Lecture 3: Double-strand DNA break repair

blunt end ligation

-cut before the end base makes a frameshift mutation (can revert but frequency is very low)

-DNA ligase an ATP can ligate DNA back together with no info lost

-cells are full of nucleases that digest the DNA ends

CRISPR

-makes double headed nuclease and cuts both strands

-usually small deletions/frameshift mutations

-takes advantage of repair mechanisms

-almost always repaired with a two ended break

DNA repair by homologous recombination

-preferred repair mechanism, no loss of info

-haploid organisms can use even though they are not diploid, but they have to be growing

-must be diploid otherwise

How E.coli uses homologous recombination even though it is haploid

-E. coli has a circular genome

-takes ~1 hour to replicate (bidirectionally), E. coli replicates in ~20min in good media

-initiates replication every 20 min, multiple replication forks

-complimentary strands made from replication forks makes it diploid

-genes near the replication fork make essentials (proteins, tRNA, etc.)

RecABCD processing of double stranded break ends

-occurs if no present ligase, CHI sites only in prokaryotes

-helicase activity of RecBCD unwinds DNA and its exonuclease activity degrades both single strands

-At the CHI site, 3’→5’ exonuclease activity decreases; continues unwinding and enhanced 5’→3’ exonuclease activity yields a single-stranded 3’ end

-RecA protein recognizes complimentary template to synthesize info back

-Rxn with CHI site regenerates previously eaten info

-3’ OH to synthesize DNA

RecBCD

-has a motor (helicase) and is a nuclease

-moves through and breaks DNA until in contact with CHI site

-eats 3’→5’ end as it moves (info lost but will regenerate later)

-stalls at CHI site and eats from 3’ end

-3’→5’ exonuclease activity decreases at CHI site, helicase activity continues

-5’→3’ exonuclease activity does not stop (makes 3’ overhang)

-now there is only a single strand of DNA

-RecA protein opens gene up so strand can enter into other strand and base pair with bottom strand

Holiday structure

-CHI M produced by RUVABC

-RUVA invades into other chromosomes, breaks off and can be replaced by DNA pol (protein crossover)

-RUVB is an ATP dependent protein motor

-RUVC is the protein cutter (of both strands) - dimer with 2 active sites, can cut vertically or horizontally, makes 3’ end and 5’ OH that can be ligated

-don’t lose info because you can polymerize the info lost due to breaks and synthesis of ends

-error prone repair because DNA pol is used

photolyse

-error free repair

-light to repair UV damage

holiday structure steps

-two homologs

-nicks made in one strand of both chromosomes

-3’ ends of cut strands cross and join 5’ end of homologous strand

-branch migration forms heteroduplex region

-rotation at crossover gives isomeric holiday structure

-cuts horizontally or vertically

-resealed

Deinococcus radiodurans (bacteria)

-gamma radiation induces point mutation and many breaks

-from high altitude deserts

-resistant to desiccation and don’t make spores

-make lots of repair enzymes

-makes lots of copies of segments of the gene during replication

-takes a lot to repair in homologous recombination

-all segments in combination must make proteins needed, must have functional genome

-high mutation rate

-get functional recombinants