RPA complexes inCaenorhabditis elegansmeiosis; unique roles in replication, meiotic recombination and apoptosis
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Abstract Replication Protein A (RPA) is critical complex that acts in replication and promotes homologous recombination by allowing recombinase recruitment to processed DSB ends. Most organisms possess three RPA subunits (RPA1, RPA2, RPA3) that form a trimeric complex critical for viability. The Caenorhabditis elegans genome encodes for RPA-1, RPA-2 and an RPA-2 paralog RPA-4. In our analysis, we determine that RPA-2 is critical for germline replication, and normal repair of meiotic DSBs. Interestingly, RPA-1 but not RPA-2 is essential for replication, contradictory to what is seen in other organisms, that require both subunits. In the germline, both RPA-1/2 and RPA-1/4 complexes form, but RPA-1/4 is less abundant and its formation is repressed by RPA-2. While RPA-4 does not participate in replication or recombination, we find that RPA-4 inhibit RAD-51 filament formation and promotes apoptosis on a subset of damaged nuclei. Altogether these findings point to sub-functionalization and antagonistic roles of RPA complexes in C. elegans .Keywords:
Replication protein A
Replication
Spermatogonial stem cells (SSCs) are the only stem cells in the body with germline potential, which makes them an attractive target for germline modification. We previously showed the feasibility of homologous recombination in mouse SSCs and produced knockout (KO) mice by exploiting germline stem (GS) cells, i.e., cultured spermatogonia with SSC activity. In this study, we report the successful homologous recombination in rat GS cells, which can be readily established by their ability to form germ cell colonies on culture plates whose surfaces are hydrophilic and neutrally charged and thus limit somatic cell binding. We established a drug selection protocol for GS cells under hypoxic conditions. The frequency of the homologous recombination of the Ocln gene was 4.2% (2 out of 48 clones). However, these GS cell lines failed to produce offspring following xenogeneic transplantation into mouse testes and microinsemination, suggesting that long-term culture and drug selection have a negative effect on GS cells. Nevertheless, our results demonstrate the feasibility of gene targeting in rat GS cells and pave the way toward the generation of KO rats.
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Replication protein A
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Abstract In Caenorhabditis elegans, germ granules called P granules are directly inherited from mother to daughter and segregate with the germ lineage as it separates from the soma during initial embryonic cell divisions. Here we define meg-1 and meg-2 (maternal-effect germ-cell defective), which are expressed in the maternal germline and encode proteins that localize exclusively to P granules during embryonic germline segregation. Localization of MEG-1 to P granules depends upon the membrane-bound protein MES-1. meg-1 mutants exhibit multiple germline defects: P-granule mis-segregation in embryos, underproliferation and aberrant P-granule morphology in larval germ cells, and ultimately, sterility as adults. The penetrance of meg-1 phenotypes increases when meg-2 is also absent. Loss of the P-granule component pgl-1 in meg-1 mutants increases germ-cell proliferation, while loss of glh-1 decreases proliferation. Because meg-1 is provided maternally but its action is required in the embryonic germ lineage during segregation from somatic lineages, it provides a critical link for ensuring the continuity of germline development from one generation to the next.
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The maternal-effect sterile (MES) proteins are maternally supplied regulators of germline development in Caenorhabditis elegans. In the hermaphrodite progeny from mes mutant mothers, the germline dies during larval development. On the basis of the similarities of MES-2 and MES-6 to known transcriptional regulators and on the basis of the effects of mes mutations on transgene expression in the germline, the MES proteins are predicted to be transcriptional repressors. One of the MES proteins, MES-3, is a novel protein with no recognizable motifs. In this article we show that MES-3 is localized in the nuclei of embryos and germ cells, consistent with its predicted role in transcriptional regulation. Its distribution in the germline and in early embryos does not depend on the wild-type functions of the other MES proteins. However, its nuclear localization in midstage embryos and its persistence in the primordial germ cells depend on wild-type MES-2 and MES-6. These results are consistent with biochemical data showing that MES-2, MES-3, and MES-6 associate in a complex in embryos. The distribution of MES-3 in the adult germline is regulated by the translational repressor GLD-1: MES-3 is absent from the region of the germline where GLD-1 is known to be present, MES-3 is overexpressed in the germline of gld-1 mutants, and GLD-1 specifically binds the mes-3 3' untranslated region (3' UTR). Analysis of temperature-shifted mes-3(bn21ts) worms and embryos indicates that MES-3 function is required in the mother's germline and during embryogenesis to ensure subsequent normal germline development. We propose that MES-3 acts epigenetically to induce a germline state that is inherited through both meiosis and mitosis and that is essential for survival of the germline.
