Disease relevance of RPA2

• Mutations in VP16 that reduce the ability of GAL4-VP16 to activate polyomavirus DNA replication also compromise the binding of VP16 to RPA [1].
• These results show that RPA is a target for autoantibodies in human autoimmune diseases, although its precise frequency, occurrence in other autoimmune diseases, and pathological significance remain to be fully elucidated [2].
• The gamma-irradiation-induced phosphorylation of RPA p34 is delayed in cells from ataxia telangiectasia, a human inherited disease conferring DNA repair defects and early-onset tumorigenesis [3].
• UV-induced phosphorylation of RPA p34 occurs less rapidly than gamma-irradiation-induced phosphorylation but is kinetically similar between ataxia telangiectasia and normal cells [3].
• In simian virus 40 (SV40) DNA replication, RPA containing either of these mutants functioned as efficiently as wild-type RPA [4].

Psychiatry related information on RPA2

• Facial dysmorphism, clinodactyly of the fifth finger, mental retardation and heart defects are, however, most frequently described, with a high-arched palate, in particular, seen in deletions distal to p34 [5].

High impact information on RPA2

• 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].
• Replication protein A (RPA), the nuclear ssDNA-binding protein in eukaryotes, is essential to DNA replication, recombination, and repair [7].
• We suggest that transcription factors may interact with RPA either to stabilize single-stranded DNA at a replication origin or to recruit DNA polymerase alpha to the replication initiation complex [1].

Chemical compound and disease context of RPA2

• A recombinant protein of zebrafish RPA p32 containing a short histidine tag at the NH(2)-terminus was overexpressed in Escherichia coli BL21(DE3) pLys using an inducible T7 expression system, and was purified by Ni-NTA affinity chromatography [8].
• Amino acid residues in the NH2-terminal region of the p32 protein exhibit similarity to glycoprotein X (gX) of pseudorabies virus (PRV) and its homolog in equine herpesvirus type 1 (EHV-1) [9].

Biological context of RPA2

• RPA2 phosphorylation in response to camptothecin required ongoing DNA replication [10].
• Keywords: camptothecin/DNA damage/DNA-dependent protein kinase/RPA2 phosphorylation[10]
• DNA-PK activation and RPA2 phosphorylation were prevented by the cell-cycle checkpoint abrogator 7-hydroxystaurosporine (UCN-01), which markedly potentiates camptothecin cytotoxicity [10].
• Camptothecin itself partially inhibited DNA synthesis, and this inhibition followed the same kinetics as DNA-PK activation and RPA2 phosphorylation [10].
• In this part of UNG a 23-amino acid sequence has a significant homology to the RPA2-binding region of XPA, a protein involved in damage recognition in nucleotide excision repair [11].

Anatomical context of RPA2

• RPA2 was mapped to chromosome 1p35 by PCR amplification of somatic cell hybrids of chromosome 1 and by fluorescence in situ hybridization [12].
• The RP-A purified from calf thymus contains 4 polypeptides with molecular weights 70kDa, 53kDa, 32kDa, and 14kDa [13].
• Replication protein A (RPA), the trimeric single-stranded DNA-binding protein complex of eukaryotic cells, is important to DNA replication and repair [3].
• Sites of UV-induced phosphorylation of the p34 subunit of replication protein A from HeLa cells [14].
• ATM immunoprecipitates from HeLa cells and the human DNA-dependent protein kinase null cell line MO59J, but not from AT cells, phosphorylated the 34-kDa subunit of replication protein A (RPA) complex in a single-stranded and linear double-stranded DNA-dependent manner [15].

Associations of RPA2 with chemical compounds

• Comparison between CPT and hydroxyurea (HU) indirectly inducing DSBs showed that RPA2 hyperphosphorylation is DNA-PK-dependent in CPT-treated cells and DNA-PK-independent in HU-treated cells [16].
• In particular, we monitored surface accessibility of RPA lysines with NHS-biotin modification in the contexts of the free protein and the nucleoprotein complex [17].
• Phosphorylation of p34 RPA occurred on threonine and serine residues [15].
• At least four of the eight serines and one threonine in the N-terminal 33 residues of RPA-p34 can become phosphorylated after UV irradiation [14].
• Both human and yeast RPA stimulated the polymerase and (at subsaturating levels of RPA) the primase activities of human DNA polymerase alpha/primase on homopolymer DNA templates [18].

