Note for: Parp-1 protects homologous recombination from interference by Ku and Ligase IV in vertebrate cells

Note: Parp-1 protects homologous recombination from interference by Ku and Ligase IV in vertebrate cells

(doi: 10.1038/sj.emboj.7601015)

Parp-1 and Parp-2 -- responsible for SSB repair but the role of these two in HR is not clear. Using DT40 as a tool to study.

DT40 has only Parp-1, no Parp-2. Parp-1(-/-) reduced the level of HR, sensitive to various DSB-inducing genotoxic agents.

More sensitive when having Ku which is the DSB-binding factor. PARP1+KU70 -- proficient in initiate HR and show resistance to DSB-inducing drugs.

In conclusion - having parp-1 suppress the Ku70 function and NHEJ on HR.

there are so many parps (poly (ADP-ribosylation) proteins -- in mammalian, it contains 17 parps, only parp1 and parp2 act on the DNA damage response. Prarp1 and 2 interact with each other and found in the large cpx with XRCC1 and DNA-pol-beta and DNA ligase III.

Evident has showed Parp1 and parp2 play role in base excision repair (BER) on damage detection, signaling and recruitment of Xrcc1 to lesion.

Parp-1 binds to BER proteins.

Parp-1 also binds Ku/DNA-PK complex (NHEJ factor) via the biochemical assay observation.

Ku/DNA-PK are the substrate of Parp-1 (adding ADP-ribose -- as poly).

the interaction mechanism is not clear how parp-1 interfere with the Ku/DNA-PK being detached from the DSB.

By this kind of interaction, the author proposed that Parp-1 and or Parp-2 involved in DSB repair.

There is the competition between HR and NHEJ - even in the S-phase, NHEJ factors are recruited to the sites of DSB. The competition between these two is still unclear.

More support evident showed that Parp should be involved with DSB repair -- deletion of Parp causing increase in sister-chromatid exchange.

cells carrying truncated BRCA2 are sensitive to inhibition of or knock down of Parp-1. Double KO between Parp-1 and wither DSB repair and checkpoint genes leading to synthetic lethality. Parp-1 partially involved in VDJ recombination -- altogether, the author points out that there was no study point out on how Parp-1 directly involves in DSB repair.

Chicken lacks the Parp2 homolog;

Parp1 - sensitive to IR, CPT and DNA-methylating agents.

Deleting Parp-1 in exon3 and 4 in DT40

Obtaining 3 clones of Parp; show similar phenotype with little clonal variation. They only measure the cell growth and perform only one clone in further experiments.

Parp inhibitor -- 3-AB

H2O2 -- could generate the SS-break.

biochemical assay for the polymer formation of sugar at the SSbreak -- using P32-NAD as the substrate to observe the affect. the output result is the protein P32-labeled PAR carrying proteins.

They start to see the interaction affect through Ju70 first due to the previous evident. They tested  the sensitivity toward several genotoxic compounds with individual KO and db KO.

Biphasic phase of Ku70 when testing with IR -- corresponding the cell phase;

G1 -- 10% --> show sensitivity

S-G2 -- showed resistant -- competition between K70 and HR pw.

Deletion of  Parp -- causing the accumulation of SSbreak and when the cell replicate causing the db strand break -- cell has to choose whether going to HR or NHEJ -- CPT show the most sensitive --> therefore , it indicate the favor to HR?

The authors mentioned that "in general", DSB repair in DT40 is similar to mammals but they are also aware that high recombination level might be irrelevant to mammalian cells - esp. balance between HR + NHEJ is under focus.

DPQ -- Parp inhibitor

DT40--Parp1(-/-) + either hParp-1 or hParp-2 rescues the phenotype of DT40-Parp1(-/-)

Parp-1 intertwines between HR and NHEJ.

DSB occurs -- compete between HR and NHEJ -- (the balance between this depending on cell type) --  Ku binds at the DSB first and the Parp1 counteracts the interaction between Ku and DNA (Parp1 bind to Ku and disrupt the interaction -- biochemical assay showed that post-modification by adding poly-sugar to Ku70 --> affect the binding of Ku to DNA).

not only Parp1 that inhibits Ku70 to prevent unwanted NHEJ but also LIG4 as indicated by the sensitivity assay toward CPT.

The authors point outs that in yeast NHEJ is very minor - therefore unwanted ligation after DSB, rarely occur, whereas in veterbrate, NHEJ is becoming more prominent due to the complexity of DNA, therefore, the vertebrate has developed mechanism which prevents unwanted NHEJ which is lethal to the cells.

High levels of SCR point out genetic instability by which DNA repair is defected and thereby increasing in HR substrates.

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