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Glycosylphosphatidylinositol (GPI)-anchor attachment is one of the most common posttranslational protein modifications. Using the nematode Caenorhabditis elegans, we determined that GPI-anchored proteins are present in germline cells and distal tip cells, which are essential for the maintenance of the germline stem cell niche. We identified 24 C. elegans genes involved in GPI-anchor synthesis. Inhibition of various steps of GPI-anchor synthesis by RNA interference or gene knockout resulted in abnormal development of oocytes and early embryos, and both lethal and sterile phenotypes were observed. The piga-1 gene (orthologue of human PIGA) codes for the catalytic subunit of the phosphatidylinositol N-acetylglucosaminyltransferase complex, which catalyzes the first step of GPI-anchor synthesis. We isolated piga-1–knockout worms and found that GPI-anchor synthesis is indispensable for the maintenance of mitotic germline cell number. The knockout worms displayed 100% lethality, with decreased mitotic germline cells and abnormal eggshell formation. Using cell-specific rescue of the null allele, we showed that expression of piga-1 in somatic gonads and/or in germline is sufficient for normal embryonic development and the maintenance of the germline mitotic cells. These results clearly demonstrate that GPI-anchor synthesis is indispensable for germline formation and for normal development of oocytes and eggs.
Caenorhabditis
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Branch migration
Replication protein A
FLP-FRT recombination
In vitro recombination
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The single-stranded DNA-binding protein, Replication Protein A (RPA), is a heterotrimeric complex with subunits of 70, 32 and 14 kDa involved in DNA metabolism. RPA may be a target for cellular regulation; the 32 kDa subunit (RPA32) is phosphorylated by several cellular kinases including the DNA-dependent protein kinase (DNA-PK). We have purified a mutant hRPA complex lacking amino acids 1–33 of RPA32 (rhRPA•32Δ1–33). This mutant bound ssDNA and supported DNA replication; however, (rhRPA•32Δ1–33) was not phosphorylated under replication conditions or directly by DNA-PK. Proteolytic mapping revealed that all the sites phosphorylated by DNA-PK are contained on residues 1–33 of RPA32. When wild-type RPA was treated with DNA-PK and the mixture added to SV40 replication assays, DNA replication was supported. In contrast, when rhRPA•32Δ1–33 was treated with DNA-PK, DNA replication was strongly inhibited. Because untreated (rhRPA•32Δ1–33) is fully functional, this suggests that the N-terminus of RPA is needed to overcome inhibitory effects of DNA-PK on other components of the DNA replication system. Thus, phosphorylation of RPA may modulate DNA replication indirectly, through interactions with other proteins whose activity is modulated by phosphorylation.
Replication protein A
Replication factor C
Origin recognition complex
Ter protein
Pre-replication complex
DNA clamp
SeqA protein domain
DNA polymerase delta
DNA polymerase II
HMG-box
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DNA double-strand break (DSB) repair by homologous recombination (HR) is crucial for the maintenance of genome stability and integrity. In this study, we aim to identify novel RNA binding proteins (RBPs) involved in HR repair because little is known about RBP function in HR. For this purpose, we carry out pulldown assays using a synthetic ssDNA/dsDNA structure coated with replication protein A (RPA) to mimic resected DNA, a crucial intermediate in HR-mediated DSB repair. Using this approach, we identify RNA-binding motif protein 14 (RBM14) as a potential binding partner. We further show that RBM14 interacts with an essential HR repair factor, CtIP. RBM14 is crucial for CtIP recruitment to DSB sites and for subsequent RPA coating and RAD51 replacement, facilitating efficient HR repair. Moreover, inhibition of RBM14 expression sensitizes cancer cells to X-ray irradiation. Together, our results demonstrate that RBM14 promotes DNA end resection to ensure HR repair and may serve as a potential target for cancer therapy.
Replication protein A
Non-homologous end joining
Homology directed repair
