Sensing DNA Damage Through ATRIP Recognition of RPA-ssDNA Complexes

  title={Sensing DNA Damage Through ATRIP Recognition of RPA-ssDNA Complexes},
  author={Lee Zou and Stephen J. Elledge},
  pages={1542 - 1548}
The function of the ATR (ataxia-telangiectasia mutated– and Rad3-related)–ATRIP (ATR-interacting protein) protein kinase complex is crucial for the cellular response to replication stress and DNA damage. Here, we show that replication protein A (RPA), a protein complex that associates with single-stranded DNA (ssDNA), is required for the recruitment of ATR to sites of DNA damage and for ATR-mediated Chk1 activation in human cells. In vitro, RPA stimulates the binding of ATRIP to ssDNA. The… 

A Novel Protein Activity Mediates DNA Binding of an ATR-ATRIP Complex*

It is found that both ATR and ATRIP associate with chromatin in vivo, and they exist as a large molecular weight complex that can bind single-stranded (ss)DNA cellulose in vitro and a distinct ATR-ATRIP complex is able to bind to DNA with lower affinity in the absence of RPA.

Reconstitution of RPA-covered single-stranded DNA-activated ATR-Chk1 signaling

An in vitro checkpoint system in which RPA-ssDNA and TopBP1 are essential for phosphorylation of Chk1 by the purified ATR-ATRIP complex, and an alternative form of RPA (aRPA), which does not support DNA replication, can substitute for the checkpoint function of R PA in vitro, thus revealing a potential role for aRPA in the activation of ATR kinase.

ATRIP associates with replication protein A-coated ssDNA through multiple interactions.

  • Y. NamikiL. Zou
  • Biology
    Proceedings of the National Academy of Sciences of the United States of America
  • 2006
It is shown that ATRIP associates with RPA-ssDNA through multiple interactions, and one internal region of ATRIP exhibited affinity to ssDNA, suggesting that AT RIP may interact with ssDNA in the ATRIP-RPA-SSDNA complex.

Ddc2ATRIP promotes Mec1ATR activation at RPA-ssDNA tracts

It is shown that Ddc2 promotes Mec1 activation independently of Ddc1/Dpb11/Dna2 function in vivo and through ssDNA recognition in vitro, which supports a model in which Ddc 2 promotes MEC1 activation at RPA-ssDNA tracts.

RPA70 depletion induces hSSB1/2-INTS3 complex to initiate ATR signaling

It is reported that the single-stranded DNA-binding protein complex, hSSB1/2-INTS3 can recruit the checkpoint complex to initiate ATR signaling.

Function of a Conserved Checkpoint Recruitment Domain in ATRIP Proteins

The results support the idea of a multistep model for ATR activation that requires separable localization and activation functions of ATRIP, and demonstrate that the CRD is critical for localization and optimal DNA damage responses.

The Basic Cleft of RPA70N Binds Multiple Checkpoint Proteins, Including RAD9, To Regulate ATR Signaling

The basic cleft of the RPA70 N-terminal OB-fold domain binds multiple checkpoint proteins, including RAD9, to promote ATR signaling, and is found to be a key determinant of checkpoint activation.

Direct binding to RPA-coated ssDNA allows recruitment of the ATR activator TopBP1 to sites of DNA damage

Protein binding assays and functional studies in Xenopus egg extracts supply a mechanism for how the critical ATR activator, TopBP1, senses DNA damage and stalled replication forks to initiate assembly of checkpoint signaling complexes.



Recruitment of Mec1 and Ddc1 Checkpoint Proteins to Double-Strand Breaks Through Distinct Mechanisms

Chromatin immunoprecipitation experiments revealed that Mec1 and Ddc1 are recruited independently to sites of DNA damage, suggesting the existence of two separate mechanisms involved in recognition ofDNA damage.

Regulation of ATR substrate selection by Rad17-dependent loading of Rad9 complexes onto chromatin.

It is shown that the human Rad17 protein recruits the Rad9 protein complex onto chromatin after damage and the phosphorylation of Rad17 requires Hus1, suggesting that the Rad1-Rad9-Hus1 complex recruited by Rad17 enables ATR to recognize its substrates.

Two checkpoint complexes are independently recruited to sites of DNA damage in vivo.

It is shown that Ddc1 or Ddc2 fused to GFP localizes to a single subnuclear focus following an endonucleolytic break, which supports a model in which assembly of multiple checkpoint complexes at DNA damage sites stimulates checkpoint activation.

Purification and characterization of human DNA damage checkpoint Rad complexes

Findings constitute biochemical support for models regarding the roles of checkpoint Rads as damage sensors in the DNA damage checkpoint response of human cells.

Preferential binding of ATR protein to UV-damaged DNA

It is found that ATR is a DNA-binding protein with higher affinity to UV-damaged than undamaged DNA, and damaged DNA stimulates the kinase activity of ATR to a significantly higher level than undAMaged DNA.

Interaction of human rad51 recombination protein with single-stranded DNA binding protein, RPA.

The interactions of human RPA with Rad51, replication proteins and DNA are suited to the linking of recombination to replication.

The ATM homologue MEC1 is required for phosphorylation of replication protein A in yeast.

The results indicate a functional similarity between MEC1 and ATM, and suggest that RPA phosphorylation is involved in a conserved eukaryotic DNA damage-response pathway defective in A-T.

ATR and ATRIP: Partners in Checkpoint Signaling

The identification of an ATR-interacting protein (ATRIP) that is phosphorylated by ATR, regulates ATR expression, and is an essential component of the DNA damage checkpoint pathway is reported.

The checkpoint protein Ddc2, functionally related to S. pombe Rad26, interacts with Mec1 and is regulated by Mec1-dependent phosphorylation in budding yeast.

The findings suggest that Ddc2 may be the functional homolog of Schizosaccharomyces pombe Rad26, strengthening the hypothesis that the mechanisms leading to checkpoint activation are conserved throughout evolution.