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And even more repair

Submitted by sjurgilewicz on Thu, 04/27/2017 - 22:39

To fix depurination and deamination, base excision repair is used. In BER, glylocalyse flips out the wrong base and cuts it off. It is called apurinic or apyrimidinic once the base has been cut out. Next, AP endonuclease cuts the sugar and the phosphate out. DNA polymerase and ligase put in the new nucleotide and seal the gap. Thymine dimers, which are covalently linked and distort the helix, are created by UV radiation. This is a bulky distortion in the DNA and DNA polymerase cannot make it through. Some special DNA polymerases can replicate using this damaged DNA, but they are error prone and not used often. Thymine dimers are fixed by nucleotide excision repair. In NER, the nuclease cleaves around the bulky lesion. Helicase is required to pull apart the DNA, but it is a specialized helicase. Clamps are needed for BER & NER to recruit proteins and active the excision.

Xeroderma Pigmentosum is a rare recessive disorder, where UV damage cannot be repaired in the DNA through NER. Skin lesions and skin cancer occur from the sun.

Double strand breaks in the DNA result in cancer, premature again, apoptosis and chromosome reengagement. The cell does do breakage on purpose during recombination and VDJ. To fix accidental breakage, non-homologous end joining and homologous rearrangement are used. Non-homologous ending joining happens by nucleases come in and create blunt ends and remove several bases and seals back together. Loss of genetic information occurs but it is very commonly used in cells during G1. Homologous rearrangement requires for a separate piece of DNA that the matches the broken on exactly. The DNA is completely repaired. The 3’ end overhangs and invades the other strand that looks like the damaged DNA and base pairs. DNA polymerase synthesized across the area using the other strand as template. Ligase fixes the nick. This repair occurs in s phase, g2 phase, m phase. Basically when another copy of the DNA is present. 

More on repair

Submitted by sjurgilewicz on Thu, 04/27/2017 - 21:12

All repair occurs post-replication. Errors and fixes always occur in the newly replicated strand. Mismatch repair must occur soon after replication or else errors will be passed along and turned into mutations. How does the cell recognized which is the template and which is the newly replicated? In E. coli, detection occurs through methylation or lack of. The adenine gets methylated on the template strand, replication occurs. An enzyme is used to recognized the methylated adenine. Therefore, it is known which is the newly synthesized. There is also a nick present. Mismatch creates a bump, the mistake is cut out and then some extra, approximately 2000 nucleotides long. Helicase, DNA pol, exonuclease, DNA ligase, ssDNA binding protein, endonuclease and clamp/clamp loader are all some of the things needed for mismatch repair to occur.

  1. Initial MMR bind proteins
  2. Endonuclease, creates nick
  3. Helicase, unwind strands
  4. Exonuclease, remove piece of strand greater than the error
  5. SSB, bind to ssDNA
  6. Clamp/clamp loader/ DNA pol synthesize across gap
  7. DNA ligase

If cells had no way to distinguish between newly synthesized DNA, how often would yo expect to see mutations from mismatch base pairing? 50% of the time.

DNA damage is caused by UV, x-ray, tobacco, reactive oxygen. Depurination occurs when entire base comes off. Deamination occurs when cytosine converts to uracil. 

Fixing DNA replication errors: PP

Submitted by sjurgilewicz on Thu, 04/27/2017 - 15:30

The error rate of DNA polymerase is 1 error in every 10^5 nucleotides added. There is a proofreading function to look for errors such as the wrong base. DNA polymerase has a 3’ to 5’ exonuclease to remove the wrong bases. An exonuclease is an enzyme that destroys polymers from the ends of the polymers as compared to an endonuclease that cuts in the middle of the polymer. Nucleases are RNA specific (RNase) and DNA specific (DNase). They can work in either 3’ to 5’ direction or vice versa. During replication the error rate is 1 in 10^7 bases. Mismatch repair occurs after replication has occurred and the error rate is 1 in 10^9. When an error is sensed, the base is flipped out and an enzyme comes and cuts it off. DNA pol 3 is the main DNA polymerase. DNA pol 1 removes primer and fills in the gap from lagging strand synthesis. DNA pol 1 must have polymerase catalytic activity and both direction exonuclease activity. 

Why does replication only go in one direction? It could be due to proofreading. Energy is still able to come in properly from a new nucleotide.

If synthesis occurred the other way, then the energy would be cut off.  Our genome is 3 billion base pairs in length, how many mistakes are being made during DNA replication each time our cells divide? 1 in a billion mistakes, 6 billon nucleotides, 6 errors on average. 

If someone has a functional proofreading but a nonfunctional mismatch repair, how many more mutations will be present compared to someone with functional mismatch repair machinery? (assuming genome is 3 billion base pairs long): 6 billion nucleotides added/ 1 in 10^7 error rate = 600 more mutations.