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Replication protein A (RPA), the eukaryotic single-stranded DNA-binding complex, is essential for multiple processes in cellular DNA metabolism. The "canonical" RPA is composed of three subunits (RPA1, RPA2, and RPA3); however, there is a human homolog to the RPA2 subunit, called RPA4, that can substitute for RPA2 in complex formation. We demonstrate that the resulting "alternative" RPA (aRPA) complex has solution and DNA binding properties indistinguishable from the canonical RPA complex; however, aRPA is unable to support DNA replication and inhibits canonical RPA function. Two regions of RPA4, the putative L34 loop and the C terminus, are responsible for inhibiting SV40 DNA replication. Given that aRPA inhibits canonical RPA function in vitro and is found in nonproliferative tissues, these studies indicate that RPA4 expression may prevent cellular proliferation via replication inhibition while playing a role in maintaining the viability of quiescent cells. Replication protein A (RPA), the eukaryotic single-stranded DNA-binding complex, is essential for multiple processes in cellular DNA metabolism. The "canonical" RPA is composed of three subunits (RPA1, RPA2, and RPA3); however, there is a human homolog to the RPA2 subunit, called RPA4, that can substitute for RPA2 in complex formation. We demonstrate that the resulting "alternative" RPA (aRPA) complex has solution and DNA binding properties indistinguishable from the canonical RPA complex; however, aRPA is unable to support DNA replication and inhibits canonical RPA function. Two regions of RPA4, the putative L34 loop and the C terminus, are responsible for inhibiting SV40 DNA replication. Given that aRPA inhibits canonical RPA function in vitro and is found in nonproliferative tissues, these studies indicate that RPA4 expression may prevent cellular proliferation via replication inhibition while playing a role in maintaining the viability of quiescent cells. Replication protein A (RPA) 3The abbreviations used are: RPA, human replication protein A; aRPA, alternative RPA; RPA1, 70-kDa subunit of RPA; RPA2, 32-kDa subunit of RPA; RPA3, 14-kDa subunit of RPA; RPA4, product of the RPA4 gene; ssDNA, single strand DNA; DBD, DNA-binding domain; AS-Ex, ammonium sulfate fractionated extract; hSSB 1, human single-stranded binding protein 1.3The abbreviations used are: RPA, human replication protein A; aRPA, alternative RPA; RPA1, 70-kDa subunit of RPA; RPA2, 32-kDa subunit of RPA; RPA3, 14-kDa subunit of RPA; RPA4, product of the RPA4 gene; ssDNA, single strand DNA; DBD, DNA-binding domain; AS-Ex, ammonium sulfate fractionated extract; hSSB 1, human single-stranded binding protein 1. is a stable complex composed of three subunits (RPA1, RPA2, and RPA3) that binds single-stranded DNA (ssDNA) nonspecifically. RPA (also referred to as canonical RPA) is essential for cell viability (1Wold M.S. Annu. Rev. Biochem. 1997; 66: 61-92Crossref PubMed Scopus (1170) Google Scholar), and viable missense mutations in RPA subunits can lead to defects in DNA repair pathways or show increased chromosome instability. For example, a missense change in a high affinity DNA-binding domain (DBD) was demonstrated to cause a high rate of chromosome rearrangement and lymphoid tumor development in heterozygous mice (2Wang Y. Putnam C.D. Kane M.F. Zhang W. Edelmann L. Russell R. Carrion D.V. Chin L. Kucherlapati R. Kolodner R.D. Edelmann W. Nat. Genet. 2005; 37: 750-755Crossref PubMed Scopus (129) Google Scholar). RPA has also been shown to have increased expression in colon and breast cancers (3Givalos N. Gakiopoulou H. Skliri M. Bousboukea K. Konstantinidou A.E. Korkolopoulou P. Lelouda M. Kouraklis G. Patsouris E. Karatzas G. Mod. Pathol. 2007; 20: 159-166Crossref PubMed Scopus (66) Google Scholar, 4Tomkiel J.E. Alansari H. Tang N. Virgin J.B. Yang X. VandeVord P. Karvonen R.L. Granda J.L. Kraut M.J. Ensley J.F. Fernandez-Madrid F. Clin. Cancer Res. 2002; 8: 752-758PubMed Google Scholar). High RPA1 and RPA2 levels in cancer cells are also correlated with poor overall survival (3Givalos N. Gakiopoulou H. Skliri M. Bousboukea K. Konstantinidou A.E. Korkolopoulou P. Lelouda M. Kouraklis G. Patsouris E. Karatzas G. Mod. Pathol. 2007; 20: 159-166Crossref PubMed Scopus (66) Google Scholar, 4Tomkiel J.E. Alansari H. Tang N. Virgin J.B. Yang X. VandeVord P. Karvonen R.L. Granda J.L. Kraut M.J. Ensley J.F. Fernandez-Madrid F. Clin. Cancer Res. 2002; 8: 752-758PubMed Google Scholar), which is consistent with RPA having a role in efficient cell proliferation. RPA is a highly conserved complex as all eukaryotes contain homologs of each of the three RPA subunits (1Wold M.S. Annu. Rev. Biochem. 1997; 66: 61-92Crossref PubMed Scopus (1170) Google Scholar). At least some plants (e.g. rice) and some protists (e.g. Cryptosporidium parvum) contain multiple genes encoding for subunits of RPA (5Ishibashi T. Kimura S. Sakaguchi K. J. Biochem. (Tokyo). 2006; 139: 99-104Crossref PubMed Scopus (40) Google Scholar, 6Rider Jr., S.D. Cai X. Sullivan Jr., W.J. Smith A.T. Radke J. White M. Zhu G. J. Biol. Chem. 2005; 280: 31460-31469Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). In rice, there is evidence for multiple RPA complexes that are thought to perform different cellular functions (5Ishibashi T. Kimura S. Sakaguchi K. J. Biochem. (Tokyo). 