The Middle Subunit of Replication Protein A Contacts Growing RNA-DNA Primers in Replicating Simian Virus 40 Chromosomes

Mass, Gilad; Nethanel, Tamar; Kaufmann, Gabriel

doi:10.1128/mcb.18.11.6399

Cited by 53 publications

(48 citation statements)

References 69 publications

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“…2). In agreement with other studies which employed different DNA-protein cross-linking agents, both RPA70 and RPA32 were cross-linked to DNA (18,21,31). Importantly, neither XPA nor XPC, DNA binding proteins with some affinity for damaged DNA, were cross-linked to psoralen.…”

Section: Resultssupporting

confidence: 89%

Molecular Anatomy of the Human Excision Nuclease Assembled at Sites of DNA Damage

Reardon

Sancar

2002

Molecular and Cellular Biology

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Human nucleotide excision repair is initiated by six repair factors (XPA, RPA, XPC-HR23B, TFIIH, XPF-ERCC1, and XPG) which sequentially assemble at sites of DNA damage and effect excision of damagecontaining oligonucleotides. We here describe the molecular anatomy of the human excision nuclease assembled at the site of a psoralen-adducted thymine. Three polypeptides, primarily positioned 5 to the damage, are in close physical proximity to the psoralen lesion and thus are cross-linked to the damaged DNA: these proteins are RPA70, RPA32, and the XPD subunit of TFIIH. While both XPA and XPC bind damaged DNA and are required for XPD cross-linking to the psoralen-adducted base, neither XPA nor XPC is cross-linked to the psoralen adduct. The presence of other repair factors, in particular TFIIH, alters the mode of RPA binding and the position of its subunits relative to the psoralen lesion. Based on these results, we propose that RPA70 makes the initial contact with psoralen-damaged DNA but that within preincision complexes, it is RPA32 and XPD that are in close contact with the lesion.The basic mechanism of nucleotide excision repair includes (i) damage recognition, (ii) assembly of repair factors at the site of damage, (iii) dual incisions that result in excision of damage-containing oligomers, (iv) resynthesis to fill in the gap, and (v) ligation. In humans excision repair is initiated by the combined action of six repair factors, XPA, RPA, XPC, TFIIH, XPG, and XPF-ERCC1, which excise the damaged nucleotide(s) in the form of 24-to 32-nucleotide (nt)-long oligomers (7,30,33,47). In yeast the same six factors are both essential and sufficient for excision of 24-to 27-nt-long oligomers (10). XPA, RPA, and XPC are involved in damage recognition, TFIIH unwinds the duplex around the lesion and stabilizes the damage recognition complex, and XPG and XPF-ERCC1 make the 3Ј and 5Ј incisions, respectively. There is general agreement about the above-stated roles of these factors, but there is limited information about the location of these proteins relative to one another and relative to the lesion within preincision and incision complexes.Previously we identified three stable DNA-protein complexes (25) which we named preincision complex 1 (PIC1) (RPA-XPA-XPC-TFIIH-DNA), PIC2 (RPA-XPA-TFIIH-XPG-DNA), and PIC3 (RPA-XPA-TFIIH-XPG-XPF-ERCC1-DNA). In this study we attempted to locate the various repair factors with a protein-DNA cross-linking assay using a furan-side psoralen-thymine adduct both as a substrate and as a cross-linker (5,16,27). Our results show that RPA is in close contact with the DNA lesion prior to unwinding by TFIIH and that following unwinding, the mode of RPA-DNA interaction changes drastically such as to affect RPA-DNA cross-linking both qualitatively and quantitatively. Surprisingly, neither XPA nor XPC are cross-linked to DNA either when bound in isolation or in the preincision complexes. In contrast, the XPD subunit of TFIIH is cross-linked to psoralen in PIC1, PIC2, and PIC3. Furthermore, we find that plac...

