SSGL1 - DNA repair mechanisms

Question 1

Mol Bio Cell pg 263-

Answer 1

DNA replication and repair

Mutation rates are very low
Mutations can be difficult to detect because they are often silent (a change of codon may not change the amino acid it specifies for: the degenerate nature of the code)
The rough rate of natural mutation is the same for all organisms and approximately occurs once in every 10(9) nucleotides

Replication

DNA is replicated semi conservatively by DNA polymerase
DNA polymerase adds a deoxyribonucleotide to the 3’ OH end of the polynucleotide chain (the primer strand)
The reaction is driven by a large favourable free energy change (pyrophosphate release and hydrolysis of 2x inorganic phosphate)
A localised region of replication moves progressively along the parental DNA double helix: this is called the replication fork
In the replication fork, a multi enzyme complex that contains DNA polymerase synthesises the DNA for both new daughter strands
Because DNA polymerase can only synthesise in the 5>3 direction, there is a leading strand and a lagging strand (Okazaki fragment)
Proofreading mechanisms prevent frequent mutation:
1. The correct nucleotide has a higher affinity for DNA polymerase than an incorrect one because a correct pairing is more energetically favourable.
2. Before the nucleotide is covalently bonded to the chain, DNA polymerase undergoes a conformational change which occurs more readily with a correct base-pairing
3. Exonucleolytic proofreading: if an incorrect nucleotide is added, DNA polymerase cannot extend the 3’OH end (because it requires a correct template). DNA polymerase has an additional catalytic site (proofreading exonuclease) removes bases until a correct base pair is found again
RNA synthesis has a higher error rate (10(4)) because these proofreading mechanisms do not exist
DNA synthesis begins at origins of replication, which contain more A-T (as this is easier to break)

Other proteins in the replication fork: 
DNA primase (makes RNA primers on the lagging strand), DNA ligase (joins 3' end of new DNA to the 5' end of the previous one), DNA helicase (unwinds the helix), Single strand DNA binding proteins (SSBs) (aid helicases by stabilising single strands so they don't hairpin and hinder DNA polymerase), Sliding clamp proteins (attaches DNA polymerase to the DNA and helps its dissociation), Clamp loader protein (hydrolyses ATP to load the clamp onto DNA), DNA Topoisomerase (breaks a single strand or a double strand to relive tension in the helix and prevent tangling)
Process: At the replication fork, DNA helicase opens the helix. Two DNA polymerases work on the fork, one on the leading strand and one on the lagging strand. The DNA polymerase on the leading strand works in a continuous fashion, and the DNA polymerase on the lagging strand restarts at short intervals using an RNA primer made by DNA primase. Close association of of the replisome proteins increases the efficiency of replication and is made possible by a folding back of the lagging strand, and also facilitates the loading of the clamp each time an Okazaki fragment is synthesised. 
Replication only takes place in S phase: eukaryotic replication forks more 10x more slowly than prokaryotic, and have many origins of replication. Eukaryotic chromosomes are activated in a sequence determined by the structure of chromatin – condensed areas replicating last. Chromatin reforms after replication through the addition of new histones to old ones. Replication of the end of the linear chromosome is achieved through the telomere, which is maintained through a telomerase. Telomerases extend one of the DNA strands at the end of a chromosome by using an RNA template that is a part of the enzyme, producing a highly repetitive sequence at the end that forms into a t-loop. 

DNA repair

the structure of DNA is ideally suited to repair: it carries two copies of all the genetic information, and the undamaged copy can be used to repair the damaged copy

Strand directed mismatch repair
detects the potential for distortion in the DNA helix caused by non complimentary base pairs
inheriting a copy of a dysfunctional mismatch repair gene results in a disposition to certain types of cancer
MSH2, MSH3, MLH1, PMS2 all cause colon cancer

DNA damage
- Depurination and deamination: depurination releases guanine, deamination converts cytosine to uracil
- DNA damage is repaired through two pathways:
Base excision repair: DNA glycosylases recognise specific altered bases and hydrolytically remove them. The missing nucleotide removed is recognised by AP endonucleases, which cut the phosphodiester backbone. DNA polymerase adds a new nucleotide and DNA ligase seals the nick.
Nucleotide excision repair: repairs bulky lesions - a large muti enzyme complex scans the helix for distortion, when it finds it DNA helicase unwinds a section of the DNA and an excision nuclease enters and removed a section of approximately 30 nucleotides, either side of the damage. DNA polymerase and DNA ligase repair the strand.
Transcription coupled repair: works with base and nucleotide excision to direct repair immediately to the cell’s most important DNA sequences (the ones that are being expressed when the damage occurs).
- RNA polymerase is linked to DNA repair: it stalls at DNA lesions and recruits repair machinery to these sites
- Special DNA polymerases are used for rapid emergency repair when extensive damage has occurred; these polymerases are less accurate

• Repair of double strand breaks:
• Non homologous end joining: broken ends are brought together and rejoined by DNA ligase (results in nucleotide loss) common in somatic cells
• Homologous recombination: used only during and after DNA replication is S and G2 (when sister chromatids can be used as templates). Breaks can occur during S as the replication fork falls apart at a lesion; strand invasion can trigger DNA polymerase to repair the strand. Recombination only takes place between sequences with high homology.
• DNA damage delays the cell cycle
• Rad51 forms a heteroduplex (D loop) but there is a loss of heterozygosity - critical in the development of some cancers