Note: RAD18 and Poly(ADP-Ribose) Polymerase Independently Suppress the Access of Nonhomologous End Joining to Double-Strand Breaks and Facilitate Homologous Recombination-Mediated Repair

Note: RAD18 and Poly(ADP-Ribose) Polymerase Independently Suppress the Access of Nonhomologous End Joining to Double-Strand Breaks and Facilitate Homologous Recombination-Mediated Repair

(doi: 10.1128/mcb.01243-06)

Rad18 in yeast involves in post-replication repair but not dsb repair. Show less sensitivity toward cpt when generating ko. In contrast, Rad18 ko in dt40 showed high sensitivity toward cpt and involve in hr to repair dsb.

The authors mentioned that when deleting another key factor of nhej in hr deficient cell, and thus restoring the activity of hr, this mean that that hr factor (Rad18) plays role in the balancing between hr and nhej.

At stalled replication fork, Rad18 and Parp1 are used to control the balance between hr and nhej, by which the suppress the nhej to prevent toxic nhej during replication folk collapse.

PRR - there are two pws, 1.translesion, 2.template switch via HR

Translesion using the specialized polymerase to bypass the damage. Rad6 and Rad18 are responsible for this. These two amino acids are very conserved from yeast to human.

Rad6 is E2 protein binding to Rad18 which have the dna binding domain, and involves in recruiting the Rad18.

Rad18 is E3 ubiquitin ligase and contains ring finger motif. The complex transfer ubiquitin to the PCNA which is the clamp loading for dna polymerase.

PCNA-ub — will load the specialize dna polymerase to bypass the damage.

They are curious on the function from yeast to higher vertebrate whether it will be conserved along the evolution. Also they mentioned that in higher eukaryote has more polymerases than in the yeast.

Rad18 showed moderately sensitive to X-ray (asynchronous) - but when Rad18-KO is synchronized and detected the sensitivity at G2 -- show more sensitivity.

Using CPT -- to perform the DSB repair during the replication; Rad18-KO showed sensitivity toward CPT--this result contradicts with yeast which its mutant showed the sensitivity.

From the sensitivity results;

Rad18 contributes DSB repair during replication (as shown sensitivity toward CPT--reflect the activity at the S-phase) and after completion of replication (G2 -- synchronize and sensitivity toward gamma-irr).

To study Rad18 control which pathway -- they generate db-KO in this case Ku70 and they used another db-KO Ku70+Rev3 as control (no relation between these two genes).

Rad18 play role in the tolerability toward a wide range of DNA damage by which 1. avoid the toxic effect of NHEJ on HR (deleting Ku70--reverse the phenotype of db KO) 2.facilitation of PRR

another approach to detect HR-dependent repair is to use I-SceI construct and introducing into the OVALBUMIN locus. This construct contained two mutant neomycin, tandemly located but complementary to each other.

I-Sce-I -- transient expression.

By this construct, it can measure the HR activity -- the phenotype that can be observed once HR is used -->neomycin resistant.

Rad18 contributes to HR-mediated DSB repair.

Rad18 facilitates HR by suppressing NHEJ factors.

Test function of Rad18 in human cells (SW480 cells - mismatch repair-proficient cell line from colon cancer) --> using siRNA to knockdown Rad18.

Rad18 - not only involves in PRR pw by which adding Ub to PCNA (loading clamp for DNA polymerase to bypass the lesion). The authors also reported that Rad18 has another function by which it facilitates HR-dependent DSB in chicken and human cells.

Proposing Rad18 suppresses inappropriate NHEJ at stalled replication fork. The molecular mechanism need to be elucidated, especially the substrate for Rad18 in controlling such suppression.

Rad18 might suppress the NHEJ by adding ubiquitin whereas Parp1 controls through adding the sugar. It has this kind of fine control in order to avoid the toxic NHEJ.

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