Datasheet

生物学的記述

Specificity	Phospho-RPA32/RPA2 (Ser8) Antibody (Rabbit mAb) [B9B12] detects endogenous levels of RPA32/RPA2 protein only when phosphorylated at Ser8.
Background	Phospho-RPA32/RPA2 (Ser8) refers to the DNA damage–inducible modification of the 32 kDa subunit of replication protein A within the heterotrimeric RPA complex, a central single-stranded DNA–binding factor required for replication, recombination, checkpoint signaling and all major DNA repair pathways. RPA32 carries multiple N‑terminal serine and threonine sites that form a phospho-code; Ser4 and Ser8, together with Ser33 and other residues, become hyperphosphorylated in response to replication stress and genotoxic insults by phosphatidylinositol 3‑kinase–related kinases including DNA‑PK, ATR and ATM, on a background of prior cell cycle–linked phosphorylation at Ser23 and Ser29 by cyclin-dependent kinases. Phosphorylation of Ser4/Ser8 by DNA‑PK occurs when RPA accumulates on single-stranded DNA at stalled forks and acts as an early replication stress signal that modifies RPA–DNA and RPA–protein interactions; cells lacking DNA‑PK activity or expressing RPA32 mutants in which Ser4/Ser8 cannot be phosphorylated show defective replication checkpoint arrest, premature restart of stalled forks, failure to block late origin firing and increased mitotic catastrophe, indicating that the phospho‑Ser4/Ser8 state is required to enforce ATR–Chk1 checkpoint outputs and protect genome integrity. In this context, replication stress–induced hyperrecombination in Ser4/Ser8 mutants is ATM‑dependent, while other defects are ATM‑independent, placing DNA‑PK–RPA32 Ser4/Ser8 phosphorylation as a key branch point coordinating ATR- and ATM-mediated responses to stalled replication. Phosphorylated RPA32 at Ser4/Ser8 also participates in the regulation of homologous recombination repair: DNA‑PK, ATR and ATM collaboratively modify the RPA32 N‑terminus and p53 in a way that releases p53–RPA complexes, facilitates Rad51 loading and promotes HR repair of double-strand breaks, providing a mechanism for crosstalk between nonhomologous end joining and homologous recombination through coordinated control of p53–RPA interaction. RPA32 Ser4/Ser8 phosphorylation in G2 cells influences TopBP1 and Rad9 accumulation on chromatin, ATM-dependent KAP1 phosphorylation and Rad51 chromatin loading, reinforcing its role as a modulator of checkpoint signaling and recombination factor recruitment beyond the S phase. Phospho-RPA32 Ser8—typically assessed in combination with Ser4—serves as a mechanistic marker of replication stress and DNA damage response, where its status reports on DNA‑PK/ATR/ATM axis activation and functionally tunes replication checkpoint enforcement, fork restart timing, origin usage and the balance between recombination and mitotic catastrophe.

Specificity

Phospho-RPA32/RPA2 (Ser8) Antibody (Rabbit mAb) [B9B12] detects endogenous levels of RPA32/RPA2 protein only when phosphorylated at Ser8.

Background

Phospho-RPA32/RPA2 (Ser8) refers to the DNA damage–inducible modification of the 32 kDa subunit of replication protein A within the heterotrimeric RPA complex, a central single-stranded DNA–binding factor required for replication, recombination, checkpoint signaling and all major DNA repair pathways. RPA32 carries multiple N‑terminal serine and threonine sites that form a phospho-code; Ser4 and Ser8, together with Ser33 and other residues, become hyperphosphorylated in response to replication stress and genotoxic insults by phosphatidylinositol 3‑kinase–related kinases including DNA‑PK, ATR and ATM, on a background of prior cell cycle–linked phosphorylation at Ser23 and Ser29 by cyclin-dependent kinases. Phosphorylation of Ser4/Ser8 by DNA‑PK occurs when RPA accumulates on single-stranded DNA at stalled forks and acts as an early replication stress signal that modifies RPA–DNA and RPA–protein interactions; cells lacking DNA‑PK activity or expressing RPA32 mutants in which Ser4/Ser8 cannot be phosphorylated show defective replication checkpoint arrest, premature restart of stalled forks, failure to block late origin firing and increased mitotic catastrophe, indicating that the phospho‑Ser4/Ser8 state is required to enforce ATR–Chk1 checkpoint outputs and protect genome integrity. In this context, replication stress–induced hyperrecombination in Ser4/Ser8 mutants is ATM‑dependent, while other defects are ATM‑independent, placing DNA‑PK–RPA32 Ser4/Ser8 phosphorylation as a key branch point coordinating ATR- and ATM-mediated responses to stalled replication. Phosphorylated RPA32 at Ser4/Ser8 also participates in the regulation of homologous recombination repair: DNA‑PK, ATR and ATM collaboratively modify the RPA32 N‑terminus and p53 in a way that releases p53–RPA complexes, facilitates Rad51 loading and promotes HR repair of double-strand breaks, providing a mechanism for crosstalk between nonhomologous end joining and homologous recombination through coordinated control of p53–RPA interaction. RPA32 Ser4/Ser8 phosphorylation in G2 cells influences TopBP1 and Rad9 accumulation on chromatin, ATM-dependent KAP1 phosphorylation and Rad51 chromatin loading, reinforcing its role as a modulator of checkpoint signaling and recombination factor recruitment beyond the S phase. Phospho-RPA32 Ser8—typically assessed in combination with Ser4—serves as a mechanistic marker of replication stress and DNA damage response, where its status reports on DNA‑PK/ATR/ATM axis activation and functionally tunes replication checkpoint enforcement, fork restart timing, origin usage and the balance between recombination and mitotic catastrophe.

使用情報

Application

WB, IF, FCM

Dilution

WB	IF	FCM
1:1000	1:200 - 1:800	1:400 - 1:1600

Reactivity

Human

Source

Rabbit Monoclonal Antibody

MW

29 kDa

Storage Buffer

PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage
(from the date of receipt)

-20°C (avoid freeze-thaw cycles), 2 years

プロトコール

References

https://pubmed.ncbi.nlm.nih.gov/22797063/
https://pubmed.ncbi.nlm.nih.gov/24819595/

Phospho-RPA32/RPA2 (Ser8) Antibody (Rabbit mAb) [B9B12]

生物学的記述

使用情報

References

Application Data