RPA2 (replication protein A2, 32kDa)

2010-04-01   Anar KZ Murphy , James A Borowiec 

Dept of Biochemistry, New York University Cancer Institute, New York University School of Medicine, New York, New York 10016, USA

Identity

HGNC
LOCATION
1p35.3
LOCUSID
ALIAS
REPA2,RP-A
FUSION GENES

DNA/RNA

Atlas Image
The sequence numbering corresponds to EMBL locus DQ001128 (26.6 kb). Exon are indicated as boxes (yellow = 5 UTR, blue = CDS, red = 3 UTR), and introns with orange lines. Two lengthy introns have been truncated (indicated with parallel diagonal lines) to improve viewability.

Description

The RPA2 gene is contained within 24.5 kb of chromosome 1. The coding sequence is contained within nine exons. There is no confirmed alternative splicing of the RPA2 gene, or differential promoter usage.

Transcription

The RPA2 mRNA transcript is 1.5 kb. The RPA2 promoter contains four E2F consensus sequences within the region about 400 bp upstream of the mRNA start site, and putative binding sites for ATF-1 and SP-1 transcription factors. Expression is upregulated 2 to 3-fold by E2F, with mutation of the three start site-proximal E2F sites causing a loss of E2F responsiveness (Kalma et al., 2001).

Pseudogene

RPA2 does not have known pseudogenes.

Proteins

Atlas Image
Upper panel: Schematic showing the key domains of RPA2. Lower panel: RPA2 phosphorylation sites are shown in bold, with the primary responsible kinases indicated above each site. Some sites can be phosphorylated by more than one kinase (e.g., T21 by ATM and DNA-PK).

Description

RPA2 is the middle subunit of the heterotrimeric Replication Protein A (RPA; (reviewed in Binz et al., 2004)). The subunit is composed of 270 residues, and has a nominal molecular weight of 29.2 kDa. RPA2 contains an N-terminal phosphorylation region with 7 phosphorylation sites, a central DNA-binding domain (termed DBD-D), and a C-terminal region that can form a three-helix bundle. One helix of the three helix bundle is contributed by each RPA subunit, with this structure responsible for supporting heterotrimerization of the RPA complex (Bochkareva et al., 2002). At least in the non-phosphorylated state, the N-terminal region is unstructured. DBD-D is constructed from an oligonucleotide/oligosaccharide binding (OB) fold (Bochkarev et al., 1999), one of six OB folds found with the RPA heterotrimer (four OB folds are located in RPA1, and one within RPA3).

Expression

RPA is an essential factor for DNA replication and repair, and hence is expressed in all tissues.

Localisation

Nuclear.

Function

General function: RPA is a heterotrimeric single-stranded DNA (ssDNA) binding protein that is essential for chromosomal DNA replication, homologous recombination, and particular DNA repair reactions (nucleotide excision repair). The apparent association constant of the RPA: ssDNA complex is 109 - 1011 M-1 (Kim et al., 1992). While RPA2 contains a central DBD (Philipova et al., 1996), the major effect of mutating DBD-D is to decrease the size of the ssDNA occluded by RPA binding, with only minor effects on RPA: ssDNA affinity (Bastin-Shanower and Brill, 2001). A key function of the RPA2 subunit is to regulate RPA activity in DNA replication and repair reactions, through the RPA2 phosphorylation state (see below).

1) RPA2 phosphorylation. The N-terminal 33 residues of RPA2 contain seven phosphorylation sites. In interphase cells, genotoxic stress (e.g., caused by chromosomal double-strand DNA breaks or DNA replication stress) induces RPA2 phosphorylation by members of the phosphatidylinositol 3-kinase-like kinase (PIKK; ATM, ATR, and DNA-PK) and cyclin-dependent kinases (CDK) families (reviewed in Binz et al., 2004). Mutation of particular RPA2 phosphorylation sites causes defects in homologous recombination (Lee et al., 2010), and Rad51 recruitment to nuclear repair foci (Anantha et al., 2008; Lee et al., 2010). Mutation of these sites also causes genomic instability in response to DNA replication stress induced by cellular treatment with hydroxyurea (Vassin et al., 2009). RPA phosphorylation also increases cell viability in response to DNA damage arising during mitosis (Anantha et al., 2008). Modification of sites in the phosphorylation region of RPA2 proceeds in a favored order in response to genotoxic stress (Anantha et al., 2007). The phosphorylation of individual RPA2 residues is dependent on the type of DNA damage or replication stress encountered (Anantha et al., 2007; Vassin et al., 2009). RPA2 is a substrate both for PP2A and PP4 phosphatases (Feng et al., 2009; Lee et al., 2010).

