Note for: Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells

Note for: Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells

(doi: 10.1093/emboj/17.18.5497)

Main points of this paper;

1. investigate two proteins; Ku70 and Rad54

2. each responsible for DSB and complement to each other.

3. both repair pw. play role in genetic maintenance

DSB -- in lower organisms has been majorly fixed by HR. But in the higher eukaryote, ike in veterbrate - favour NHEJ to fix the DSB (my guess would be the cell is not divided anymore)

3 components important for NHEJ;

1.DNA-PK

2.XRCC4

3.DNA ligase IV

Advantage of DT40;

1. high homologous recombination - easy to generate knockout

2.stable phenotype -easy to compare phenotype of the effect genes

Reason of this study;

no one studies these two genes (KU70 and Rad54) on DSB repair in higher eukaryote except in yeast.

In yeast, HR is much preferred to fix DSB in yeast.

Chicken showed 70% homolog to human Ku70.

Cell count every 24 hr with optimum growth between 10^5-10^6 cell/ml.

Plating efficiencies in methyl cellulose;

WT -100%

Ku70 - 100%

Rad54 - 80%

Rad54 and Ku70 - 50%

Proliferation rate effect may come from;

1. change in cell cycle length

2. spontaneous cell death

Rad54, Rad54Ku70 -- the slower proliferation is likely to cause by spontaneous cell death.

growth curve result averages from two separate clones Rad54(-/-)#c.1 and c.2

For yeast; unrepaired DSB induces cell death. So DT40 is investigated further, esp., for the clones that defect in DNA repair pathways by karyotype.

Investigate on chromosome by karyotyping on meta-pahase-arrested cells. DT40 wild type has 80 chromosomes (11 autosomal macromasomes, zw sex chromosome, 67 microchromosomes)

Only the macrochromosome and Z is examined by karyotyping at metaphase due to the mini is difficult.

chromosomal aberration;

1.isochromatid-type -- both sister chromatids broken at same locus

2.chromatid type -- single chromatid broken

Rad54-KO showed chromatid-type break whereas Ku70 and WT -very few.

Rad54-KO shows most sensitive to ionizing radiation and MMS.

Rad54+Ku70 -- even worst comparing to individual KO.

The degree of sensitivity toward the DNA damage challenge --> reflect which pw is preferred to fix DNA. In this Rad54 KO is more sensitive than Ku70 --> meaning HR is more favorable to fix the break generated by IR/MMS rather than Ku70.

Ku70 is less sensitive toward the IR/MMS comparing to the WT.

If we would like to investigate repair pw which relates to cell cycle state --> we have to synchronize the cell.

nocodazole can stop the cell at G2-M phase.

elutriation enriches the cell at G1 phase.

Rad54(-/-) -->has higher spontaneously chromosomal breaks.

Rad54 and Rad51 expressed only in cycling cell but not the resting cell.

In yeast species, lack of Rad51 and Rad54 --> the yeasts still survives but the growth rate is slow and the plating efficiency is very poor.

In higher eukaryote, the genome is much higher complex, therefore, HR is becoming important to maintain genome integrity.

Comparing between DT40 and mouse ES cell -- it was claimed that HR in DT40 is more important to fix DSB rather than in ES.

This study points out the preferential using of HR over NHEJ that is consistent  with the higher ratio of targeted over randomly integration comparing to other vertebrate ES cells.

Suggestion of this study for DT40 - effect of HR in DT40 giving the similar phenotypes with murine ES --> author suggest this DT40 is suitable model for studying HR in higher eukaryote.

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