DNA Replication
List the steps in synthesizing the lagging strand.
1) After the helicase creates an origin of replication a primer is made by DNA primase. 2) DNA Polymerase synthesizes a segment of DNA initiated at the RNA primer. 3) RNA primer then degraded and replaced with DNA 4) The 'nick' between the two Okazaki fragments is sealed with the enzyme DNA ligase requires ATP.
What is the direction of DNA replication?
5' to 3' only
What is a Primer, why is a primer necessary in DNA replication, what is the name of the enzyme that makes the primer?
A primer is a 10 base pair segment of RNA necessary for DNA polymerase to initiate the synthesis of DNA. It is necessary because DNA polymerase requires an available 3' oxygen to from a phosphodiester bond. The enzyme that makes the primer is DNA primase.
What is a transposon? What transposon makes up 10% of our DNA?
A transposon is a transposable element which is a sequence of DNA that can replicate and insert itself in another location in the genome. The Alu element is the most common transposon in humans making-up 10% of all human DNA.
What is the function of telomerase? Why is it important? Describe what telomerase accomplishes.
At the end of the eukaryotic chromosome, there is no place to lay down the lagging strand RNA primer. In order to complete DNA synthesis at the very end of a eukaryotic chromosome there has to be DNA extending beyond the DNA to be copied Telomerase will synthesize GGGGTTA multiple times. This ensures the lagging strand synthesis stops short prior to the end of the Chromosome. This is also critical because it prevents the chromosomes from degrading from the telomeres.
Exonuclease
Breaks phosphodiester bonds. Will degrade DNA or RNA. The E-site in DNA polymerase is an exonuclease.
What enzyme is responsible for synthesizing DNA?
DNA polymerase
How does DNA polymerase edit mistakes? What is exonuclease activity?
DNA polymerase has in addition to polymerase activity (P-site), it also has editing activity (E-site). After a nucleotide is added to the growing DNA, if the nucleotide is correct, it will enter the P-site of the polymerase and the next correct nucleotide will be added. However if there is a mismatch such as G to T, the newly added nucleotide (G in this example) will enter the E site and will be cleaved off (this is exonuclease activity). The phosphodiester bond is cleaved 3' to 5', and a new nucleotide will enter the P-site, if it is correct (A in this example) synthesis will continue uninterrupted.
What type of enzyme is DNA primase?
DNA primase is actually and RNA polymerase that makes the RNA primer.
DNA Repair Polymerase
Degrades RNA Primer (RNA Exonuclease) and replaces the short segment of RNA with DNA.
Where does the energy come from to synthesize DNA?
During DNA replication, each incoming nucleotide is a triphosphate. ATP, GTP, CTP and TTP. (Generally referred to as nTP) The hydrolysis of the terminal two phosphates (pyrophosphate) is exergonic and provides the energy of synthesis.
semi-conservative DNA replication
Each strand of the parental DNA acts as a template to build the other half of the DNA molecule. So in this process, half of the DNA is conserved from the parent DNA and half of the DNA is newly synthesized.
Which nitrogen bases pair bond and how many hydrogen bonds are in each of the pair bonds?
Guanine forms 3 hydrogen bonds with Cytosine. Adenine forms 2 hydrogen bonds with Thymine.
How is DNA replication initiated?
Initiator proteins recognize the origin of replication by a consensus sequence of nucleotides. These initiator proteins will then recruit the other enzymes and proteins necessary to form the DNA replication complex. A helicase will separate the double helix and form a replication fork. DNA replication then proceeds in both directions from the origin. Replication is bidirectional.
DNA Ligase
Joins the 5' end of one piece of DNA with the 3' end of the adjacent piece of DNA. It seals nicks and requires ATP as an energy source.
What is the fidelity of DNA replication and why is it almost error free?
One mistake for 1 x 109 nucleotides. (DNA acts as its own template ensuring the correct sequence of nucleotides and there is editing to correct mistakes during DNA replication.
DNA replication and repair
Precise process that ensures a cell can produce two genetically identical daughter cells. DNA replication is the duplication process DNA must undergo in order for cells to divide producing two genetically identical daughter cells. Mutations do occurring during replication. Cells have continual surveillance to prevent these mutations. They will often time catch the mistakes and fix it or repair it. DNA replication is near flawless because of this.
How is the RNA primer removed? Include the function of the three important enzymes
Primer Removal Three things need to occur to replace RNA primers These act quickly to remove RNA and replace it with DNA 1) RNA nuclease breaks apart the RNA primer 2) DNA Repair Polymerase replaces the RNA with DNA (uses Okazaki fragment as primer). 3) DNA Ligase joins the 5' end of the new DNA fragment to the 3' hydroxyl end of the next Okazaki fragment.
