Replication
How does DNA ligase repair the DNA?
3 step process: formation of a covalently linked enzyme-AMP intermediate through a Lysine side chain, then transfer of the AMP to the 5' phosphate group of the nicked strand, attack of the AMP-DNA bond by the 3'-OH group and displacement of AMP.
What is the function of the SPO11 gene?
Spo11 introduces ds breaks in chromosomal DNA during the early stages of meiosis in eukaryotes. It cuts with little to no specificity, cuts at a very specific time, and cuts at locations that are not tightly packed into nucleosomes. The Spo11 dimer creates a ds cut in the DNA through a Tyrosine intermediate. Spo11 then recruits MRX (RecBCD equivalent) and the production of the 3' ssDNA ends facilitate the binding of RecA like proteins (Rad51/Dmc1).
MMR Process
MutS (as a protein dimer) is recruited to the replication fork by the sliding clamp and scans the DNA looking for a mismatch. When it finds a mismatch, it changes conformation and binds to ATP. This recruits MutL and endonuclease MutH. MutH creates a nick on one side of MutS, and UvrD (helicase) binds and unwinds the DNA. One of three different exonucleases then comes in, binds, and removes ssDNA. DNA pol III fills in the gap and ligase closes the nick. The strand that is removed is the unmethylated strand.
How incredible is it that we have all this replication going on in our cells and yet we still maintain such a low mutation rate from generation-to-generation??
*insert yellow thumbs up emoji here*
What are some of the possible effects of a transposon?
Can cause the protein product to not be functional, can cause transcriptional activation in other cell types, and it could have no effect.
What drives replication/addition of dNMP to 3' end? What kind of reaction is it?
Addition of a base releases PPi (and H+ ion). The PPi is then broken down into two inorganic molecules of P, which is a highly favorable reaction. The reaction of adding a base and releasing PPi is coupled to the reaction of breaking down PPi to 2 Ps by pyrophosphatase. It is a substitution nucleophilic bimolecular reaction where the OH group at the 3' end of the primer acts as a nucleophile that attacks alpha-phosphate group of incoming dNTP.
What is the role of cohesin? How do the sister chromatids fall apart? Why do sister chromatids stay together during meiosis I but not II (what is the protection mechanism)?
Cohesin holds the sister chromatids together (also important in spindle attachment, repair via recombo, dsDNa break repair, and regulation of transcription). Cohesin forms during the S phase as the sister chromatids are being replicated. The cohesin (Rec8) is completely phosporylated by kinases. Before anaphase, separase (a protease) is inhibited by securin. The anaphase promoting complex (APC) then mediates the destruction of securin through a ubiquitin dependent process, which releases separase. Separase then targets phosphorylated cohesin subunits for cleavage. In meiosis I, shugoshin (Sgo1) protects the cohesin complexes near the centromere from getting degraded. Then, during meoisis II, Sgo1 is removed, allowing for the cohesin complex to be cleaved.
What does BER fix and how does it do it?
BER typically fixes non-bulky lesions involving a single base. In this process, a glycosylase, traveling along the minor groove and flipping bases out, will remove the base by breaking the glycosidic bond between the sugar and the base. An AP endonuclease will cut next to the AP site on the 5' side, while a 5'-3' exonuclease will remove the AP site, and DNA Pol and ligase will repair the break.
What are the three things that could occur if an incorrect base in incorporated during replication?
Proofreading 3-5' exonuclease removes misincorporated base and correct base is added. The mismatched base in repaired. The mismatched bp is replicated in the daughter cell, resulting in granddaughter cells that have the fixed mutation
What are some of the characteristics of transposons and how does it transpose?
They include flanking inverted repeats, recombination sites, a transposase gene, and duplication sites. A cut and paste transposon is the simplest form of transposition, where a transposase binds to the inverted repeats. The 3'-OH groups attack the target DNA and facilitate strand movement. A new duplication site is created and the dsDNA breaks in the host DNA are repaired. Replicative transposons occur when one strand of the transposon is cut, the free 3' ends of the transposon attacks the target DNA and replicates itself.
Why does the polymerase include dNTP when NTP is so much more prevelent?
DNA pol has a discriminator site that includes amino acid side chains that differentiate between dNTPs and NTPs. The amino acids are primarily hydrophobic, non polar, and neutral that establish van der Waals contacts with ribose ring of dNTPs. Those interactions don't occur with NTPs due to the presence of the OH group. There is also steric interference where the binding pocket is too small for NTPs to fit.
What is the difference between a splice and a patch product during recombination?
A splice product has recombination on each strand. A patch product has recombination on only 2 strands.
What does NER fix and how does it do it?
