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Molecular Biology

The gene- vet techs

What makes DNA acidic? -the phosphate groups
Why in DNA do we use 2-deoxy-D-ribose as a sugar not ribose? This is because the 2 OH of ribose makes it less stable due to the fact that it can be attacked by a nucleophile in the presence of an OH group from phosphorus which breaks the phosphodiester link to form a 2', 3' cyclic phosphate
How long is DNA in humans and how is it compacted? -it is 1-2 metres long and is compacted with histone proteins into chromatins
What is a gene? -segment of DNA coding for a single polypeptide chain
What are the nucleotides in DNA? A, T, G, C
What are purines? -A and G, two compartments
What are pyrimidines? -T and C, one compartment
What nucleotides pair with what? - A to T - G to C
DNA melting? -at 95 degrees celcius, a short DNA piece will have its two stands separated due to heat breaking the hydrogen bonds
DNA annealing? -when solution cools after melting, stands will slowly rejoin eachother
If DNA has more G-C nucleotides, will its structure be stronger or weaker? -stronger due to more bonds than A-T, 3 compared to 2 bonds
When do base pairs collapse on top of each other in a helical formation? -to avoid exposure of their hydrophobic faces to water
5' and 3' ends of DNA? -5' end the OH group is bound (o-p-) compared to the 3' end OH which is free - first strand downwards from 5' to 3' and the second stand goes upwards from 5' to 3'
human genome compared to ecoli and viruses? human- 4 billion bp e-coli - 2 billion bp viruses - few thousand bp
How many chromosomes in human DNA? -46 23 pairs
Where is DNA in humans found? -in the nucleus of cells
What are histones made up of? -arginine and lysine they give it a positive charge so that it can form ionic bonds with negative phosphate groups of the outside helix of DNA
What is a chromosome? -double stranded DNA
DNA replication in a prokaryotic? -semi conservative -parental strands separate and acts as a template for synthetizing new strands, each has one old and one new
DNA replication in an eukaryotic? -much more complex due to complex cell cycle -G1, Synthesis phase, G2, mitosis phase
What is transcription? -DNA copied into mRNA that tells how to make proteins -DNA is unwinded in a short region -copying DNA to RNA by RNA polymerase -copying DNA to DNA by DNA polymerase
What is replication? -DNA is copied from one strand to another just before mitosis occurs and the cell splits into two
Ecoli chromosomes and replication? -chromosomes are circular -separated at a point of origin and two replication forks moving in opposite directions synthesis DNA until the meet at opposite sides of the circle
Human chromosomes and replication? -linear -same as ecoli replication but more replication origins and replication forks can work in opposite directions -replicon is a section of DNA when replication is initiated by a centre of origin; and their are multiple of them
Control of initiation in ecoli? -origin of DNA= OriC, it is rich in A-T pairs as less H bonds to break apart -DnaA binds to multiple OriC and causes their separation -it permits the unwinding enzyme DnaB (helicase)works at replication forks to unwind strands both directions
supercoiling in ecoli? -closed circular relaxed state acts as a clamp and strand separation in DNA replication causes the formation of positive supercoils (tightened, more twist and less bp/turn), gyrase then cuts strands and duplex forms over front side forming -ve supercoil
Supercoiling in human DNA? -duplex rotates causing overwinding and +ve supercoils to form -ahead of replication forks tighten as further stranding separation is resisted, this must be relieved
Topoisomerases? -act on the DNA to isomerize of change its topology
Topoisomerase 1? -breaks one strand of a supercoiled DNA double helix which permits it to rotate on phophodiester bond, after rotation it reseals the complex -does not need ATP, does not hydrolyse-transfers bond to itself
Topoisomerase 2? -breaks two strands of DNA double hexlix -makes breakage reversible -called a gyrase in ecoli -uses ATP -relaxes supercoils to allow DNA synthesis to occur
Difference between eukaryotic and e-coli topoisomerase I? ecoli-cuts one strand- relaxes -ve supercoils eukaryote-cuts one strand- relaxes both -ve and +ve supercoil
