coli mismatch repair mechanism
MutS: A protein dimer that scans the DNA for regions that are readily distorted (detection mechanism) (I.e. Have incorrect base pairing)
- if DNA is disordered MutS binds and stays and changes conformation
MutL: Binds MutS and recruits MutH
MutH: When bound to MutL makes a nick in the weakly synthesized
- nick can be up or downstream
After mismatch has been identified and marked
UvrD helicase: Unwinds DNA in direction of MutS
Exonuclease: Degrades the unwound DNA pass site of MutS binding
DNA Pol 1-2 and ligase: Binds to new 3’ OH and synthesizes across the gap
- DNA ligase repairs the remaining nick
Diff exonuclease for diff directions
If nick is on 5’ end: Degraded by Exo V11 OR RECJ exonuclease (5’ to 3’ direction)
On 3’ end: Degraded by Exo1 (3’ to 5’)
how does Ecoli know which stand is right
needs memory mechanism
- know which is OG strand
- if there’s a mismatch assume old strand is correct and new strand needs change
DNA methylation
- Ecoli methylates its DNA for other reasons hut evolve to be the way to tell which is the “new” strand
Enzyme responsible:
- DNA methyl transferase
After rep:
- once a new strand is methylation…
- really the cell has seconds to bind and repair the error before possible paramount mutation
mismatch in euks
Homologs of..
MutS: MSH proteins
MutL; MLH proteins
No MutH
Lack MutH and methylation:
Recent evidence sighed that euks use the nicks between Okazaki fragments as markers of parent and daughter strand
Forms of DNA Damage
DNA damage can have two effects:
1) base pair dimers, nicks or breaks in the backbone can create blocks that stop DNA replication or transcription (structured changes and “certain death”)
2) Mutations that changes the DNA sequence (“possible death”)
Types of DNA damage
Deamination’s: Loss of Nh3 from the nitro base ( replaced by oxygen)
1) Cytokine to Uracil
Result: GC pair became an AT pair
2) Adenine to Hypoxanthine
Result: Hypoxanthine binds w/cytosine
AT pair becomes GC pair
3) Guanine to Xanthine
Result: Xanthine pairs/binds w/cytosine
- if unfixed, xanthine and cytosine only bind w/ 2 H bonds not 3
Depurinations:
1) N’ glycosylic link in purines broken
Result: Nitro base removed and becomes an “abasic” base ( no nitro base is a sugar phosphidier bond)
even more sources of mutation
3) UV light causes fusion of adjacent pyrimidines
Mutagen: UV light
Result: Adjacent pyrimidines become crosslinked (form covalent bonds w/eachother)
- most common example: Thymine dimera
Gamma & X-ray irradiation causes ds breaks
Mutagen: Gamma xrays and Xray exposure
Result: Cause double strand breaks and certain death not fixed
Intercalating chemicals inserting into DNA
Mutagen: Flat, planar molecules that insert into the DNA
Result: Cause polymerase to skip ahead or behind during sythnesis
How Cells Can Repair DNA Damage
3 Basic Strategies
1) Direct Repair:
Cell recognized mutation and specifically fixes it
Double Strand Break Repair:
Also a direct pair mechanism
when both strands are broken, sequence information can be obtained from the undamaged copy of the chromosome
Bypassing the problem:
Goes from certain to potential death
when polymerase is blocked, a translesion polymerase synthesizes across the error site
Direct repair systems
1) Photoreactivation: Repairs pyrimidine dimers
- proks have “DNA photolyase” which observes visable light and breaks dimers
Removing alkyl groups: methyl from O6-methylguanine removed by methyltransferase (Turnover: Removes methyl group off O6 - methylguanine turnover =0 when it puts on amino acid its done so not really enzyme
3) Base Excision Repair:
General repair pathway for identifying and removing unnatural nt in genome
Base excision repair
1) Glycosylase binds to the unnatural nuceotide cuts off the nitrogenous base and creates abasic base ( nitro bade is now OH)
2) Exonuclease cuts the phosphoiester bond st the 5’ end of the abasic base
b))leaves a 1 nt gap
3) DNA POL 1 fills 1 nt gap and ligase makes the last phosphidiester bond
Nucleotide excision repair
1) UvrAB kermit
Recognizes DNA distortions from a mismatch and binds that region of DNA
2) UvrA Kermits eyes
Helps bind mutated DNA then leaves once bound
3) UvrB kermits mouth
Bins mutated sequence andrecruits UvrC
4) UvrC earmuffs
Bind UvrB and makes two nicks in DNA one at either side of UvrB
5) UvrD
Binds UvrB and unwinds DNA between two stands ( release cut ss fragments)
6) DNA poly and ligase
Pol fills in gap and ligase makes last bond
IN euks
Have basically the same pathway
- mutations in pathway lead to Xerderma pigmatosa a very high sensitivity to light
Recombinational Repair
Double strand break repair:
-a diploid organism has 2 copies of each chromosome
-cells can use homologous recombination to fix the broken chromosome using the unbroken chromosome as a template
- yeast (simple eukaryotes) can do this but humans can not & fix with no errors (original State)
Non-homologues end joining
- Humans do this
- chop off DNA & put back together but now the original state can not be achieved
- repairing breaks path way in multi
cell euks
- aligns 3’ends, trims them repairs
- 100 percent mutagenic
- certain death to possible
Tranleision DNA synth.
Replication can bypass DNA damage
- POl 4 & 5 are translesion Polymerases
- causes stop in synthesis (dimers) are good example
- becomes a potential but replication can continue
- pathway of last resort when polymerase encounters a block jn DNA during replication
- performed by Translesion polymerases
- syths across the block in the template strand using an internal RNA template subunit
- guaranteed to be mutagenic and common source of mutations in organisms
- changes the result of the mutation from certain to uncertain death
- translesion polymerases are very well conserved (Y family of polymerases)