There is an end replication problem: when the primer is removed, there is no 3’ end to synthesize and add to that end. The cell will chew back on itself on the 5’ end and the telomere region will shorten each time. The telomere has repeating nucleotides without coding for anything. If the cell doesn’t have telomeres and is naked, DNA damage repair will be activated. To prevent this, DNA bends back on itself and binds proteins. Most cells get shorter each time they divide. There is a way to fix using telomeres: DNA protein structures, prevent cell from generating DNA damage response. Once telomere is too short, apoptosis occurs. This is associated with aging. Cancer cells over express telomerase (who to keep telomeres longer).

Fixing DNA replication errors

Submitted by sjurgilewicz on Thu, 04/27/2017 - 14:57

The error rate of DNA polymerase is 1 error in every 10^5 nucleotides added. There is a proofreading function to look for errors such as the wrong base. DNA polymerase has a 3’ to 5’ exonuclease to remove the wrong bases. An exonuclease is an enzyme that destroys polymers from the ends of the polymers as compared to an endonuclease that cuts in the middle of the polymer. Nucleases are RNA specific (RNase) and DNA specific (DNase). They can work in either 3’ to 5’ or vice versa. During replication the error rate is 1 in 10^7. Mismatch repair occurs after replication has occurred and the error rate is 1 in 10^9. When an error is sensed, the base is flipped out and an enzyme comes and cuts it off. DNA pol 3 is the main DNA polymerase. DNA pol 1 removes primer and fills in the gap from lagging strand synthesis. DNA pol 1must have polymerase catalytic activity and both direction exonuclease activity. 

Why does replication only go in one direction? Could be due to proofreading. Energy is still able to come in properly from new nucleotide. If synthesis occurred the other way, then the energy would be cut off.  Our genome is 3 billion base pairs in length, how many mistakes are being made during DNA replication each time our cells divide? 1 in a billion mistakes, 6 billon nucleotide, 6 errors on average. If someone has a functional proofreading but a nonfunctional mismatch repair, how many more mutations will be present compared to someone with functional mismatch repair machinery? (assume genome is 3 billion base pairs long): 6 billion nucleotides added/ 1 in 10^7 error rate = 600 more mutations. End replication problem: primer is removed, no 3’ end to synthesize and add at the end. Cell will chew back on itself on the 5’ end and the telomere will shorten each time. The telomere has repeating nucleotides without coding nucleotides. If the cell doesn’t have telomere and is naked, DNA damage repair will be activated. To prevent, DNA bends back on itself and binds proteins. Most cells get shorter each time they divide. There is a way to fix. Telomeres: DNA protein structures, prevent cell from generating DNA damage response. Once telomere is too short, apoptosis, associated with aging. Cancer cells over express telomerase (who to keep telomeres longer) 

Mechanism of DNA rep

Submitted by sjurgilewicz on Wed, 04/26/2017 - 13:26

Replication starts at the origins of replication. The pre-RC is made of 6 subunit heterohexamer found at the origins of replication. Replication happens bi-directionally, two replication forks go in different directions. Humans have 6 billion base pairs of DNA with many origin of replications to have replication happen in a timely manner. Origin firing is where replication starts, some fire early in S phase while others fire in late S phase. Each strand of DNA is used as a template and a new strand is synthesized from 5’ to 3’. The new nucleotide is added at the 3’ end to the previous nucleotide. DNA polymerase cannot make a strand without a previous nucleotide to add to with the free 3’ OH group. Primase adds an RNA primer that is complementary to the template strand of DNA, which supplies the 3’ end for the DNA to build off of. The incoming nucleotide is a triphosphate, which supplies energy for the reaction. A pyrophosphate is released and gets recycled when the bases pair. The H from the 3’ OH is removed and a bond is formed between this oxygen and the previous 3’ OH. This is call a phosphodiester bond. The leading strand can be synthesized continuously, but the lagging strand is replicated in pieces called Okazaki fragments. DNA polymerase cant attach the fragments, so DNA ligase seals the gap.

The lagging strand loops back on itself, 180 degrees, which allows for the same direction synthesis as the leading strand. There are specific proteins present during replication, helicase (pulls apart DNA), DNA polymerase, sliding clamp (keeps DNA pol. from falling off of DNA so replication can continue) and sliding clamp loader.

DNA replication: PP

Submitted by sjurgilewicz on Fri, 04/21/2017 - 11:17

When protiens interact with DNA, they interact on the major groove because the minor groove is too small. Nucleotides make up the DNA strand and form a polymer. Each nucleotide contains a phosphate group, a sugar, and a nitrogenous base. Base pairing occurs where A attaches to T and G attaches to C. The 1’ carbon is attached to base all the way to 5’ carbon at the phosphate in the sugar. This is important to the directionality of the polymer. The 3’ end of DNA/polymer is on the sugar, 5’ is where the phosphate is exposed. Polymers attach by hydrogen (non-covalent) bonding to create the DNA strand. The DNA runs antiparallel fashion. A primer is required for DNA polymerase to begin adding nucleotides.

