Disease relevance of RPA1

• We observed no significant difference with the unphosphorylated and phosphorylated forms of HSSB in the simian virus 40 DNA replication or nucleotide excision repair systems reconstituted with purified proteins [1].
• Taken together, we conclude that RPA1 has multiple roles in vivo and functions in DNA replication, repair, and recombination, like the single-stranded DNA-binding proteins of bacteria and phages [2].
• Reconstitution of functional human single-stranded DNA-binding protein from individual subunits expressed by recombinant baculoviruses [3].
• Escherichia coli single stranded DNA binding protein at concentrations that coat the single stranded regions had no influence on DNA unwinding by RP-A suggesting that RP-A binds fast and tightly to single-stranded DNA [4].
• There have been only three reported cases of anti-RPA in systemic lupus erythematosus (SLE) and Sjögren syndrome (SjS) [5].

High impact information on RPA1

• Phosphorylation of RPA may play a role in coordinating DNA metabolism in the cell [6].
• In cells, RPA is phosphorylated by DNA-dependent protein kinase when RPA is bound to single-stranded DNA (during S phase and after DNA damage) [6].
• RPA may also have a role in modulating gene expression [6].
• The NBSp26 protein is not physically associated with the MRE11 complex, whereas the p70 species is physically associated with it [7].
• In eukaryotic cells, the origin recognition complex (ORC) is the initiator protein and replication protein A (RPA; ref. 3) is the single-stranded DNA-binding protein [8].

Chemical compound and disease context of RPA1

• RhIL-12 p70 enhanced (P < .05) the secretion of IFN-gamma in controls, while it was ineffective in LPS-stimulated whole blood from critically ill patients [9].
• Room temperature fluorescence and low-temperature phosphorescence studies of the association of p10, a basic low molecular weight single-stranded DNA binding protein isolated from murine leukemia viruses, point to the involvement of its single tryptophan residue in a close-range interaction with single-stranded polynucleotides [10].
• As a first step towards manipulating the tropism of tetraviral nanoparticles (Capsivectors), a (His)6-tag was inserted into the GH loop (between Ala 378 and Gly 379) of the surface-exposed Ig-like domain of NomegaV capsid protein (p70) [11].
• Using [35S] methionine-labeled cell lines and immunoprecipitation, we have demonstrated the constitutive expression of p70 as well as a major component at p58 and a number at lower molecular weights in the myeloid leukemia cell lines HL60, KG1, K562 and U937, but not in the lymphocytic leukemia cell lines Molt-4, Hut-78 and CEM [12].

Biological context of RPA1

• Cells lacking RPA1 accumulate as multiply budded cells with a single nucleus suggesting a defect in DNA replication [13].
• RNA interference-mediated suppression of RPA1 caused a slowing of S phase progression, G2/M cell cycle arrest, and apoptosis in HeLa cells [14].
• Differential expression during the life cycle stages in three apicomplexan parasites suggests that the two RPA1 types exercise specialized biological functions [15].
• Apicomplexan RPA1 proteins are phylogenetically more related to plant homologues and probably arose from a single gene duplication event prior to the expansion of the apicomplexan lineage [15].
• The phosphorylation status of the p34 subunit of HSSB was unchanged during the reactions [1].

Anatomical context of RPA1

• Replication factor A (RPA) is a protein that binds single-stranded DNA in eukaryotic cells; it participates in replication, repair, and recombination of DNA [16].
• In human umbilical vein endothelial cells, inhibition of RPA1 expression using antisense oligonucleotide restored transcription driven by the mutated promoter sequence, whereas, conversely, overexpression of RPA1 further reduced it [17].
• RPA1 thus apparently functions as a repressor protein in the -786T-->C mutation-related reduction of eNOS gene transcription associated with the development of coronary artery disease [17].
• RPA1 was similarly detected in placenta and eNOS mRNA levels in placentas carrying the -786T-->C mutation were significantly lower than in placentas without it [17].
• These results demonstrate that in HL-60 cells, Hoechst 33342-induced apoptosis is associated with a rapid loss of the binding capacity of RPA to oligo(dT)(30) as well as immunoactive RPA-70 and RPA-32 [18].

Associations of RPA1 with chemical compounds

• Together, these data demonstrate that RPA and the MRN complex co-localize and interact after HU- or UV-induced replication stress and suggest that protein phosphorylation may play a role in this interaction [19].
• X-ray fluorescence spectra revealed that RPA70-CTD possesses a coordinated Zn(II) [20].
• Western blotting results showed that both RPA-32 and RPA-70 were decreased significantly in a time-dependent manner after 1 h of incubation with Hoechst 33342 [18].
• In contrast, the RPA70 subunit does not require DTT for its DNA binding activity and is not affected by the redox condition [21].
• Loss of RPA1 induces Chk2 phosphorylation through a caffeine-sensitive pathway [22].

