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Producing recombinant human replication protein A 70 kDa DNA-binding subunit (RPA1) in E. coli involves co-cloning the target gene (2-616aa of human RPA1) into an expression vector with an N-terminal 6xHis-SUMO-tag gene, which is introduced into E. coli cells. The cells are cultured under conditions that promote protein expression. After sufficient growth, the cells are lysed to release the recombinant RPA1 protein. The collected proteins undergo affinity chromatography purification. The purity of the recombinant RPA1 protein is assessed using SDS-PAGE, exceeding 90%. Human RPA1 is a crucial component of the Replication Protein A (RPA) complex, which is a heterotrimeric single-stranded DNA-binding protein essential for various DNA processes in eukaryotic cells. RPA1 possesses strong single-stranded DNA binding activity. It plays a critical role in DNA metabolism, including processes such as replication, recombination, and repair [1-3]. RPA1 is crucial for maintaining genomic stability and ensuring accurate DNA replication [5]. Additionally, RPA1 is phosphorylated to facilitate mitotic exit in response to DNA damage during cell division [6]. The complex nature of RPA1, with its multiple functional domains, underscores its importance in DNA replication and repair processes [4] [5]. References:[1] G. Dodson, Y. Shi, & R. Tibbetts, Dna replication defects, spontaneous dna damage, and atm-dependent checkpoint activation in replication protein a-deficient cells, Journal of Biological Chemistry, vol. 279, no. 32, p. 34010-34014, 2004. https://doi.org/10.1074/jbc.c400242200[2] H. He, J. Wang, & T. Liu, Uv-induced rpa1 acetylation promotes nucleotide excision repair, Cell Reports, vol. 20, no. 9, p. 2010-2025, 2017. https://doi.org/10.1016/j.celrep.2017.08.016[3] E. Bochkareva, L. Frappier, A. Edwards, & A. Bochkarev, The rpa32 subunit of human replication protein a contains a single-stranded dna-binding domain, Journal of Biological Chemistry, vol. 273, no. 7, p. 3932-3936, 1998. https://doi.org/10.1074/jbc.273.7.3932[4] H. Kim and S. Brill, Rfc4 interacts with rpa1 and is required for both dna replication and dna damage checkpoints in saccharomyces cerevisiae, Molecular and Cellular Biology, vol. 21, no. 11, p. 3725-3737, 2001. https://doi.org/10.1128/mcb.21.11.3725-3737.2001[5] S. Binz and M. Wold, Regulatory functions of the n-terminal domain of the 70-kda subunit of replication protein a (rpa), Journal of Biological Chemistry, vol. 283, no. 31, p. 21559-21570, 2008. https://doi.org/10.1074/jbc.m802450200[6] R. Anantha, E. Sokolova, & J. Borowiec, Rpa phosphorylation facilitates mitotic exit in response to mitotic dna damage, Proceedings of the National Academy of Sciences, vol. 105, no. 35, p. 12903-12908, 2008. https://doi.org/10.1073/pnas.0803001105
Producing recombinant human replication protein A 70 kDa DNA-binding subunit (RPA1) in E. coli involves co-cloning the target gene (2-616aa of human RPA1) into an expression vector with an N-terminal 6xHis-SUMO-tag gene, which is introduced into E. coli cells. The cells are cultured under conditions that promote protein expression. After sufficient growth, the cells are lysed to release the recombinant RPA1 protein. The collected proteins undergo affinity chromatography purification. The purity of the recombinant RPA1 protein is assessed using SDS-PAGE, exceeding 90%.
Human RPA1 is a crucial component of the Replication Protein A (RPA) complex, which is a heterotrimeric single-stranded DNA-binding protein essential for various DNA processes in eukaryotic cells. RPA1 possesses strong single-stranded DNA binding activity. It plays a critical role in DNA metabolism, including processes such as replication, recombination, and repair [1-3].
RPA1 is crucial for maintaining genomic stability and ensuring accurate DNA replication [5]. Additionally, RPA1 is phosphorylated to facilitate mitotic exit in response to DNA damage during cell division [6]. The complex nature of RPA1, with its multiple functional domains, underscores its importance in DNA replication and repair processes [4] [5].
