Part:BBa_K4814005

RPA1 (human)

To further design a system that can detect DNA breaks in vivo, we proposed a FRET (Förster resonance energy transfer) system using the interaction between ATRIP and RPA1.

Due to the double-strand breaks (DSBs), replication protein A (RPA) will bind to the single-stranded DNA (ssDNA) and recruit ATRIP complex. The RPA70 (RPA1) N-terminus interacts with the ATRIP. (Bhat, K.P. and Cortez, D, 2018) (Maréchal, A. and Zou, L., 2015)

Dueva, R., & Iliakis, G. (2020) stated that ATRIP is one of the prominent RPA-interacting partners, playing a crucial role in the repair mechanism of DNA repairing and DNA damage checkpoints (Zou, Y., Liu, Y., Wu, X., & Shell, S. M., 2006). Moreover, the N-terminus of RPA1 (RPA70) is shown to be binding to the ATRIP protein (Xu, X., et al, 2008). Considering the protein size, we did not use the entire RPA, instead, we chose RPA1, the interacting domain, to design the FRET system.

• Click here for the experiment data: RPA1-mCherry BBa_K4814007 and RPA1-EYFP BBa_K4814009

References:

Bhat, K.P., & Cortez, D. (2018). RPA and RAD51: fork reversal, fork protection, and genome stability. Nature Structural & Molecular Biology, 25(6), 446-453. https://doi.org/10.1038/s41594-018-0075-z

Maréchal, A., & Zou, L. (2015). RPA-coated single-stranded DNA as a platform for post-translational modifications in the DNA damage response. Cell Research, 25(1), 9-23. https://doi.org/10.1038/cr.2014.147

Dueva, R., & Iliakis, G. (2020). Replication protein A: a multifunctional protein with roles in DNA replication, repair and beyond. NAR cancer, 2(3), zcaa022. https://doi.org/10.1093/narcan/zcaa022

Zou, Y., Liu, Y., Wu, X., & Shell, S. M. (2006). Functions of human replication protein A (RPA): from DNA replication to DNA damage and stress responses. Journal of cellular physiology, 208(2), 267–273. https://doi.org/10.1002/jcp.20622

Xu, X., Vaithiyalingam, S., Glick, G. G., Mordes, D. A., Chazin, W. J., & Cortez, D. (2008). The basic cleft of RPA70N binds multiple checkpoint proteins, including RAD9, to regulate ATR signaling. Molecular and cellular biology, 28(24), 7345–7353. https://doi.org/10.1128/MCB.01079-08

Sequence derived from: https://www.uniprot.org/uniprotkb/P27694/entry

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 922
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1735