2) Involvement of RPA2 in protein-protein interactions. RPA2 interacts with the nucleotide excision repair factor XPA (He et al., 1995), base excision repair enzyme UNG2 (Mer et al., 2000), homologous recombination (HR) factor Rad52 (Mer et al., 2000), replication checkpoint protein Tipin (Unsal-Kacmaz et al., 2007), and the annealing helicase HARP/SMARCAL1 (Bansbach et al., 2009; Ciccia et al., 2009; Yuan et al., 2009). These interactions likely aid the multiple roles of RPA in facilitating DNA repair.

Homology

A close homolog of RPA2, termed RPA4, is located on Xq21.33 (Haring et al., 2010).

Mutations

Note

Naturally-occurring mutations of human RPA2 have not yet been described. A small number of genetic polymorphisms have been described in SNP datasets (Y14S, G15R, and N203S), but these have not yet been reported to have any biological effects (NIEHS SNPs program).

Implicated in

Entity name
Colorectal adenocarcinoma
Disease
Overexpression of the RPA2 (and RPA1) proteins have been found to be prognostic indicator of colon cancer. Strong associations between RPA2 expression and disease stage, lymph node metastasis, and the histological grade of carcinomas have been observed.
Prognosis
In addition, RPA2 protein expression correlates with poor survival of stage II and III patients (Givalos et al., 2007).
Entity name
Ductal breast carcinoma
Disease
Levels of anti-RPA2 antibodies was observed to be significantly higher in sera from breast cancer patients (10.9%; n = 801) as compared to normal controls (0.0%; n = 221). Examining individuals with early stage intraductal in situ carcinomas, 10.3% (n = 39) similarly showed the presence of high levels of anti-RPA2 antibodies. Even so, follow-up studies indicated that there were no apparent differences in mean survival, occurrences of a second primary tumor, or metastasis frequency between breast cancer patients that were positive or negative for anti-RPA2 sera. Although RPA is a nuclear protein, RPA was seen to be localized to both nuclei and cytoplasm in the cells of at least one breast tumor, with RPA also over-expressed (Tomkiel et al., 2002).
Entity name
Non-small cell carcinoma
Disease
A fraction of individuals with squamous cell lung cancer were found to have significant levels of anti-RPA2 antibodies (9.1%; n = 22) (Tomkiel et al., 2002).
Entity name
Laryngeal tumors
Disease
One patient (out of 35; 2.9%) with head and neck tumors tested positive for the presence of anti-RPA2 sera (Tomkiel et al., 2002).
Disease
A derivative of the human HL-60 promyelocytic leukemia cell line (HL-60/P1), selected for its decreased sensitivity to undergo apoptosis in response to TNF-related apoptosis-inducing ligand (TRAIL), was found to have decreased (2-fold) expression of RPA2 (Petrak et al., 2009).
Entity name
Sjögren syndrome
Disease
Serum from a patient with Sjögren syndrome was found to have high levels of anti-RPA2 antibodies. A higher rate of non-Hodgkin lymphoma, and lymphoid malignancies, is seen in individuals with Sjögren syndrome, compared to normal individuals (Garcia-Lozano et al., 1995).
Entity name
Systemic lupus erythematosus (SLE)
Disease
One out of 55 individuals with autoimmune disorders was found to test positive for anti-RPA2 antibodies (1.8%). This individual had SLE, and secondary Sjögren syndrome (Garcia-Lozano et al., 1995).
Entity name
Rheumatoid arthritis (RA)
Note
Fibroblast-like synoviocytes (FLSs) are a cell type whose invasive properties provide an indicator of RA severity. Microarray studies from FLSs in DA rats (arthritis-susceptible inbred model) show a modest increase in the level of RPA2 mRNA, compared to back-crossed arthritis-resistant DA.F344 (Cia5d) congenic strains (Laragione et al., 2008).