Physical interactions of RPA1

• DNA-PKcs interacted directly with RPA1 in vitro [23].
• Partial proteolytic digests revealed that p14 and p32 together stabilize the C terminus of p70 against degradation [24].
• The complex of hRPA14 and hRPA32-(43-171) in turn formed a trimeric complex with the C-terminal region of hRPA70 (amino acids 436-616) [25].
• It appears that the RPA32CTD can substitute for RPA70 in binding Rad52 [26].
• We found that RPA interacts with xeroderma pigmentosum group A complementing protein (XPAC), a protein that specifically recognizes UV-damaged DNA [27].

Enzymatic interactions of RPA1

• Both Cdc2 kinase and the DNA-dependent protein kinase (DNA-PK) phosphorylate HSSB-p34 in vitro [28].
• While ATR phosphorylates the N-terminus of RPA70, Chk1 preferentially phosphorylates RPA's major ssDNA binding domain [29].

Co-localisations of RPA1

• Werner's syndrome protein (WRN) migrates Holliday junctions and co-localizes with RPA upon replication arrest [30].

Regulatory relationships of RPA1

• Fractionation of cyclin A-activated G1 extract identified two kinases involved in the hyperphosphorylation of HSSB p34: cdk-cyclin A complex and DNA-dependent p350 protein kinase (DNA-PK) [31].
• The stimulatory effect of HSSB was markedly inhibited by the combined action of A1 and PCNA [32].
• The 3'-5' exonuclease activity was stimulated 8-10 fold by the addition of HSSB, and this stimulatory effect was preferential to HSSB since other SSBs from E. coli, T4 or adenovirus, had a little or no effect [32].
• The N-terminal 168 residues of RPA70 form a structurally distinct domain that stimulates DNA polymerase alpha activity, interacts with several transcriptional activators including tumor suppressor p53, and during the cell cycle it signals escape from the DNA damage induced G2/M checkpoint [33].
• Both wild-type p53 and a transactivation-deficient p53 mutant (L22Q/W23S) suppressed HR and prevented RPA binding to ssDNA in vitro and in vivo [34].

Other interactions of RPA1

• In this study, we investigated the effect of phosphorylation of p34 by these kinases on the replication and repair function of HSSB [1].
• Addition of p21cip1, a specific inhibitor of cdk-cyclin A but not DNA-PK, nearly abolished the hyperphosphorylation of HSSB p34 in G1 extract preincubated with cyclin A. This suggests a requirement of the cdk-cyclin A activity for the phosphorylation of p34 by DNA-PK in G1 extract [31].
• Here, we demonstrate that the ATR checkpoint kinase and RPA1 are required for efficient FANCD2 monoubiquitination [35].
• We examined the effect of this XPAC-RPA interaction on in vitro simian virus 40 (SV40) DNA replication catalyzed by the monopolymerase system [27].
• In addition, the consistent formation of phospho-Nbs1 foci in all of the treatment groups suggests that the MRN complex may play a more universal role in the recognition and response to DNA lesions of all types, whereas the role of RPA may be limited to certain subsets of lesions [36].

Analytical, diagnostic and therapeutic context of RPA1

• Gel filtration analyses of cellular extracts showed that endogenous (HSA)kin17 protein co-eluted with both replication proteins RPA32 and RPA70 in a fraction containing complexes of M(r) 600,000 [37].
• Using a gel retardation assay, we show here that nuclear extracts of C. fasciculata contain a protein factor(s) that binds specifically to RNA from 5'-UTRs of TOP2 and RPA1 genes [38].
• We report here that, by using affinity chromatography and immunoprecipitation, we found that human RPA bound specifically and directly to two excision repair proteins, the xeroderma pigmentosum damage-recognition protein XPA (refs 8, 9) and the endonuclease XPG (refs 10-13) [39].
• Thus, one fraction carries out nick-directed and mismatch-dependent excision; the second carries out DNA repair synthesis and DNA ligation; and the third provides hRPA, which plays multiple roles in human MMR by protecting the template DNA strand from degradation, enhancing repair excision, and facilitating repair synthesis [40].
• In vitro dissection of the MMR reaction and crucial intermediates elucidated biochemical functions of individual fractions in human MMR and identified hitherto unknown functions of human replication protein A (hRPA) in MMR [40].

References