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Section: Resultssupporting

confidence: 89%

Molecular Anatomy of the Human Excision Nuclease Assembled at Sites of DNA Damage

Reardon

Sancar

2002

Molecular and Cellular Biology

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“…RPA participates in the synthesis and processing of Okazaki fragments during DNA replication in viruses and in yeast (Mass et al, 1998;Bae et al, 2001). In multicellular organisms, however, its participation in the preinitiation complex is currently unclear.…”

Section: Introductionmentioning

confidence: 99%

A hypophosphorylated form of RPA34 is a specific component of pre-replication centers

Françon

Lemaı̂tre

Dreyer

et al. 2004

Journal of Cell Science

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IntroductionReplication protein A (RPA) is a stable complex of three different subunits (p70, p34 and p11) that participates in different cellular processes: DNA replication, recombination and repair (Wold, 1997). The RPA70 subunit has a high affinity for single-stranded DNA, but a DNA-binding activity that is associated with the RPA34 and RPA11 subunits (Bochkareva et al., 1998). Cell-cycle-regulated phosphorylation of RPA34 at the G1-S transition has been described (Din et al., 1990;Fang and Newport, 1993;Pan et al., 1994). However, it is not yet clear whether phosphorylation of RPA is involved in the onset of DNA replication (Pan et al., 1995;Philipova et al., 1996), as DNA replication efficiency is not affected by DNAdependent protein kinase (Brush et al., 1994;Lee and Kim, 1995; Pan et al., 1995) or Cdc2 (Henricksen andWold, 1994), two kinases involved in RPA modification.The RPA70 large subunit alone is not sufficient for DNA replication, and the RPA complex cannot be replaced by E. coli single-stranded DNA-binding protein SSB (Dornreiter et al., 1992;Walter and Newport, 2000), suggesting that the RPA34 and RPA11 subunits are necessary for the function of RPA in DNA replication. RPA participates in the synthesis and processing of Okazaki fragments during DNA replication in viruses and in yeast (Mass et al., 1998;Bae et al., 2001). In multicellular organisms, however, its participation in the preinitiation complex is currently unclear. Notably, sites of DNA synthesis are detected as replication foci that co-localize with RPA and the DNA polymerase δ processivity factor PCNA (Adachi and Laemmli, 1992;Dimitrova et al., 1999), but none of the proteins forming the pre-replication complex (e.g. ORC, cdc6, cdt1, MCMs) exhibits such clear localization. The significance of RPA foci is therefore debated.In Xenopus in vitro systems, RPA is present on chromatin before initiation of DNA replication. It first localizes at distinct foci that might be pre-replication centers, but appears to be relocalized evenly throughout the nucleus after initiation of DNA replication (Adachi and Laemmli, 1992). In mammalian cells, RPA localizes to replication foci at the onset of S phase (Brenot-Bosc et al., 1995;Murti et al., 1996), but RPA foci were not observed in early G1 (Dimitrova et al., 1999;Dimitrova and Gilbert, 2000). These observations have led to the proposal that RPA foci in Xenopus may be unrelated to initiation of DNA synthesis and may represent a nonspecific storage of RPA bound to chromatin (Dimitrova et al., 1999).We have investigated the association of the regulatory subunit RPA34 with chromatin during the cell cycle and its participation in pre-replication complexes. We show that the regulatory RPA34 subunit is rapidly dephosphorylated upon mitosis exit, and then associates with chromatin prior to the initiation of DNA replication. RPA34 is detected in its hypophosphorylated form during the whole of S phase and assembles into detergent-resistant foci. Mitosis results in phosphorylation of RPA34 and its loss of chro...

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“…Once a 3Ј-NT-5Ј or 3Ј-NTT-5Ј initiation signal is encountered by the Pol ␣-primase complex, synthesis of a primer-RNA/DNA molecule is initiated. Previous studies indicate that RPA(HSSB) (87), particularly the 32-kDa subunit, is also required for the synthesis of primer-RNA/DNA (47,50) and for coordinating a Pol switch with Pol ␦ (76,92). Based on observations made in prokaryotic (30) and human systems (92), it is proposed that the synthesis of the origin proximal primer-RNA/DNA molecules is a prerequisite for the recruitment of RFC, PCNA, and Pol ␦ and the subsequent initiation of leading-strand synthesis.…”