Physical interactions of RPA2

• Partial proteolytic digests revealed that p14 and p32 together stabilize the C terminus of p70 against degradation [19].

Enzymatic interactions of RPA2

• Although ataxia telangiectasia-mutated (ATM) and DNA-dependent protein kinase (DNA-PK) phosphorylate RPA2 in vitro, their role in vivo remains uncertain, and contradictory results have been reported [20].
• We have previously shown that the phosphorylation of p34 is catalyzed by both cyclin-dependent kinase-cyclin A complex and DNA-dependent protein kinase [21].
• RPA32 was cleaved rapidly to a approximately 28-kDa polypeptide containing the C-terminus that was partially resistant to further digestion [22].
• It has been shown previously that RPA32 is phosphorylated both during the S-phase of a normal cell cycle and in response to DNA damage [23].
• RPA was phosphorylated in vitro by the cell cycle-regulated cdc2 protein kinase [24].

Regulatory relationships of RPA2

• Multiple changes in the digestion pattern were observed when RPA bound single-stranded DNA: degradation of the approximately 52-kDa domain of RPA70 was inhibited while proteolysis of RPA32 was stimulated [22].
• ATR knock-down with siRNA suppressed CPT-induced RPA2 hyperphosphorylation and focus formation [16].
• Taken together, these results suggest that 53BP1 is involved in DNA damage-induced RPA2 hyperphosphorylation, and inhibition of 53BP1 function may sensitize cancer cells to camptothecin treatment [25].
• However, the relative roles of ATM and DNA-PK in the site-specific DNA damage-induced phosphorylation of RPA32 have not been reported [26].
• Further mutational studies indicated that, although the N-terminal 32-amino acid peptide of C/EBPepsilon isoform p32 did not greatly influence the transactivation activity compared with p30 isoform, this peptide does modulate transactivation activity [27].

Other interactions of RPA2

• In this study, we investigated the effect of phosphorylation of p34 by these kinases on the replication and repair function of HSSB [21].
• In an in vitro assay for DNA replication, DNA-PK is the sole kinase phosphorylating RPA2, indicating that processes not reproduced in the in vitro assay are required for RPA2 phosphorylation by ATM [20].
• Limited proteolysis of the hRPA14.32 complex defined a core dimer composed of the central region of hRPA32 (amino acids 43-171) and RPA14 [28].
• The menin-RPA2 interaction was confirmed in a conventional yeast two-hybrid system and by direct interaction between purified proteins [29].
• UCN-01, an inhibitor of protein kinase C, Chk1, and cyclin-dependent kinases, has no effect on IR-induced RPA2 phosphorylation [20].

Analytical, diagnostic and therapeutic context of RPA2

• Northern (RNA) blot analysis suggest that there are alternatively processed forms of the RPA4 mRNA, and Southern blot analysis indicates that beside RPA4 there may be other members of the RPA2 gene family [30].
• Here we show that RPA2 phosphorylation is delayed in cells deficient in one of these kinases and completely abolished in wild-type, ATM, or DNA-PK-deficient cells after treatment with wortmannin at a concentration-inhibiting ATM and DNA-PK [20].
• A sandwich enzyme-linked immunosorbent assay with trimeric RPA and the two-hybrid system both demonstrated that the interaction depends on a region in UNG localized between amino acids 28 and 79 in the open reading frame [11].
• In this work, we employed a mass spectrometric protein footprinting method of single amino acid resolution to investigate the interactions of the entire heterotrimeric hRPA with ssDNA [17].
• In female patients but not in male patients, the body mass index z score decreased from 0.60 in group RPA0 to 0.51 in group RPA2 (P<.001) [31].

References