Nuclear import of RPA in Xenopus egg extracts requires a novel protein XRIPalpha but not importin alpha.

Replication protein A (RPA) is a eukaryotic single-stranded (ss) DNA-binding protein that is essential for general DNA metabolism. RPA consists of three subunits (70, 33 and 14 kDa). We have identified by two-hybrid screening a novel Xenopus protein called XRIPalpha that interacts with the ssDNA-binding domain of the largest subunit of RPA. XRIPalpha homologues are found in human and in Drosophila but not in yeast. XRIPalpha is complexed with RPA in Xenopus egg extracts together with another 90 kDa protein that was identified as importin beta. We have demonstrated that XRIPalpha, but not importin alpha, is required for nuclear import of RPA. Immunodepletion of XRIPalpha from the egg extracts blocks nuclear import of RPA but not that of nucleoplasmin, a classical import substrate. RPA import can be restored by addition of recombinant XRIPalpha. Conversely, depletion of importin alpha blocks import of nucleoplasmin but not that of RPA. GST-XRIPalpha pull-down assay shows that XRIPalpha interacts directly with recombinant importin beta as well as with RPA in vitro. Finally, RPA import can be reconstituted from the recombinant proteins. We propose that XRIPalpha plays the role of importin alpha in the RPA import scheme: XRIPalpha serves as an adaptor to link RPA to importin beta.     

ATR kinase activity regulates the intranuclear translocation of ATR and RPA following ionizing radiation

Upon damage of DNA in eukaryotic cells, several repair and checkpoint proteins undergo a dramatic intranuclear relocalization, translocating to nuclear foci thought to represent sites of DNA damage and repair. Examples of such proteins include the checkpoint kinase ATR (ATM and Rad3-related) as well as replication protein A (RPA), a single-stranded DNA binding protein required in DNA replication and repair. Here, we used a microscopy-based approach to investigate whether the damage-induced translocation of RPA is an active process regulated by ATR. Our data show that in undamaged cells, ATR and RPA are uniformly distributed in the nucleus or localized to promyelocytic leukemia protein (PML) nuclear bodies. In cells treated with ionizing radiation, both ATR and RPA translocate to punctate, abundant nuclear foci where they continue to colocalize. Surprisingly, an ATR mutant that lacks kinase activity fails to relocalize in response to DNA damage. Furthermore, this kinase-inactive mutant blocks the translocation of RPA in a cell cycle-dependent manner. These observations demonstrate that the kinase activity of ATR is essential for the irradiation-induced release of ATR and RPA from PML bodies and translocation of ATR and RPA to potential sites of DNA damage.     

Novel Function of the Fanconi Anemia Group J or RECQ1 Helicase to Disrupt Protein-DNA Complexes in a Replication Protein A-stimulated Manner

Understanding how cellular machinery deals with chromosomal genome complexity is an important question because protein bound to DNA may affect various cellular processes of nucleic acid metabolism. DNA helicases are at the forefront of such processes, yet there is only limited knowledge how they remodel protein-DNA complexes and how these mechanisms are regulated. We have determined that representative human RecQ and Fe-S cluster DNA helicases are potently blocked by a protein-DNA interaction. The Fanconi anemia group J (FANCJ) helicase partners with the single-stranded DNA-binding protein replication protein A (RPA) to displace BamHI-E111A bound to duplex DNA in a specific manner. Protein displacement was dependent on the ATPase-driven function of the helicase and unique properties of RPA. Further biochemical studies demonstrated that the shelterin proteins TRF1 and TRF2, which preferentially bind the telomeric repeat found at chromosome ends, effectively block FANCJ from unwinding the forked duplex telomeric substrate. RPA, but not the Escherichia coli single-stranded DNA-binding protein or shelterin factor Pot1, stimulated FANCJ ejection of TRF1 from the telomeric DNA substrate. FANCJ was also able to displace TRF2 from the telomeric substrate in an RPA-dependent manner. The stimulation of helicase-catalyzed protein displacement is also observed with the DNA helicase RECQ1, suggesting a conserved functional interaction of RPA-interacting helicases. These findings suggest that partnerships between RPA and interacting human DNA helicases may greatly enhance their ability to dislodge proteins bound to duplex DNA, an activity that is likely to be highly relevant to their biological roles in DNA metabolism.     