Section: Discussionmentioning

confidence: 99%

“…It was previously demonstrated that the Pol ␣-primase complex initiates DNA replication by synthesizing a small RNA/DNA hybrid that has been termed primer-RNA/DNA (16,(56)(57)(58). Primer-RNA/DNA is formed exclusively from lagging-strand DNA templates, initially in the vicinity of the SV40 origin and, at later stages of synthesis, at progressively distal locations (13,25,50). Once synthesized, primer-RNA/DNA molecules are extended by a second PCNA-dependent polymerase to form full-length Okazaki fragments (16,(56)(57)(58)78).…”

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confidence: 99%

In the Simian Virus 40 In Vitro Replication System, Start Site Selection by the Polymerase α-Primase Complex Is Not Significantly Altered by Changes in the Concentration of Ribonucleotides

Purviance

Prack

Barbaro

et al. 2001

J Virol

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The simian virus 40 (SV40) in vitro replication system was previously used to demonstrate that the human polymerase (Pol) ␣-primase complex preferentially initiates DNA synthesis at pyrimidine-rich trinucleotide sequences. However, it has been reported that under certain conditions, nucleoside triphosphate (NTP) concentrations play a critical role in determining where eukaryotic primase initiates synthesis. Therefore, we have examined whether increased NTP concentrations alter the template locations at which SV40 replication is initiated. Our studies demonstrate that elevated ribonucleotide concentrations do not significantly alter which template sequences serve as initiation sites. Of considerable interest, the sequences that serve as initiation sites in the SV40 system are similar to those that serve as initiation sites for prokaryotic primases. It is also demonstrated that regardless of the concentration of ribonucleotides present in the reactions, DNA synthesis initiated outside of the core origin. These studies provide additional evidence that the Pol ␣-primase complex can initiate DNA synthesis only after a considerable amount of single-stranded DNA is generated.Studies employing the simian virus 40 (SV40) in vitro replication system have been used to establish much of what is known about the enzymology of eukaryotic DNA replication (10,40,81). This system has also been used to study the reaction mechanisms that take place during particular stages in the replication process; for instance, during the initiation of DNA replication. SV40 replication is initiated when T antigen (T-ag), the single viral protein necessary for replication, site specifically binds to the viral origin (reviewed in references 8, 11, and 31). Upon binding, T-ag assembles into a double hexamer (21,23,51,61,77) that is able to function as a helicase (22,34,66,68,84). Owing to its helicase activity, T-ag is capable of catalyzing origin-specific unwinding, provided replication protein A (RPA) and topoisomerase I are present in the reaction (15,22,27,88).Recent insights into initiation of DNA replication at the SV40 origin include images of T-ag double hexamers assembled on the viral core origin (77). Additional studies have helped to define the minimal core origin sequences necessary for T-ag double hexamer formation (41,42,67). Further insights into T-ag's interactions with the central region of the core origin, site II, were provided by the solution structure of the T-ag origin binding domain (T-ag-obd 131-260 ) (49). These studies established that a pair of loops is utilized to make sequence-specific contacts with site II (49); a similar surface is used by the DNA-binding domain of papillomavirus to bind to DNA (29). Recent experiments have also helped to unravel how T-ag's interactions with the core origin are controlled by the cell cycle machinery (5; reference 83 and references therein).The SV40 replication system has also been used to investigate the formation of nascent DNA in the vicinity of the SV40 origin following T-ag cataly...

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The Middle Subunit of Replication Protein A Contacts Growing RNA-DNA Primers in Replicating Simian Virus 40 Chromosomes

Cited by 53 publications

References 69 publications

Molecular Anatomy of the Human Excision Nuclease Assembled at Sites of DNA Damage

Molecular Anatomy of the Human Excision Nuclease Assembled at Sites of DNA Damage

A hypophosphorylated form of RPA34 is a specific component of pre-replication centers

In the Simian Virus 40 In Vitro Replication System, Start Site Selection by the Polymerase α-Primase Complex Is Not Significantly Altered by Changes in the Concentration of Ribonucleotides

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