Difference between eukaryotic and e-coli topoisomerase II? ecoli-cuts 2 strands, ATP dependent eukaryotic-cuts 2 strands, ATP dependent ecoli-relaxes +ve supercoils and inserts -ve supercoils eukaryotic-relaxes +ve supercoils but cannot insert -ve supercoils
DNA replication happens by what? -DNA polymerase -in ecoli replication occurs at the replication fork by DNA polymerase III but polymerase I also plays a role in replication and repair
Substrates for DNA polymerases? -deoxyribonucleotides: DATP, DGTP, DCTP and DTTP
DNA polymerases? -can only extend or elongate existing primers (20 nucleosides), attaches to the 3' end of the OH group of the primer and releases a pyrophosphate (ppi) in the process
How do eukaryotes insert a negative supercoil? -DNA winds around nucleosome so region in contact with protein is underwound or -ve supercoil achieved by left handed coil -to compensate a positive supercoil is formed, giving overall zero charge, but this can be relaxed by topoisomerase I or II
Where is DNA synthesized at? -each replication fork in ecoli 2 DNA polymerase III at each strand are linked by single holoenzyme dimer which moves in a single direction
Which direction can polymerase only synthesis in? 5' to 3' direction , so the new strands being made must go in the opposite way to the synthesis dimer direction
leading strand? -left hand strand -primase lays down a primer and DNA polymerase then takes lead synthesizing DNA
Lagging strand? -right hand strand -as unwinds, repeated primases lay down many primers and DNA polymerase extends a short bit of each leaving behind short lengths of DNA fragments called okazaki fragments
How is DNA synthesized in the lagging strand without going backwards? -DNA is looped so that for a short distance the strand is in same 3' to 5' direction as leading strand template -achieved by regular intervals where the loop is reformed and enlarged and adds a new primer each time and is how okazaki fragments are formed
helicase? -unwinds DNA, is ATP driven -primase is attached to it and in e-coli this complex is known as a primasome
SSB in DNA? -single stranded binding protein which stabilizes single strands -it has a high affinity for single strands but not base sequences
Sliding Clamp? -ring like protein structure that surrounds DNA -hole big enough for DNA sliding but not large enough that it will fall off -ecoli-beta protein, dimer (2 subunits) -eukaryotes-PCNA (proliferating cell nuclear antigen), trimer (3 subunits)
Sliding clamp has two sites what are they for? -one for DNA polymerase III attachment and one for RNA primer/DNA hybrid structure -polymerase III must detach and reattach for each new okazki fragment in the lagging strand
DNA polymerase I role? -exonuclease activity -attaches to the spaces between the okazaki fragments and synthesizes DNA and then gets rid of the primers by hydrolysis which is replaced by DNA by another pol I, once reaches DNA is detached and leaves a nick repaired by ligase
Ecoli methyl-directed mismatch-identification? -every A in a GTAC sequence is methylated in the parent strand and this doesn't happen straight away in the new strand-allowing the new strand to be identified for an incorrectly processed mismatch
Ecoli methy-directed mismatch-steps? MutS-identifies the mismatch MutL-connects MutS to MutH (an endonuclease) MutH-identifies the first unmethylated GTAC from the mismatch and makes a nick exonucleas, helicase, SSB remove this segment polymerase III and ligase repair the section
Why is there a t instead of a U in DNA? -because otherwise it would be hard to distinguish between a U and an improper U formed by deamination of cytosine
How is a lesion like a thymine dimer (2 t nucleotides instead of 1) removed? -exonuclease cuts the DNA before and after the dimer and removes it -DNA polymerase III and ligase repair the section
How is a uracil, formed by deamination of cytosine, removed? -removed by glycosylase to form AP site -endonuclease identifies area and makes a nick -polymerase I and ligase replace and repair area by hydrolysis
New DNA chromosomes synthesized are shorter due to the removal of primers, how is this overcome? -by telomeric DNA adding telomeres to lengthen it
Created by: sherloki



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