RNA has a difference in its sugar as compared to DNA. The 2’ carbon has OH coming off of it, DNA has 2’ H. RNA is less stable, the oxygen allows it to act as a catalyst in chemical reactions. RNA can make intramolecular reactions on itself, like a protein, but structures are limited (ribosomes & splicasome). RNA has uracil instead of thymine. When DNA is damaged, cytosine can change to uracil and base paring then does not occur properly. It is possible for double stranded RNA to be formed and DNA can bind with it. It is a hybrid double helix, a slightly different shape. DNA replication is semi-conservative (a mix). Originally there were three methods thought up, Semi-conserve, Conservative and dispersive. 

DNA replication

Submitted by sjurgilewicz on Wed, 04/19/2017 - 20:22

Protiens interact with DNA on the major groove because the minor groove is too small. A attaches to T and G attaches to C. Nucleotide structure contains a phosphate group, a sugar, and a nitrogenous base. The 1’ carbon is attached to base all the way to 5’ carbon at the phosphate in the sugar. This is important to the directionality of the polymer. The 3’ end of DNA/polymer is on the sugar, 5’ is where the phosphate is exposed. Polymers attach by hydrogen (non-covalent) bonding to create DNA. The DNA runs antiparallel fashion. Watson and Crick said bases on the structure you can guess how DNA is replicated. A primer is required for DNA polymerase to begin adding nucleotides. RNA has a difference in its sugar. The 2’ has OH, DNA has 2’ H. RNA is less stable, the oxygen allows it to act as a catalyst in chemical reactions. RNA can make intramolecular reactions on itself, like a protein, but structures are limited (ribosomes & splicasome). RNA has uracil instead of thymine. When DNA is damaged, cytosine can change to uracil. Double stranded RNA can be formed and DNA can bind with it. It is a hybrid double helix, a slightly different shape. DNA replication is semi-conservative (a mix). Originally there were three methods thought up, Semi-conserve, Conservative and dispersive. 

Microtubule drugs

Submitted by sjurgilewicz on Wed, 04/19/2017 - 17:13

Microtubule destabilizers include colchicine and vinblastine. They prevent polymerization. In early m-phase microtubules fall apart. Plant derived medicines include Taxol, colchicine and vinblastine. Taxol and vinblastine are both used in cancer treatment. Colchicine is used to treat gout, as an anti-inflammatory. MAPs (microtubule associated proteins) and motors are ATPases and “walk”. Tau is an example of a MAP which is involved in neurodegenerative diseases. Tau is located along axons of neurons and stabilizes microtubules along the axon and stabilizes. It is heat stable, not easily damaged or denatured and is soluble. Tau is regulated by phosphorylation. Tauopathies are associated with Alzheimer’s and trauma, where tau gets hyper-phosphorylated and aggregates, microtubules destabilize and plaques form that are similar to prions. Kinesins are plus end directed and walk toward the minus end of the microtubule. Dynein is minus end directed and walks toward the plus end. There are multiple families of kinesin. 

More microtubules

Submitted by sjurgilewicz on Wed, 04/19/2017 - 16:04

The centrosome is a microtubule organizing center (MTOC) with two centrioles. This is not the place where microtubules are made from. Rather, the nucleating site is where microtubules grow from the minus end and builds on the plus end. MT must interact at many interfaces. The gamma complex is the mold for tubulin. The alpha and beta tubulin build upon the gamma tubulin ring complex. Dynamic instability is governed by GTP hydrolysis. Only beta tubulin hydrolyzes in this case. The minus end is at the ring complex, the plus end is where tubulin is being added. Any tubulin that is added is bound to GTP and will continue to grow once bound to GDP & embedded. The GDP/embedded end catches up to the GTP and disassembly occurs. Nothing will be added because there is nothing to add to. There needs to be sufficient GTP bound to beta or no growth occurs. This is dependent on local concentrations and diffusion. Proteins bind to microtubules and regulate. When the microtubules hits the kinetochore, the microtubule is stable and wont peel away. Individual microtubules grow and shrink independently from each other. Using In vitro microtubule assembly assay, it is possible to track microtubule polymerization over time by using a spectrophotometer. Light shines through the tube, and measures how much is diffracted. The more “stuff” that is present, the higher the absorption and more microtubules present. In the first minutes, there is only free tubulin with no gamma ring present. Once the ring is present, building takes off. Eventually, microtubules will depolymerize. If you put non-hydrolyzable GTP into this experiment, the microtubules would continue to grow at a greater slope and then plateau once tubulin runs out. If a gamma ring was present to begin with, the lag phase would be shorter. 

Microtubule formation

Submitted by sjurgilewicz on Wed, 04/19/2017 - 13:19

Alpha and Beta tubulin structures come together to form a dimer. There is a GTP binding site where they come together. Taxol is a cancer drug that forms a dimer. It is a microtubule stabilizer which means that the spindles cannot form properly due to depolymerization not occurring. A protofilament is made of alpha and beta subunits connected. Once there are 13 protofilaments connected, the microtubule is created. The microtubule grows more on the “+” end where the beta is exposed as opposed to the “-” end where alpha is exposed. Microtubules have certain properties: they are composed of a heterodimer of alpha and beta tubulin. They are naturally unstable and they are naturally hollow.

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