Why can DNA only be synthesized from 3' to 5' direction?
Proof reading could not function, If DNA was growing 3' to 5', and a nucleotide was edited by DNA polymerase, the high energy bond in the nTP could not be hydrolyzed and no energy would be provided. Synthesis would stop cold.
Single Stranded DNA Binding Protein
Stabilizes short segments of single stranded DNA in the lagging strand to allow for the synthesis of Okazaki fragments
What are the steps in DNA repair?
Survey enzymes recognize a mismatch of a base pair and will nick the DNA upstream of the mismatch. A DNA repair enzyme will recognize the nick and mismatch and degrade that segment of DNA (exonuclease). DNA polymerase will then fill the space with a new correct segment of DNA then DNA ligase will seal the nicks.
DNA Polymerase
Synthesizes DNA. This enzyme needs an RNA primer to begin DNA synthesis. The enzyme has a polymerase site (Where DNA is synthesized) and an edit site. The P-site synthesizes DNA in the 5' to 3' direction. The edit site will recognize a base pair mismatch and remove the incorrect nucleotide in the 3' to 5' direction.
What is an Okazaki fragment?
The Okazaki fragments are each of the discontinuous fragments of DNA synthesized on the lagging strand.
What is the difference in the initiation of DNA replication in bacteria compared to eukaryotic cells?
The circular prokaryotic chromosome only has one origin of replication; chromosomes in eukaryotic cells have hundreds of origins of replication.
What is the leading strand and lagging strand during DNA replication? Why can only one strand be synthesized continuously?
The leading strand is the strand on DNA that can be synthesized continuously in the 5' to 3' direction. However, the lagging strand is built in the opposite direction so as the helicase moves the lagging strand is build a short bit, then another short bit, then another short bit. This is because the other strand of DNA is in the opposite direction and the new strand can only be built in the 5' to 3' direction.
What is the replication origin? Why is it rich in A and T?
The origin of replication is the location on DNA where replication starts and it is a sequence of nucleotides recognized by initiator proteins. It is rich in A and T because these have fewer hydrogen bonds requiring less energy to separate the DNA to initiate DNA replication.
What is topoisomerase? Why is it critical in DNA replication?
The topoisomerase will nick the DNA upstream of the direction of the replication fork allowing the DNA to rotate. This eliminates torsional stress that, if not relieved would cause supercoiling of the DNA and stop DNA replication.
List and describe three spontaneous mutations in DNA. What are the consequences of each?
There is a depurination- spontaneous removal of the guanine base. This will result in the loss of the base causing a frame-shift which is a significant mutation. There is deamination- removal of an amine from Cysteine converting it to Uracil. This will cause an Adenine to be in the place of Guanine resulting in a point mutation. There is the creation of a thymine dimer, where exposed to UV light two adjacent thymines will covalently bind with each other. This will prevent correct pair bonding during DNA replication and cause a frame-shift mutation.
Initiator Proteins
These recognize the origin of replication in DNA and recruit all the other proteins of the DNA replication complex.
Helicase
This enzyme is responsible for opening and unwinding DNA creating the replication fork. This exposes the bases to act as templates during DNA replication. Special helicases survey and identify mismatched DNA to initiate DNA repair.
Topoisomerase
This enzyme nicks the DNA ahead of the replication fork and allows the DNA to twist relieving torsional stress. This enzyme prevents torsional stress and super-coiling during DNA replication.
Telomerase
This enzyme will add multiple GGGGTTA repeated sequences to the ends of a chromosome. This allows the lagging strand to lay down a final primer to finish the lagging strand. This ensures the lagging strand stops short prior to the end of the chromosome. Telomerase also add critical stability to the chromosome and prevents degradation of the DNA at the ends of the chromosome.
What is a phosphodiester bond? Where is it found? And why is it important?
This is an ester bond between phosphate and carbon. P - O - C . This bond is found in polymers of nucleic acids (both DNA and RNA). It is a rather stable covalent bond providing stability to the DNA molecule.
How was the origin of replication discovered?
Yeast that could not make a critical nutrient His were made to take up a plasmid lacking an origin of replication that had the gene to make his. Random DNA was put into the plasmids. The yeast were then grown on media lacking the His nutrient. Colonies of yeast could only grow in those yeast that had the plasmid with a functional origin of replication. These plasmids were then purified and the sequence of the origin was determined.