Bibliography

Pubmed IDLast YearTitleAuthors
187236752008RPA phosphorylation facilitates mitotic exit in response to mitotic DNA damage.Anantha RW et al
197938612009The annealing helicase SMARCAL1 maintains genome integrity at stalled replication forks.Bansbach CE et al
114792962001Functional analysis of the four DNA binding domains of replication protein A. The role of RPA2 in ssDNA binding.Bastin-Shanower SA et al
152797882004Replication protein A phosphorylation and the cellular response to DNA damage.Binz SK et al
104494151999The crystal structure of the complex of replication protein A subunits RPA32 and RPA14 reveals a mechanism for single-stranded DNA binding.Bochkarev A et al
119275692002Structure of the RPA trimerization core and its role in the multistep DNA-binding mechanism of RPA.Bochkareva E et al
197938622009The SIOD disorder protein SMARCAL1 is an RPA-interacting protein involved in replication fork restart.Ciccia A et al
197040012009Protein phosphatase 2A-dependent dephosphorylation of replication protein A is required for the repair of DNA breaks induced by replication stress.Feng J et al
77614581995Presence of antibodies to different subunits of replication protein A in autoimmune sera.Garcia-Lozano R et al
173612042007Replication protein A is an independent prognostic indicator with potential therapeutic implications in colon cancer.Givalos N et al
199426842010A naturally occurring human RPA subunit homolog does not support DNA replication or cell-cycle progression.Haring SJ et al
77003861995RPA involvement in the damage-recognition and incision steps of nucleotide excision repair.He Z et al
113138812001Expression analysis using DNA microarrays demonstrates that E2F-1 up-regulates expression of DNA replication genes including replication protein A2.Kalma Y et al
13201951992Binding properties of replication protein A from human and yeast cells.Kim C et al
187060932008Cia5d regulates a new fibroblast-like synoviocyte invasion-associated gene expression signature.Laragione T et al
201547052010A PP4 phosphatase complex dephosphorylates RPA2 to facilitate DNA repair via homologous recombination.Lee DH et al
110816312000Structural basis for the recognition of DNA repair proteins UNG2, XPA, and RAD52 by replication factor RPA.Mer G et al
198349052009Identification of molecular targets for selective elimination of TRAIL-resistant leukemia cells. From spots to in vitro assays using TOP15 charts.Petrak J et al
88043161996A hierarchy of SSB protomers in replication protein A.Philipova D et al
118959052002Autoimmunity to the M(r) 32,000 subunit of replication protein A in breast cancer.Tomkiel JE et al
172967252007The human Tim/Tipin complex coordinates an Intra-S checkpoint response to UV that slows replication fork displacement.Unsal-Kaçmaz K et al
198435842009Human RPA phosphorylation by ATR stimulates DNA synthesis and prevents ssDNA accumulation during DNA-replication stress.Vassin VM et al
197938642009The annealing helicase HARP protects stalled replication forks.Yuan J et al

Other Information

Locus ID:

NCBI: 6118
MIM: 179836
HGNC: 10290
Ensembl: ENSG00000117748

Variants:

dbSNP: 6118
ClinVar: 6118
TCGA: ENSG00000117748
COSMIC: RPA2

RNA/Proteins

Gene IDTranscript IDUniprot
ENSG00000117748ENST00000313433P15927
ENSG00000117748ENST00000373909P15927
ENSG00000117748ENST00000373912P15927
ENSG00000117748ENST00000419958Q5TEJ0
ENSG00000117748ENST00000444045Q5TEJ7

Expression (GTEx)