Functions of the Epstein-Barr virus EBNA1 protein in viral reactivation and lytic infection

EBNA1 is the only nuclear Epstein-Barr virus (EBV) protein expressed in both latent and lytic modes of infection. While EBNA1 is known to play several important roles in latent infection, the reason for its continued expression in lytic infection is unknown. Here we identified two roles for EBNA1 in the reactivation of latent EBV to the lytic cycle in epithelial cells. First, EBNA1 depletion in latently infected cells was shown to positively contribute to spontaneous EBV reactivation, showing that EBNA1 has a role in suppressing reactivation. Second, when the lytic cycle was induced, EBNA1 depletion decreased lytic gene expression and DNA amplification, showing that it positively contributed to lytic infection. Since we have previously shown that EBNA1 disrupts promyelocytic leukemia (PML) nuclear bodies, we investigated whether this function could account for the effects of EBNA1 on lytic infection by repeating the experiments with cells lacking PML proteins. In the absence of PML, EBNA1 did not promote lytic infection, indicating that the EBNA1-mediated PML disruption is responsible for promoting lytic infection. In keeping with this conclusion, PML silencing was found to be sufficient to induce the EBV lytic cycle. Finally, by generating cells with single PML isoforms, we showed that individual PML isoforms were sufficient to suppress EBV lytic reactivation, although PML isoform IV (PML IV) was ineffective because it was most efficiently degraded by EBNA1. Our results provide the first function for EBNA1 in lytic infection and show that EBNA1 interactions with PML IV lead to a loss of PML nuclear bodies (NBs) that promotes lytic infection.     

Contribution of the C-terminal Regions of Promyelocytic Leukemia Protein (PML) Isoforms II and V to PML Nuclear Body Formation

Promyelocytic leukemia protein (PML) nuclear bodies are dynamic and heterogeneous nuclear protein complexes implicated in various important functions, most notably tumor suppression. PML is the structural component of PML nuclear bodies and has several nuclear splice isoforms that share a common N-terminal region but differ in their C termini. Previous studies have suggested that the coiled-coil motif within the N-terminal region is sufficient for PML nuclear body formation by mediating homo/multi-dimerization of PML molecules. However, it has not been investigated whether any of the C-terminal variants of PML may contribute to PML body assembly. Here we report that the unique C-terminal domains of PML-II and PML-V can target to PML-NBs independent of their N-terminal region. Strikingly, both domains can form nuclear bodies in the absence of endogenous PML. The C-terminal domain of PML-II interacts transiently with unknown binding sites at PML nuclear bodies, whereas the C-terminal domain of PML-V exhibits hyperstable binding to PML bodies via homo-dimerization. This strong interaction is mediated by a putative α-helix in the C terminus of PML-V. Moreover, nuclear bodies assembled from the C-terminal domain of PML-V also recruit additional PML body components, including Daxx and Sp100. These observations establish the C-terminal domain of PML-V as an additional important contributor to the assembly mechanism(s) of PML bodies.     

Replication protein A promotes 5'-->3' end processing during homology-dependent DNA double-strand break repair

Replication protein A (RPA), the eukaryotic single-strand deoxyribonucleic acid (DNA [ss-DNA])-binding protein, is involved in DNA replication, nucleotide damage repair, mismatch repair, and DNA damage checkpoint response, but its function in DNA double-strand break (DSB) repair is poorly understood. We investigated the function of RPA in homology-dependent DSB repair using Xenopus laevis nucleoplasmic extracts as a model system. We found that RPA is required for single-strand annealing, one of the homology-dependent DSB repair pathways. Furthermore, RPA promotes the generation of 3' single-strand tails (ss-tails) by stimulating both the Xenopus Werner syndrome protein (xWRN)-mediated unwinding of DNA ends and the subsequent Xenopus DNA2 (xDNA2)-mediated degradation of the 5' ss-tail. Purified xWRN, xDNA2, and RPA are sufficient to carry out the 5'-strand resection of DNA that carries a 3' ss-tail. These results provide strong biochemical evidence to link RPA to a specific DSB repair pathway and reveal a novel function of RPA in the generation of 3' ss-DNA for homology-dependent DSB repair.     

Interaction of the adenovirus type 5 E4 Orf3 protein with promyelocytic leukemia protein isoform II is required for ND10 disruption

Nuclear domain 10 (ND10s), or promyelocytic leukemia protein (PML) nuclear bodies, are spherical nuclear structures that require PML proteins for their formation. Many viruses target these structures during infection. The E4 Orf3 protein of adenovirus 5 (Ad5) rearranges ND10s, causing PML to colocalize with Orf3 in nuclear tracks or fibers. There are six different PML isoforms (I to VI) present at ND10s, all sharing a common N terminus but with structural differences at their C termini. In this study, PML II was the only one of these six isoforms that was found to interact directly and specifically with Ad5 E4 Orf3 in vitro and in vivo; these results define a new Orf3 activity. Three of a series of 18 mutant Orf3 proteins were unable to interact with PML II; these were also unable to cause ND10 rearrangement. Moreover, in PML-null cells that contained neoformed ND10s comprising a single PML isoform, only ND10s formed of PML II were rearranged by Orf3. These data show that the interaction between Orf3 and PML II is necessary for ND10 rearrangement to occur. Finally, Orf3 was shown to self-associate in vitro. This activity was absent in mutant Orf3 proteins that were unable to form tracks and to bind PML II. Thus, Orf3 oligomerization may mediate the formation of nuclear tracks in vivo and may also be important for PML II binding.     

