Note for: RAD18 and RAD54 cooperatively contribute to maintenance of genomic stability in vertebrate cells

Note for: RAD18 and RAD54 cooperatively contribute to maintenance of genomic stability in vertebrate cells

(doi: 10.1093/emboj/cdf534)

Major pathway for post-replcational DNA damage;

1. TLS

2. HR

Rad18- conserved from lower eukaryotes to vertebrates.

double KO, synthetic lethal --> ach has nearly normal kinetic (in term of proliferation).

Gap in this study; no one study the role of TLS in maintenance of chromosomal DNA.

In yeast, S. cerevisiae;

RAD6/RAD18 epistasis group (all in;

1. RAD6(UBC2)

2. RAD18

3. REV1

4. REV3

5. REV7

Rad6 -- E2 conjugating enzyme

Rad18 -- has the DNA binding domain

it is proposed that Rad18 recruited Rad6 and transferring Ub to the targeted proteins.

Another gap --> so far, the targeted for Rad18+Rad6 is still unk, but it has been proposed that it could bind to DNA polymerase that play activity in TLS.

Mammals,

2 homologs of RAD6 --> HR6A and HR6B

1 homolog RAD18 --> RAD18

AA comparison;

Rad18

- human/mouse and chicken

50% identity

- human and mous

65%

- yeast and chicken

25%

RING-domain

-human and mouse

70%

All five of RAD18-KO showed identical phenotype - in the paper therefore show only two representative.

To investigate how the DNA damaging kill the cell -- they perform the chromosomal aberration test after exposing to the drug-->bc of the chromosomal breakage was detected in higher level in KO --> authors assume that the sensitivity is caused by this mechanism.

To test whether this gene is related to post-replication repair;

1. synchronize the cell to G1 phase

2. damage with UV with various time point after release

3. colony survival assay

PRR;

1. TLR

2. HR

Way to test hyperactivation of HR --> targeted integration.

Lethal when generating - RAD18+RAD54, therfore, they have to generate the conditional KO with RAD54 in RAD18 KO.

Transient expression of Cre-->can get rid of loxP fragment within 3 days in DT40 cells.

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