2006; 139: 99-104Crossref PubMed Scopus (40) Google Scholar). In contrast, only a single alternative form of RPA2, called RPA4, has been identified in humans (7Keshav K.F. Chen C. Dutta A. Mol. Cell. Biol. 1995; 15: 3119-3128Crossref PubMed Scopus (40) Google Scholar). RPA4 was originally identified as a protein that interacts with RPA1 in a yeast two-hybrid screen (7Keshav K.F. Chen C. Dutta A. Mol. Cell. Biol. 1995; 15: 3119-3128Crossref PubMed Scopus (40) Google Scholar). The RPA4 subunit is 63% identical/similar to RPA2. Comparison of the sequences of RPA4 and RPA2 suggests that the two proteins have a similar domain organization. 4S. J. Haring, T. D. Humphreys, and M. S. Wold, submitted for publication.4S. J. Haring, T. D. Humphreys, and M. S. Wold, submitted for publication. RPA4 appears to contain a putative core DNA-binding domain (DBD G) flanked by a putative N-terminal phosphorylation domain and a C terminus containing a putative winged-helix domain (Fig. 1A). The RPA4 gene is located on the X chromosome, intronless, and found mainly in primates. 4S. J. Haring, T. D. Humphreys, and M. S. Wold, submitted for publication. Initial characterization of RPA4 by Keshav et al. (7Keshav K.F. Chen C. Dutta A. Mol. Cell. Biol. 1995; 15: 3119-3128Crossref PubMed Scopus (40) Google Scholar) indicated that either RPA2 or RPA4, but not both simultaneously, interacts with RPA1 and RPA3 to form a complex. This analysis also showed that RPA4 is expressed in placenta and colon tissue but was either not detected or expressed at low levels in most established cell lines examined (7Keshav K.F. Chen C. Dutta A. Mol. Cell. Biol. 1995; 15: 3119-3128Crossref PubMed Scopus (40) Google Scholar). These studies describe the purification and functional analysis of an alternative RPA (aRPA) complex containing RPA1, RPA3, and RPA4. The aRPA complex is a stable heterotrimeric complex similar in size and stability to the canonical RPA complex (RPA1, RPA3, and RPA2). aRPA interacts with ssDNA in a manner indistinguishable from canonical RPA; however, it does not support DNA replication in vitro. Mixing experiments demonstrate that aRPA also inhibits canonical RPA from functioning in DNA replication. Hybrid protein studies paired with structural modeling have allowed for the identification of two regions of RPA4 responsible for this inhibitory activity. Data presented here are consistent with recent analyses of RPA4 function in human cells, 4S. J. Haring, T. D. Humphreys, and M. S. Wold, submitted for publication. and we conclude that RPA4 has anti-proliferative properties and has the potential to play a regulatory role in human cell proliferation through the control of DNA replication. Materials—HI buffers contain 30 mm HEPES (diluted from 1 m stock at pH 7.8), 1 mm dithiothreitol, 0.25 mm EDTA, 0.5% (w/v) inositol, and 0.01% (v/v) Nonidet-P40. HI was supplemented with different salt concentrations as indicated. Creatine phosphokinase from rabbit skeletal muscle and creatine phosphate disodium salt were purchased from Calbiochem. [γ-32P]ATP (250 μCi) and [α-32P]dCTP (250 μCi) were purchased from PerkinElmer Life Sciences. Construction of aRPA and aRPA Hybrid Expression Plasmids—To purify RPA4 in a complex with RPA1 and RPA3, a PCR fragment containing RPA4 cDNA was amplified using primers 5′-CACCTGACGTCAAAAAACCCCTCAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTATTATCAATCAGCAGACTTAAAATGCTC-3′ and 5′-TTGATGGATCCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGAGTAAGAGTGGGTTTGGGAGC-3′. This fragment was then cloned into the BamHI-AatII sites of pET16b-hSSB (9Zhang D. Frappier L. Gibbs E. Hurwitz J. O'Donnell M. Nucleic Acids Res. 1998; 26: 631-637Crossref PubMed Scopus (46) Google Scholar), replacing the RPA2 cDNA. Subsequently, a BsrGI-ScaI fragment containing the 3′ end of RPA3 and the entire RPA4 coding region was cloned into p11d-tRPA(10Henricksen L.A. Umbricht C.B. Wold M.S. J. Biol. Chem. 1994; 269: 11121-11132Abstract Full Text PDF PubMed Google Scholar) to generate the plasmid p11d-aRPA. Plasmids for expressing RPA4 alone or with RPA3 were generated by inserting the BamHI-AatII fragment into pET-11d or pET16b-RPA32/his14 (a derivative of pET16b-hSSB in which RPA1 has been deleted), respectively. A His10 tag was added to the N terminus of RPA4 and cloned into pET11d using the same method with the primer 5′-TTGATGGATCCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGGGCCATCATCATCATCATCATCATCATCATCACAGCAGCGGCCATATCGAAGGTCGTCATATGAGTAAGAGTGGGTTTGGGAGC-3′. All plasmids were confirmed by restriction digest and DNA sequencing. Hybrid constructs were amplified from their corresponding pEGFP plasmids 4S. J. Haring, T. D. Humphreys, and M. S. Wold, submitted for publication. using either primer 5′-TCTCGAGGTGGATTAATGAGTAAGAGT-3′ or 5′-CTCGAGGTGGATTAATGTGGAACAGT-3′ and 5′-AGATCCGGTGGATCCCGGGCCCGC-3′. The fragment was digested with AseI and KpnI and then cloned into the NdeI and KpnI sites of pRSF. All plasmids were confirmed by restriction analysis and DNA sequencing. Protein Expression and Purification—RPA, aRPA, and aRPA hybrids were expressed in BL21 (DE3) cells and purified as described previously (10Henricksen L.A. Umbricht C.B. Wold M.S. J. Biol. Chem. 1994; 269: 11121-11132Abstract Full Text PDF PubMed Google Scholar, 11Binz S.K. Dickson A.M. Haring S.J. Wold M.S. Methods Enzymol. 