References:[1] G. Dodson, Y. Shi, & R. Tibbetts, Dna replication defects, spontaneous dna damage, and atm-dependent checkpoint activation in replication protein a-deficient cells, Journal of Biological Chemistry, vol. 279, no. 32, p. 34010-34014, 2004. https://doi.org/10.1074/jbc.c400242200[2] H. He, J. Wang, & T. Liu, Uv-induced rpa1 acetylation promotes nucleotide excision repair, Cell Reports, vol. 20, no. 9, p. 2010-2025, 2017. https://doi.org/10.1016/j.celrep.2017.08.016[3] E. Bochkareva, L. Frappier, A. Edwards, & A. Bochkarev, The rpa32 subunit of human replication protein a contains a single-stranded dna-binding domain, Journal of Biological Chemistry, vol. 273, no. 7, p. 3932-3936, 1998. https://doi.org/10.1074/jbc.273.7.3932[4] H. Kim and S. Brill, Rfc4 interacts with rpa1 and is required for both dna replication and dna damage checkpoints in saccharomyces cerevisiae, Molecular and Cellular Biology, vol. 21, no. 11, p. 3725-3737, 2001. https://doi.org/10.1128/mcb.21.11.3725-3737.2001[5] S. Binz and M. Wold, Regulatory functions of the n-terminal domain of the 70-kda subunit of replication protein a (rpa), Journal of Biological Chemistry, vol. 283, no. 31, p. 21559-21570, 2008. https://doi.org/10.1074/jbc.m802450200[6] R. Anantha, E. Sokolova, & J. Borowiec, Rpa phosphorylation facilitates mitotic exit in response to mitotic dna damage, Proceedings of the National Academy of Sciences, vol. 105, no. 35, p. 12903-12908, 2008. https://doi.org/10.1073/pnas.0803001105
| Cat.No | ACP04142 | Target Name | RPA1 |
|---|---|---|---|
| Form | Liquid or Lyophilized powder | Expression System | E.coli |
| Expression Range | 2-616aa | Mol Weight | 84.0kDa |
| Protein Length | Full Length of Mature Protein | Purity | Greater than 90% as determined by SDS-PAGE. |
| Storage Buffer | 5%-50% glycerol. Lyophilized powder form: the buffer before lyophilization is Tris/PBS-based buffer, 6% Trehalose, Liquid form: default storage buffer is Tris/PBS-based buffer, pH 8.0. |
| Target Species | Human | Uniprot ID | P27694 |
|---|
Uniprot Id
P27694
Target Species
Human
Target Name
RPA1
Target Full Name
Replication protein A 70 kDa DNA-binding subunit
Target Function
As part of the heterotrimeric replication protein A complex (RPA/RP-A), binds and stabilizes single-stranded DNA intermediates, that form during DNA replication or upon DNA stress. It prevents their reannealing and in parallel, recruits and activates different proteins and complexes involved in DNA metabolism. Thereby, it plays an essential role both in DNA replication and the cellular response to DNA damage. In the cellular response to DNA damage, the RPA complex controls DNA repair and DNA damage checkpoint activation. Through recruitment of ATRIP activates the ATR kinase a master regulator of the DNA damage response. It is required for the recruitment of the DNA double-strand break repair factors RAD51 and RAD52 to chromatin in response to DNA damage. Also recruits to sites of DNA damage proteins like XPA and XPG that are involved in nucleotide excision repair and is required for this mechanism of DNA repair. Plays also a role in base excision repair (BER) probably through interaction with UNG. Also recruits SMARCAL1/HARP, which is involved in replication fork restart, to sites of DNA damage. May also play a role in telomere maintenance. As part of the alternative replication protein A complex, aRPA, binds single-stranded DNA and probably plays a role in DNA repair. Compared to the RPA2-containing, canonical RPA complex, may not support chromosomal DNA replication and cell cycle progression through S-phase. The aRPA may not promote efficient priming by DNA polymerase alpha but could support DNA synthesis by polymerase delta in presence of PCNA and replication factor C (RFC), the dual incision/excision reaction of nucleotide excision repair and RAD51-dependent strand exchange.
Target Subcellular Location
Nucleus. Nucleus, PML body.
Target Protein Families
Replication factor A protein 1 family
Target Research Area
Epigenetics and Nuclear Signaling
Target Synonyms
Dmrpa1; Drosophila Replication Protein A; DRPA; HSSB; Human single stranded DNA binding protein ; MST075; MSTP075; p70; REPA1; Replication factor A; Replication factor A protein 1; Replication protein A 70 kDa DNA-binding subunit; Replication protein A 70kDa DNA binding subunit; Replication protein A1 70kDa; Replication protein A1; RF A; RF-A protein 1; RFA; RFA1_HUMAN; RP A; RP-A p70; RPA 70; RPA; rpa1; Single stranded binding protein 70; Single-stranded DNA-binding protein
Target Background
This gene encodes the largest subunit of the heterotrimeric Replication Protein A (RPA) complex, which binds to single-stranded DNA (ssDNA), forming a nucleoprotein complex that plays an important role in DNA metabolism, being involved in DNA replication, repair, recombination, telomere maintenance, and co-ordinating the cellular response to DNA damage through activation of the ataxia telangiectasia and Rad3-related protein (ATR) kinase. The nucleoprotein complex protects the single-stranded DNA from nucleases, prevents formation of secondary structures that would interfere with repair, and co-ordinates the recruitment and departure of different genome maintenance factors. This subunit contains four oligonucleotide/oligosaccharide-binding (OB) domains, though the majority of ssDNA binding occurs in two of these domains. The heterotrimeric complex has two different modes of ssDNA binding, a low-affinity and high-affinity mode, determined by which ssDNA binding domains are utilized. The different binding modes differ in the length of DNA bound and in the proteins with which it interacts, thereby playing a role in regulating different genomic maintenance pathways.
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