Notes

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If damage occurs at same place in both strands of DNA recombination repair in E.coli
Holiday junctions: must be resolved same way as for recombination in meiosis

Error-prone repair(s)
Non homologous end joining
SOS translesion repair

Nonhomologous end joining
- Happens when both starnds in DNA are broken at the same time
- No undamaged complementary strand available
- Excision + repair approach is not going to work here
- Fail safe system - 1 in 1000 survival when chromosome break happens in yeast,
- Last resort
- Two ends of broken DNA are directly ligated together - error prone repair
- Ku70/Ku80 dimer, recognizes broken ends; forms scaffold to hold ends together
- DNA-dependent protein kinase brings and phosphorylates protein Artemis
- Artemis - has exo- and endo- nuclease activities when activated; trims overhangs, cleaves hairpins
- DNA polymerase fills in
- DNA ligase IV, XRCC4 joins ds ends
Translesion repair (7)
Highly error-prone because polymerase add nucleotides randomly without proper base pairing = low fidelity DNA synthesis
Introduces mutations but still enables complete chromosomes replication
Proteins/polymerase not present under normal conditions

Bypass DNA polymerases
- Highly conserved across 3 domains of life
- UmuC - part of Pol V of E.coli, high error rate
- Low processivity polymerases
- Eukaryotiic polymerase are also able to bypass damage
Both NHEJ and Recombination repair repair DSB, difference is the accuracy of the repair


DNA reaarangement by genetic recombination
Recombination; exchange of genetic material between two DNA molecules causes DNA rearrangement
Roles/uses - depends on who/what is causing it; biological or experimental
Biological Roles of Recombination
1. DNA repair
2. Creation of new gene/allele combinations
3. Formation of new genes
4. Integration of a specific DNA element
Experimental Uses of Recombination
- Gene mapping (distance between genes estimated through recombinatino frequency)
Genetic Recombination
- General or homologous recombination
○ Genetic exchanges between pair of homologous DNA sequences
- Site-specific recombination
○ Sequences with a limited stretch of similarity
- Transportation
○ Mobile DNA element
1. General or homologous recombination
Genetic exchange between a pair of homologous DNA sequences

Key steps in ss break model:
Alignment of two homologous DNA molecules
Introduction of the break in one strand
Strand invasion, ss region from one parental strands pairss with the complementary strand from homologous DNA molecule
Outcome: two molecules become connected through crossing DNA strands
Cross structure = holiday junction
Branch migration - movement of holiday junction
Cleavage of holiday junction - resolution
Homoduplex part

- Look at the recombination between genes A and C
○ Splice or cross over products
○ Patch or no-crossover products
Model for homologous recombination
Similar steps in prokaryotic and eukaryotic DSB replicatioin repair
1. Szostak model, double strand break
2. Exonuclease extend the gap producing 3' hangs
3. Exposed 3' end pairs with its complement in th eintact homolog
4. Extension at 3' end via DNA polymerase & Branch-migration and formation of 2nd crossover
5. Cleavage of holiday junction
- Follow where things will get cut and where they'll be splyced back together

Homologous recombination in E.coli
Chi sequences in E.coli
Crossover - hotpspot - instigator = "Chi"
Role: enhance recombination frequency
Consensus sequence: 5' - GTGGTGG - 3'
Overrepresented > 1000 chi sequences in E.coli genome
Recombination enhancers also exist in other organisms

Rec/Ruv - E.coli enzymes involved in homologous recombination
RecBCD - nuclease/helicase; binds to duplex DNA and processes it into a substate for recombination; requires ATP
RecA starts recombination by facilitating pairing of homologous DNAs involved in strand invasion
RuvAB complex with helicase activity recognizes holliday junctions, catalyses branch migration
RuvC - endonuclease that catalyses the reaction of Holliday junctions
RecBCD/chi mechanism
1) RecBCD complex enters the DNA at site of ds break
2) Unwinds and degrades both strands of DNA in presence of ATP
3) Upon reaching a chi sequence activity changes - chi sequence controls its activity
4) Degradation of 3' end stops
5) RecA binds to the overhang and initiates strand exchange
6) Branch migration by RuvAB and holliday junction resolution by RuvC
Homologous recombination in Eukaryotes
Programmed generation of DSBs occur during meiosis
Homologous recombination:
Required for proper chromosome pairing during meiosis
Gene reshuffling
First step: DSBs are formed at recombination hotspots

Enzymes:
Spo 11 may be Topo II-like enzyme as it generates doublestrand breaks
Mre11 is nuclease
Dmc1 and Rad1 are RecA type strand exhcnage proteins

Possibility: the sequence used as a template for repair by homologous recombination is different from the sequence to be repaired
Mismatch repair system recognizes mispaired bases in heteroduplex
- It excises and replaces one of the strands to restore complementarility
- Conversion of one allee into another
- Transfer of genetic information is nonreciprocal gene conversion
Possible outcome of the meisois due to gene conversion, for example: 3 red alleles and 1 blue allel in the daughter cells

Occurs between sequences with a limited stretch of similarity involves specific sites
Three functions:
1. Inversion (e.g expression of alternative genes - inversion of DNA sequence/gene by DNA invertases)
2. Insertion (e.g infection)
3. Deletions (e.g mediation of programmed DNA rearrangement)

1. Rearrangement enzymes - recombinases, site -specific recombinases cleave and rejoin DNA using a covalent protein-DNA intermediate
A) Site-specific endonuclease activity (cleavage)
B) DNA ligase activity (rejoining)

2. Recombination site places for DNA exchange
A) Specifically recognized by recombinase
B) Partially asymmetric

Same orientation: two different DNA molecules: insertion
Same orientation; same DNA molecule: deletion
Inverted orientation: same DNA molecule: inversion

DNA deletions and insertions
Used to recycle genes
- Recombination sites are recognized by specific recombinase
- Recombination sites are flanking the gene in direct orientation and on the same DNA molecule
STEPS: recombination sites align next to each other recombinase revolves the structure and cuts out the gene
Both the insertion of the lambda phage DNA into bacterial genome and its deletion from it are accomplished by a site-specific recombination event, catalyzed by the lambda integrase enzyme, insertion involves some bacterial enzyme as well.
- When lambda DNA enters ends form circular DNA molecule
- Lambda bacteriophage can multiply in E.coli by lytic pathway
- Prophage can exit the host chromosome and shift to lytic growth
Hin recombinase in Salmonela - invertase, inverts segment of the bacterial genome

Transposition
Nonhomologous recombination: transposable elements insert into DNA that has no sequence homology with the transposon
Donor site: contains a transposable element
Target site: usually ranodm, but there are hot spots: preferred sequences that are targetted
Autonomous vs nonautomonmous elements
Autonomous encode all the enzymes necessary to move,
Nonautonomous have no coding capacity
Transposons - called jumping DNA however do not always leave one place for another
Nonreplicative transposition
- Cut and pastte
- Both strands of original transposon DNA move together from 1 place to another without replicating
Replicative transposition
- Copy and paste
- Involves DNA replication phase -> 1 copy of a transposon remains at original site New copy inserts at the new site