2006; 409: 11-38Crossref PubMed Scopus (54) Google Scholar). RPA4/3 complex was purified as described. 5A. M. Dickson, Y. Krasikova, P. Pestryakov, O. Lavrik, and M. S. Wold, submitted for publication. When dual vectors were used, both ampicillin (120 μg/ml) and kanamycin (30 μg/ml) were used for colony selection and growth. DNA Binding Assays—Gel mobility shift assays were carried out as described previously (11Binz S.K. Dickson A.M. Haring S.J. Wold M.S. Methods Enzymol. 2006; 409: 11-38Crossref PubMed Scopus (54) Google Scholar). Briefly, indicated amounts of protein and radiolabeled oligonucleotide were incubated for 20 min at 25 °C in filter binding buffer (30 mm HEPES (diluted from 1 m stock at pH 7.8), 100 mm NaC1, 5 mm MgC12, 0.5% inositol, and 1 mm dithiothreitol). Reaction mixtures were separated on a 1% agarose gel in 0.1× Tris acetate-EDTA running buffer. Bound and free DNA from gel mobility shift experiments were quantitated using a Packard Instant Imager. Apparent affinity constants were calculated by nonlinear least squares fitting of the data to the Langmuir binding equation using KaleidaGraph (Synergy Software) as described previously (13Kim C. Paulus B.F. Wold M.S. Biochemistry. 1994; 33: 14197-14206Crossref PubMed Scopus (202) Google Scholar). SV40 Replication and Elongation Assays—SV40 reactions were carried out in 25 μl. Standard reactions contained 30 mm HEPES (pH 7.5); 7 mm MgCl2;50 μm dCTP with 2.5 μCi (92.5 kBq) of [α-32P]dCTP; 100 μm each of dATP, dGTP, and dTTP; 200 μm each of CTP, GTP, and UTP; 4 mm ATP; 40 mm creatine phosphate; 2.5 μg of creatine kinase; 15 mm potassium phosphate; and 50 ng of pUC.HSO DNA template. RPA, usually 300 ng, was added as indicated. Each reaction also contained 100 μg of HeLa cell cytoplasmic extract and 0.2-0.5 μg of SV40 T-antigen. SV40 T-antigen was purified by immunoaffinity chromatography from Sf9 cells infected with a baculovirus vector containing the T-antigen gene as described previously (14Brush G.S. Kelly T.J. Stillman B. Methods Enzymol. 1995; 262: 522-548Crossref PubMed Scopus (45) Google Scholar). Complementation assays were carried out using a 35-65% ammonium sulfate fraction of HeLa cell extract (11Binz S.K. Dickson A.M. Haring S.J. Wold M.S. Methods Enzymol. 2006; 409: 11-38Crossref PubMed Scopus (54) Google Scholar). Briefly, 1 ml of complete extract was precipitated by the gradual addition of ammonium sulfate to 35%. The supernatant was further precipitated with 65% ammonium sulfate. The resulting precipitant was dissolved in one-fifth of the initial volume of 50 mm Tris-HCl, pH 7.8, 1 mm dithiothreitol, 0.1 mm EDTA, 10% glycerol and dialyzed to remove any residual ammonium sulfate. All reaction mixtures were assembled on ice and incubated at 37 °C for 2 h. The reactions were analyzed on gels as described previously (11Binz S.K. Dickson A.M. Haring S.J. Wold M.S. Methods Enzymol. 2006; 409: 11-38Crossref PubMed Scopus (54) Google Scholar) or quantitated by precipitation by trichloroacetic acid; reactions were quenched by the addition of 0.1 m sodium pyrophosphate to a final concentration 80 mm and precipitated with 500 μl of 10% trichloroacetic acid. The reaction mixtures were filtered through glass microfiber filters and radioactive DNA quantitated by liquid scintillation. SV40 T-antigen dependent elongation assays (15Walther A.P. Bjerke M.P. Wold M.S. Nucleic Acids Res. 1999; 27: 656-664Crossref PubMed Scopus (19) Google Scholar) were done as described in the SV40 replication assay with the following modifications. Reactions were assembled as above except the [α-32P]dCTP was excluded from stage I. After incubation at 37 °C for 2 h, [α-32P]dCTP and RPA or RPA variants were added, and a stage II incubation was carried out for an additional hour at 37 °C. Products were analyzed as described above. RPA4 Forms a Stable, Functional ssDNA-binding Complex—Recombinant RPA4 was produced using methodology previously described to generate recombinant canonical RPA (10Henricksen L.A. Umbricht C.B. Wold M.S. J. Biol. Chem. 1994; 269: 11121-11132Abstract Full Text PDF PubMed Google Scholar). The cDNA encoding RPA4 was cloned into a bacterial expression vector either alone, with RPA3, or with RPA1 and RPA3 and expressed in Escherichia coli. Overall, RPA4 had properties that were similar to those of recombinant RPA2 (10Henricksen L.A. Umbricht C.B. Wold M.S. J. Biol. Chem. 1994; 269: 11121-11132Abstract Full Text PDF PubMed Google Scholar). A His-tagged RPA4 gene expressed alone was predominantly insoluble (data not shown). When RPA4 was expressed with RPA3, both proteins were predominantly soluble and could be purified as a stable RPA4/3 complex (Fig. 1B and data not shown). When all three genes (RPA1, RPA4, RPA3) were expressed simultaneously, all three polypeptides were substantially soluble, and a complex, aRPA, could be purified to near homogeneity following the purification procedure used for canonical RPA (11Binz S.K. Dickson A.M. Haring S.J. Wold M.S. Methods Enzymol. 