0
50
100
150

Pathways

PathwaySourceExternal ID
DNA replicationKEGGko03030
Nucleotide excision repairKEGGko03420
Mismatch repairKEGGko03430
Homologous recombinationKEGGko03440
DNA replicationKEGGhsa03030
Nucleotide excision repairKEGGhsa03420
Mismatch repairKEGGhsa03430
Homologous recombinationKEGGhsa03440
Fanconi anemia pathwayKEGGko03460
Fanconi anemia pathwayKEGGhsa03460
RPA complexKEGGhsa_M00288
RPA complexKEGGM00288
Gene ExpressionREACTOMER-HSA-74160
Generic Transcription PathwayREACTOMER-HSA-212436
Transcriptional Regulation by TP53REACTOMER-HSA-3700989
Cell CycleREACTOMER-HSA-1640170
Cell Cycle CheckpointsREACTOMER-HSA-69620
G2/M CheckpointsREACTOMER-HSA-69481
G2/M DNA damage checkpointREACTOMER-HSA-69473
Activation of ATR in response to replication stressREACTOMER-HSA-176187
Cell Cycle, MitoticREACTOMER-HSA-69278
Mitotic G1-G1/S phasesREACTOMER-HSA-453279
G1/S TransitionREACTOMER-HSA-69206
Activation of the pre-replicative complexREACTOMER-HSA-68962
S PhaseREACTOMER-HSA-69242
Synthesis of DNAREACTOMER-HSA-69239
DNA strand elongationREACTOMER-HSA-69190
Lagging Strand SynthesisREACTOMER-HSA-69186
Processive synthesis on the lagging strandREACTOMER-HSA-69183
Removal of the Flap IntermediateREACTOMER-HSA-69166
M/G1 TransitionREACTOMER-HSA-68874
DNA Replication Pre-InitiationREACTOMER-HSA-69002
Chromosome MaintenanceREACTOMER-HSA-73886
Telomere MaintenanceREACTOMER-HSA-157579
Extension of TelomeresREACTOMER-HSA-180786
Telomere C-strand (Lagging Strand) SynthesisREACTOMER-HSA-174417
Processive synthesis on the C-strand of the telomereREACTOMER-HSA-174414
Removal of the Flap Intermediate from the C-strandREACTOMER-HSA-174437
MeiosisREACTOMER-HSA-1500620
Meiotic recombinationREACTOMER-HSA-912446
DNA ReplicationREACTOMER-HSA-69306
DNA RepairREACTOMER-HSA-73894
Base Excision RepairREACTOMER-HSA-73884
Resolution of Abasic Sites (AP sites)REACTOMER-HSA-73933
Resolution of AP sites via the multiple-nucleotide patch replacement pathwayREACTOMER-HSA-110373
PCNA-Dependent Long Patch Base Excision RepairREACTOMER-HSA-5651801
DNA Damage BypassREACTOMER-HSA-73893
Recognition of DNA damage by PCNA-containing replication complexREACTOMER-HSA-110314
Translesion synthesis by Y family DNA polymerases bypasses lesions on DNA templateREACTOMER-HSA-110313
Translesion synthesis by REV1REACTOMER-HSA-110312
Translesion Synthesis by POLHREACTOMER-HSA-110320
Translesion synthesis by POLKREACTOMER-HSA-5655862
Translesion synthesis by POLIREACTOMER-HSA-5656121
Termination of translesion DNA synthesisREACTOMER-HSA-5656169
Mismatch RepairREACTOMER-HSA-5358508
Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha)REACTOMER-HSA-5358565
Mismatch repair (MMR) directed by MSH2:MSH3 (MutSbeta)REACTOMER-HSA-5358606
Cellular responses to stressREACTOMER-HSA-2262752
Cellular response to heat stressREACTOMER-HSA-3371556
HSF1 activationREACTOMER-HSA-3371511
Regulation of HSF1-mediated heat shock responseREACTOMER-HSA-3371453
DNA Double-Strand Break RepairREACTOMER-HSA-5693532
Homology Directed RepairREACTOMER-HSA-5693538
HDR through Homologous Recombination (HR) or Single Strand Annealing (SSA)REACTOMER-HSA-5693567
Processing of DNA double-strand break endsREACTOMER-HSA-5693607
HDR through Homologous Recombination (HRR)REACTOMER-HSA-5685942
Homologous DNA Pairing and Strand ExchangeREACTOMER-HSA-5693579
Presynaptic phase of homologous DNA pairing and strand exchangeREACTOMER-HSA-5693616
HDR through Single Strand Annealing (SSA)REACTOMER-HSA-5685938
Nucleotide Excision RepairREACTOMER-HSA-5696398
Global Genome Nucleotide Excision Repair (GG-NER)REACTOMER-HSA-5696399
Formation of Incision Complex in GG-NERREACTOMER-HSA-5696395
Dual Incision in GG-NERREACTOMER-HSA-5696400
Gap-filling DNA repair synthesis and ligation in GG-NERREACTOMER-HSA-5696397
Transcription-Coupled Nucleotide Excision Repair (TC-NER)REACTOMER-HSA-6781827
Dual incision in TC-NERREACTOMER-HSA-6782135
Gap-filling DNA repair synthesis and ligation in TC-NERREACTOMER-HSA-6782210
Fanconi Anemia PathwayREACTOMER-HSA-6783310
Regulation of TP53 ActivityREACTOMER-HSA-5633007
Regulation of TP53 Activity through PhosphorylationREACTOMER-HSA-6804756

Protein levels (Protein atlas)

Not detected
Low
Medium
High

References

Pubmed IDYearTitleCitations
179282962007Sequential and synergistic modification of human RPA stimulates chromosomal DNA repair.85
170352312006RPA2 is a direct downstream target for ATR to regulate the S-phase checkpoint.80
201547052010A PP4 phosphatase complex dephosphorylates RPA2 to facilitate DNA repair via homologous recombination.55
193867202009Homologous recombinational repair factors are recruited and loaded onto the viral DNA genome in Epstein-Barr virus replication compartments.47
157935852005Insights into hRPA32 C-terminal domain--mediated assembly of the simian virus 40 replisome.46
202019262010Human variation in alcohol response is influenced by variation in neuronal signaling genes.45
117314422001Replication protein A2 phosphorylation after DNA damage by the coordinated action of ataxia telangiectasia-mutated and DNA-dependent protein kinase.44
201300192010The role of RPA2 phosphorylation in homologous recombination in response to replication arrest.44
251130312014Phosphorylated RPA recruits PALB2 to stalled DNA replication forks to facilitate fork recovery.40
285756582017RFWD3-Mediated Ubiquitination Promotes Timely Removal of Both RPA and RAD51 from DNA Damage Sites to Facilitate Homologous Recombination.36

Citation

Anar KZ Murphy ; James A Borowiec

RPA2 (replication protein A2, 32kDa)

Atlas Genet Cytogenet Oncol Haematol. 2010-04-01

Online version: http://atlasgeneticsoncology.org/gene/42146/rpa2