NER typically fixes bulky lesions of DNA. UvrA identifies the distortion, and then, along with UvrB and the help of ATP, binds to the bulky lesion. UvrB melts the DNA. UvrA then falls off while UvrB remains attacked. UvrC then binds to the lesion and creates two nicks (1: 8bps from distortion on 5' side, 2: 4-5bps from the distortion on the 3' side). UvrC falls off and UvrD (helicase) removes ssDNA. DNA pol I and DNA ligase then comes in to fill in the gap.
What is one example of a deamination occurrence, and why is it such an issue?
When 5-methyl cytosine becomes deaminated, it looks like a thymine. Methylated CpG islands are associated with transcriptional silencing. This is a problem because MMR does not know which base it should remove as both strands are methylated.
What is MAT and how does it work?
MAT is the mating type loci in yeast, and is an example of how recombination can control the expression of certain genes. The HMR (a-cell type), and HML (alpha-cell type) loci are silent markers that facilitate switching from 1 haploid type to the other through recombination. The HO endonuclease generates a DSB at the MAT locus where the new cassette can be put in.
It uses an assay where there is one short, labeled ssDNA molecule and one circular, unlabeled ssDNA molecule. You allow the ssDNA to bind to the circular ssDNA. You then use a restriction endonuclease to cleave the labeled ssDNA asymmetrically. The helicase is then loaded onto the ssDNA from the unlabeled part and ATP is added. When ATP is hydrolyzed to ADP, the helicase becomes active and can start unwinding dsDNA. Depending on the direction of the helicase, one of the two labeled strands would fall off. You run this out on a gel, and can see which part fell off.
What are the structures and functions of RuvA, B, and C?
RuvA is a tetrameric protein that recognizes the Holliday junction. RuvB is a hexameric helicase that promotes branch migration. It also includes an ATPase activity that promotes movement. RuvC resolvase binds as a dimer and cleaves symmetrically (strands on opposite sides of the Holliday join, and with opposite polarities). It cuts with moderate sequence specificity.
How do pyrimidine dimers form under UV radiation and how can they be fixed?
UV radiation causes pyrimidine dimers to form between adjacent pyrimidines. This causes a distortion in the DNA double helix where the bond is now covalent (~1.6A between rings) while the normal spacing is ~3.4A. Two thymine rings can form cyclobutane ring. These dimers can be fixed using photoreactivation catalyzed by a photolyase. The photolyase captures the energy from visible light and uses it to break the cyclobutane ring.
What does N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) do and how will it get fixed?
MNNG is an alkylating agent that could transfer a methyl group to the oxygen on the 6 carbon of guanine. This will cause it to bp with thymine and could lead to an A:T fixed mutation if not fixed. It is fixed by O6-alkylguanine DNA alkyltransferase that removes the mutagenic methyl group by transferring it to an amino acid (cysteine residue) on the enzyme itself. This is a suicide mechanism, as once the enzyme has a methyl group on the cysteine, it is not active anymore.
What are some features of retrotransposition, and how does it work?
How does the sliding clamp get loaded onto the DNA?
How is a 3' ended ssDNA generated in E. coli?
RecBCD binds to the ds DNA breaks. RecB is a slow helicase that moves in the 3-5' direction. It also has a domain that acts as an endonuclease that cleaves the ssDNA generated by the helicases. RecD is a fast helicase that moves in the 5'-3' direction. A loop is formed due to the difference in speeds of the two helicases. RecC recognizes the Chi (crossover hotspot instigator) and changes the activities of the helicases and endonuclease. RecB is now faster than RecD, and the endonuclease stops cutting the 3' strand, and cleaves the 5' strand more readily. You now have a ssDNA which RecA can then bind in a 5-3' direction and helps facilitate homologous base pairing and strand invasion.
What happens when DNA replication has not occurred and/or the homologous pair is not available for recombination?
Non-homologous end joining, which can lead to the loss of DNA.
What are intercalating agents and how can they damage the DNA?
Intercalating agents are planar molecules that insert themselves between the bp of the DNA. Examples include ethidium, acridine orange, and proflavin. They can cause either insertions or deletions. Insertions are caused when the intercalating agent serves as a substrate for a dNTP to bind in the active site of the pol and add an extra nucleotide base. Deletions are caused when the agent creates a distortion in the replicating DNA that causes the polymerase to miss the nucleotide addition. Both insertions or deletions can cause a frame shift mutation that can knock out the activity/expression of the gene.
What are the features of non-viral retrotransposons and how do they work?
A non-viral retrotransposon has a flanking untranslated region, ORF1 (RNA binding protein) and ORF2 (RT and RE). This type of transposition uses target site primed reverse transcription mechanism. LINE TEs autonomously transpose while SINE TEs transpose non-autonomously. In this case, the transposon is transcribed into RNA. The ORF1 and ORF2 enzymes are translated, and the RNA binding protein holds the complex together and finds a poly-T region of homology. The RE makes the first nick in the target DNA and the RT then synthesizes cDNA.