2006; 409: 11-38Crossref PubMed Scopus (54) Google Scholar). The expression of RPA4 in E. coli and the yield of aRPA complex after purification were similar to that for RPA (∼0.8 mg/liter of culture). The purified aRPA contained three intense bands of 70, 34, and 14 kDa (Fig. 1B). Although RPA4 has nine fewer amino acids than RPA2 and a predicted pI (6.07) slightly more basic than RPA2 (5.75), the RPA4 subunit consistently migrated slower in SDS-PAGE gels. We examined the hydrodynamic properties of aRPA and found them to be nearly indistinguishable from those of the canonical RPA complex; the sedimentation and Stokes' radius of aRPA were determined to be 5.0 s and 52.0 Å (versus 5.0 s and 51.2 Å for canonical RPA; Fig. 1C). The mass calculated for aRPA is in close agreement to that predicted from the amino acid sequence (Fig. 1C) and indicates RPA4 is forming a heterotrimeric complex with RPA1 and RPA3. The frictional coefficients for aRPA and RPA are both consistent with an elongated shape (16Gomes X.V. Wold M.S. Biochemistry. 1996; 35: 10558-10568Crossref PubMed Scopus (90) Google Scholar), which suggests that when RPA4 is substituted for RPA2, the overall shape of the complexes in solution is similar. The predicted sequence of RPA4 is 63% identical/similar to RPA2. 4S. J. Haring, T. D. Humphreys, and M. S. Wold, submitted for publication. This similarity allows homology modeling to be used to predict the structure of the putative domains of RPA4. The known structure of the DNA-binding domain of RPA2 (DBD D; 2PI2.pdb) is shown in Fig. 2A. The shallow, putative DNA-binding cleft between the L12 and L45 loops is indicated (17Deng X. Habel J.E. Kabaleeswaran V. Snell E.H. Wold M.S. Borgstahl G.E. J. Mol. Biol. 2007; 374: 865-876Crossref PubMed Scopus (35) Google Scholar). Two other prominent features of the structure are the flexible L34 loop (at the top of structure) and the C-terminal α helix, which has been shown to be part of the subunit interface of RPA2 (right side of structure) (17Deng X. Habel J.E. Kabaleeswaran V. Snell E.H. Wold M.S. Borgstahl G.E. J. Mol. Biol. 2007; 374: 865-876Crossref PubMed Scopus (35) Google Scholar, 18Bochkareva E. Korolev S. Lees-Miller S.P. Bochkarev A. EMBO J. 2002; 21: 1855-1863Crossref PubMed Scopus (229) Google Scholar). The known structure for DBD D of RPA2 was used to model DBD G of RPA4 (Fig. 2A). The predicted structure of DBD G is very similar to that of DBD D, suggesting that the two domains may assume similar structures (Fig. 2A). However, comparison of the predicted surface charge of the DBDs of RPA2 and RPA4 indicates that the surface of RPA2 is much more acidic than that of RPA4 (Fig. 2A, lower row, see also below). In canonical RPA, two domains in RPA1 (DBD A and B) are both necessary and sufficient for high affinity DNA binding, and RPA2 contributes little to the overall affinity of the complex for ssDNA (19Walther A.P. Gomes X.V. Lao Y. Lee C.G. Wold M.S. Biochemistry. 1999; 38: 3963-3973Crossref PubMed Scopus (79) Google Scholar, 20Sibenaller Z.A. Sorensen B.R. Wold M.S. Biochemistry. 1998; 37: 12496-12506Crossref PubMed Scopus (65) Google Scholar, 21Fanning E. Klimovich V. Nager A.R. Nucleic Acids Res. 2006; 34: 4126-4137Crossref PubMed Scopus (399) Google Scholar). Therefore, aRPA, which contains RPA1, RPA4, and RPA3, was expected to bind ssDNA with high affinity. We analyzed the binding affinity of purified aRPA to (dT)30 by gel mobility shift assays. The binding of canonical RPA and aRPA is very similar; nearly equivalent concentrations of protein were needed to form a complex, and only one protein-DNA species was observed (Fig. 3A). Quantitation of the titrations demonstrated that both RPA complexes have high affinity for ssDNA; Kd equals 7.5 × 10-9 m for RPA and 20 × 10-9 m for aRPA (Fig. 3B). Binding was also examined with longer oligonucleotides, (dT)50 and (dT)70. Only one protein-DNA species was observed with (dT)50, whereas two distinct protein-DNA bands were observed with dT70 for both aRPA and RPA (Fig. 3A), suggesting that for high concentrations of both proteins, two molecules bind to dT70. Together these data indicated that the occluded binding site of aRPA is 25-35 nucleotides, which is comparable with the binding site size of RPA (13Kim C. Paulus B.F. Wold M.S. Biochemistry. 1994; 33: 14197-14206Crossref PubMed Scopus (202) Google Scholar). The occluded binding site size was confirmed with stoichiometric reverse titrations monitoring changes in intrinsic protein fluorescence as described by Kim et al. (13Kim C. Paulus B.F. Wold M.S. Biochemistry. 