Identification of nuclear pre-replication centers poised for DNA synthesis in Xenopus egg extracts: immunolocalization study of replication protein A

We demonstrate by immunofluorescence that a 70-kD protein (P70) purified from Xenopus egg extracts is associated with subnuclear foci (about 200) which we propose to be an assembly of DNA pre-replication centers (preRCs). A cDNA encoding this protein reveals that P70 is the Xenopus homologue of replication protein A (RPA also called RF-A). RPA is know to be a cellular, three-subunit single-stranded DNA binding protein, which assists T-antigen in the assembly of the pre-priming complex in the SV40 replication system. The punctated preRCs exist transiently; they form post-mitotically during the period of nuclear membrane breakdown and disappear during ongoing DNA replication. P70 is homogeneously associated with chromatin at the later stages of the S- phase and is displaced from chromatin post replication, so that P70 cannot be detected on mitotic chromosomes. Double-immunofluorescence studies using biotin-dUTP demonstrate that initiation of DNA synthesis is confined to preRCs, resulting in the punctated replication pattern observed previously by others. PreRCs form efficiently on decondensed chromatin in membrane-free egg extracts if ATP and divalent cations are present. Our results suggest that preRCs are composed of an assembly of a large number of pre-initiation replication complexes poised for initiation at discreet subnuclear regions prior to nuclear reconstruction and initiation of DNA synthesis.     

Study of the cell cycle-dependent assembly of the DNA pre-replication centres in Xenopus egg extracts.

RPA is a cellular, three-subunit, single-stranded (ss) DNA binding protein, which assists T-antigen in the assembly of the pre-priming complex in the SV40 replication system. By immunodepletion and complementation, we have identified RPA as an essential factor for cellular DNA replication in Xenopus extracts. RPA assembles post-mitotically on the decondensing chromosomes into numerous subnuclear pre-replication centres (preRCs) which serve, upon formation of the nuclear membrane, as RCs for the initiation of DNA synthesis. By a variety of experiments including the use of isolated components, we demonstrate that an inactive cdc2-cyclin B kinase complex is essential to allow post-mitotic assembly of the preRCs. In contrast, the active cdk2-cyclin A kinase does not impede or facilitate the assembly of preRCs. Digestion analysis using the single-strand-specific P1 nuclease as well as competition experiments with ssDNA, reveal that replication-associated unwinding of the DNA, assisted by RPA, requires the formation of the nuclear membrane. The p21 cdk-interacting protein Cip1 appears to inhibit DNA replication prior to the unwinding DNA step, but after assembly of preRC and nuclear reconstruction.     

Targeting of adenovirus E1A and E4-ORF3 proteins to nuclear matrix- associated PML bodies

The PML protein was first identified as part of a fusion product with the retinoic acid receptor alpha (RAR alpha), resulting from the t(15;17) chromosomal translocation associated with acute promyelocytic leukemia (APL). It has been previously demonstrated that PML, which is tightly bound to the nuclear matrix, concentrates in discrete subnuclear compartments that are disorganized in APL cells due to the expression of the PML-RAR alpha hybrid. Here we report that adenovirus infection causes a drastic redistribution of PML from spherical nuclear bodies into fibrous structures. The product encoded by adenovirus E4- ORF3 is shown to be responsible for this reorganization and to colocalize with PML into these fibers. In addition, we demonstrate that E1A oncoproteins concentrate in the PML domains, both in infected and transiently transfected cells, and that this association requires the conserved amino acid motif (D)LXCXE, common to all viral oncoproteins that bind pRB or the related p107 and p130 proteins. The SV-40 large T antigen, another member of this oncoprotein family is also found in close association with the PML nuclear bodies. Taken together, the present data indicate that the subnuclear domains containing PML represent a preferential target for DNA tumor viruses, and therefore suggest a more general involvement of the PML nuclear bodies in oncogenic processes.     

Functional studies on the interaction between human replication protein A and Xeroderma pigmentosum group A complementing protein (XPA).