1994; 33: 14197-14206Crossref PubMed Scopus (202) Google Scholar) (data not shown). These analyses also indicate that aRPA binds with low cooperativity similar to RPA because if aRPA bound with high cooperativity, a single transition would have been observed with dT70 rather than a gradual transition between single- and double-liganded species (Fig. 3A). We conclude that aRPA has ssDNA binding properties indistinguishable from canonical RPA; it binds ssDNA with high affinity and low cooperativity. aRPA Function in SV40 Replication—RPA was originally identified as a protein essential for simian virus 40 (SV40) DNA replication (22Wold M.S. Kelly T. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2523-2527Crossref PubMed Scopus (369) Google Scholar); therefore, we examined whether aRPA could support SV40 DNA replication. Cell extracts derived from human tissue culture cells contain all of the cellular proteins required for SV40 replication, except the viral protein large T antigen (Tag) (23Kelly T.J. J. Biol. Chem. 1988; 263: 17889-17892Abstract Full Text PDF PubMed Google Scholar). RPA is required for SV40 replication and is present in the cell extracts (22Wold M.S. Kelly T. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2523-2527Crossref PubMed Scopus (369) Google Scholar); however, the extracts can be depleted of RPA using ammonium sulfate fractionation, making the DNA synthesis dependent on both Tag and RPA (11Binz S.K. Dickson A.M. Haring S.J. Wold M.S. Methods Enzymol. 2006; 409: 11-38Crossref PubMed Scopus (54) Google Scholar). RPA-depleted, ammonium sulfate-fractionated extract (AS-Ex) is unable to support DNA synthesis in the presence of Tag unless the reaction is also supplemented with RPA (Fig. 4A, bars 1-3). A complete reaction with canonical RPA gives robust DNA synthesis (Fig. 4A, bar 3). In contrast, supplementation with aRPA results in only background levels of DNA synthesis (Fig. 4A, bar 4). Background synthesis was also observed when purified RPA4/3 complex was added in place of RPA (data not shown). Replication of the SV40 origin containing DNA occurs by two mechanisms in these reactions: circle-to-circle and rolling circle. These mechanisms produce different products, circles and long linear DNA, respectively (Fig. 4B) (15Walther A.P. Bjerke M.P. Wold M.S. Nucleic Acids Res. 1999; 27: 656-664Crossref PubMed Scopus (19) Google Scholar). Analysis of the products by gel electrophoresis showed that aRPA did not support the formation of either type of product (Fig. 4B, left panel). We conclude that although aRPA binds ssDNA with high affinity, it is unable to support SV40 DNA replication. Interestingly, the addition of aRPA to unfractionated extracts also showed only background levels of synthesis (data not shown). This is surprising because canonical RPA is present in these unfractionated extracts and normally supports replication. Reactions containing both purified aRPA and purified RPA were analyzed. Additional canonical RPA (double the normal amount) in the reaction results in a modest increase in DNA synthesis (Fig. 4A, bar 10). When aRPA was added in the presence of equal amounts of RPA, no DNA synthesis was observed (Fig. 4A, bar 11). This demonstrates that aRPA has a dominant negative effect on the function of canonical RPA in SV40 DNA replication. This effect does not appear to be caused by dissociation of the aRPA complex or subunit exchange because the addition of the purified RPA4/3 complex had no effect on a complete SV40 replication containing canonical RPA (data not shown). RPA2 and RPA4 are both composed of three distinct functional domains: the phosphorylation domain, a DBD, and a winged-helix domain (Fig. 1A). To determine what region(s) of RPA4 is responsible for the properties of aRPA in DNA replication, three hybrid proteins were generated in which the phosphorylation domain, the DBD, or the C terminus of RPA2 was replaced with the corresponding domain of RPA4, named RPA2 (422), RPA2 (242), and RPA2 (224), respectively (Fig. 2B). These domain hybrid proteins were expressed with RPA1 and RPA3, and the resulting complexes were purified. All three complexes purified with a yield similar to RPA and bound (dT)30 with an affinity equivalent to wild-type RPA (data not shown). When the trimeric complexes, RPA·2 (422), RPA·2 (242), and RPA·2 (224), were examined for the ability to support DNA synthesis, only the RPA·2 (422) hybrid complex was able to support wild-type levels of DNA synthesis (Fig. 4A, bars 5-7). RPA·2 (242) and RPA·2 (224) both supported levels of synthesis that were slightly above background and aRPA levels. We conclude that the phosphorylation domain of RPA4 is not responsible for the phenotype observed with aRPA. These data also indicate that both the DBD and the winged-helix domains of RPA2 are necessary for RPA function in SV40 DNA replication and that both of these domains of RPA4 are contributing to the aRPA phenotype. Mixing experiments were also carried out with the RPA2-RPA4 hybrids. RPA·2 (422) did not inhibit the function of RPA and showed levels of synthesis comparable with that of RPA alone. Both RPA·2 (242) and RPA·2 (224) showed levels of DNA synthesis that were significantly reduced from that of RPA (t test; p < 0.005 and p < 0.001, respectively) but greater than that of aRPA (Fig. 4A, bars 12-14). RPA·2 (224) consistently showed more inhibition than RPA·2 (242), suggesting that the two domains may have different effects on SV40 DNA replication. We conclude that both the DBD and the winged-helix domain of aRPA are contributing to the inhibitory effect of RPA4. Mechanism of aRPA Inhibition of SV40 Replication—RPA is required for both initiation and elongation phases of DNA replication. To examine which phase of replication is being affected by aRPA, two-stage elongation assays were carried out. Time course experiments have shown that in the SV40 replication reaction, initiation predominantly occurs during early times (stage I), and at later times (stage II), only elongation synthesis on rolling-circle intermediates is occurring (15Walther A.P. Bjerke M.P. Wold M.S. Nucleic Acids Res. 1999; 27: 656-664Crossref PubMed Scopus (19) Google Scholar). It is therefore possible to examine aRPA function in elongation in a two-stage reaction. Stage I contains all the components necessary for initiation and elongation of SV40 origin-containing DNA except for the radioactive dCTP tracer. This stage is incubated for 2 h at 37 °C, during which normal initiation and elongation occur, but the DNA synthesized is not labeled. In stage II, [α-32P]dCTP and various forms of RPA are added, the incubation is continued for 1 h, and the elongation DNA synthesis is quantitated. This assay measures DNA synthesis occurring during the elongation phase and is independent of the initiation processes (15Walther A.P. Bjerke M.P. Wold M.S. Nucleic Acids Res. 1999; 27: 656-664Crossref PubMed Scopus (19) Google Scholar). Substantial elongation synthesis was observed in the stage II elongation phase (Fig. 5A, bar 2). This synthesis was dependent on the presence of RPA from the start of the reaction and could be stimulated by additional RPA at the beginning of stage II (Fig. 5A, bars 1-3). aRPA strongly inhibits elongation synthesis, demonstrating that aRPA inhibits the normal function of canonical RPA at the pre-existing replication fork (Fig. 5A, bar 4). This strong inhibition was not observed with the hybrid subunits (Fig. 5A, bars 5-7). RPA·2 (422) causes a slight increase in elongation synthesis similar to the addition of canonical RPA (t test; p < 0.0005) and consistent with its ability to promote replication. RPA·2 (224) had no effect on elongation synthesis (t test; p < 0.11), whereas RPA·2 (242) showed slightly reduced levels of DNA synthesis (t test; p < 0.001). Together these experiments indicate that the putative phosphorylation domain of RPA4 has no role in inhibiting elongation synthesis, whereas DBD G of RPA4 inhibits elongation synthesis. Interestingly, the putative winged helix of RPA4 appears to have a separation of function phenotype. Although this region results in inhibition of the complete SV40 DNA synthesis, it does not affect elongation synthesis. This suggests that the putative winged helix-containing C terminus of RPA4 is defective for replication initiation only. Structural Basis of RPA4 Inhibition—The DNA-binding domains of RPA2 and RPA4 are predicted to have similar structures but very different electrostatic surface potentials (Fig. 2A). Because the solution structure of the C-terminal region of RPA2 is known (24Arunkumar A.I. Klimovich V. Jiang X. Ott R.D. Mizoue L. Fanning E. Chazin W.J. Nat. Struct. Mol. Biol. 2005; 12: 332-339Crossref PubMed Scopus (67) Google Scholar), we used homology modeling to predict the structure of the C terminus of RPA4. Fig. 2C shows that the predicted structure for the winged helix of RPA4 is very similar to the known structure of the RPA2 winged helix. The predicted surface potential is predominantly acidic for both winged-helix domains; however, the N terminus of the predicted winged helix of RPA4 is much more acidic than the equivalent region of RPA2 (Fig. 2C). The inhibition studies discussed above suggest that the putative winged-helix domain of RPA4 is inhibiting initiation; RPA·2 (224) inhibits the complete reaction but has no effect on elongation synthesis. This is consistent with a previous analysis that described an important role for the winged-helix domain of RPA2 in initiation of SV40 replication (24Arunkumar A.I. Klimovich V. Jiang X. Ott R.D. Mizoue L. Fanning E. Chazin W.J. Nat. Struct. Mol. Biol. 2005; 12: 332-33
Replication protein A
Replication factor C
Origin recognition complex
Licensing factor
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