The human replication protein A (RPA; also known as human single-stranded DNA binding protein, HSSB) is a multisubunit complex (70, 34 and 11 kDa subunits) involved in the three processes of DNA metabolism; replication, repair, recombination. We found that both 34 and 70 kDa subunits (p34 and p70, respectively), of RPA interacts with the Xeroderma pigmentosum group A complementing protein (XPA), a protein that specifically recognizes UV-damaged DNA. Our mutational analysis indicated that no particular domains of RPA p70 were essential for its interaction with XPA. We also examined the effect of this XPA-RPA interaction on in vitro simian virus 40 (SV40) DNA replication catalyzed by the crude extract and monopolymerase system. XPA inhibited SV40 DNA replication in vitro through its interaction with RPA. Taken together, these results suggest that there is a role for RPA in the regulation of DNA metabolism through its ability to modulate the interactions of proteins involved in the processes of DNA metabolism.     

Cell cycle specific plasmid DNA replication in the nuclear extract of Saccharomyces cerevisiae: modulation by replication protein A and proliferating cell nuclear antigen

Plasmid DNA replication in nuclear extracts of Saccharomyces cerevisiae in vitro has been shown to be S-phase specific, similar to that observed in vivo. We report here a reconstituted in vitro system with partially purified replication proteins, purified replication protein A (RPA), and recombinant proliferating cell nuclear antigen (PCNA). Nuclear extracts from S-phase, G(1)-phase, and unsynchronized yeast cells were fractionated by phosphocellulose chromatography. Protein fraction (polymerase fraction) enriched with replication proteins, including DNA polymerases (alpha, delta, etc.), was isolated, which was not capable of in vitro replication of supercoiled plasmid DNA. However, when purified yeast RPA and recombinant PCNA together were added to the polymerase fraction obtained from S-phase synchronized cells, in vitro plasmid DNA replication was restored. In vitro plasmid DNA replication with polymerase fractions from unsynchronized and G(1)-phase cells could not be reconstituted upon addition of purified RPA and PCNA. RPA and PCNA isolated from various phases of the cell cycle complemented the S-phase polymerase pool to the same extent. Reconstituted systems with the S-phase polymerase pool, complemented with either the RPA- and PCNA-containing fraction or purified RPA and recombinant PCNA together, were able to produce replication intermediates (ranging in size from 50 to 1500 bp) similar to that observed with the S-phase nuclear extract. Results presented here demonstrate that both RPA and PCNA are cell cycle-independent in their ability to stimulate in vitro plasmid DNA replication, whereas replication factors in the polymerase fractions are strictly S-phase dependent.     

Herpesvirus protein ICP27 switches PML isoform by altering mRNA splicing

Viruses use alternative splicing to produce a broad series of proteins from small genomes by utilizing the cellular splicing machinery. Since viruses use cellular RNA binding proteins for viral RNA processing, it is presumable that the splicing of cellular pre-mRNAs is affected by viral infection. Here, we showed that herpes simplex virus type 2 (HSV-2) modifies the expression of promyelocytic leukemia (PML) isoforms by altering pre-mRNA splicing. Using a newly developed virus-sensitive splicing reporter, we identified the viral protein ICP27 as an alternative splicing regulator of PML isoforms. ICP27 was found to bind preferentially to PML pre-mRNA and directly inhibit the removal of PML intron 7a in vitro. Moreover, we demonstrated that ICP27 functions as a splicing silencer at the 3′ splice site of the PML intron 7a. The switching of PML isoform from PML-II to PML-V as induced by ICP27 affected HSV-2 replication, suggesting that the viral protein modulates the splicing code of cellular pre-mRNA(s) governing virus propagation.     

Replication protein A as a major eukaryotic single-stranded DNA-binding protein and its role in DNA repair

Replication protein A (RPA) is a key regulator of eukaryotic DNA metabolism. RPA is a highly conserved heterotrimeric protein and contains multiple oligonucleotide/oligosaccharide-binding folds. The major RPA function is binding to single-stranded DNA (ssDNA) intermediates forming in DNA replication, repair, and recombination. Although binding ssDNA with high affinity, RPA can rapidly diffuse along ssDNA and destabilizes the DNA secondary structure. A highly dynamic RPA binding to ssDNA allows other proteins to access ssDNA and to displace RPA from the RPA–ssDNA complex. As has been shown recently, RPA in complex with ssDNA is posttranslationally modified in response to DNA damage. These modifications modulate the RPA interactions with its protein partners and control the DNA damage signaling pathways. The review considers up-to-date data on the RPA function as an active coordinator of ssDNA intermediate processing within DNA metabolic pathways, DNA repair in particular.