Phosphorylation of the replication protein A large subunit in the Saccharomyces cerevisiae checkpoint response

The checkpoint mechanisms that delay cell cycle progression in response to DNA damage or inhibition of DNA replication are necessary for maintenance of genetic stability in eukaryotic cells. Potential targets of checkpoint-mediated regulation include proteins directly involved in DNA metabolism, such as the cellular single-stranded DNA (ssDNA) binding protein, replication protein A (RPA). Studies in Saccharomyces cerevisiae have revealed that the RPA large subunit (Rfa1p) is involved in the G1 and S phase DNA damage checkpoints. We now demonstrate that Rfa1p is phosphorylated in response to various forms of genotoxic stress, including radiation and hydroxyurea exposure, and further show that phosphorylation of Rfa1p is dependent on the central checkpoint regulator Mec1p. Analysis of the requirement for other checkpoint genes indicates that different mechanisms mediate radiation- and hydroxyurea-induced Rfa1p phosphorylation despite the common requirement for functional Mec1p. In addition, experiments with mutants defective in the Cdc13p telomere-binding protein indicate that ssDNA formation is an important signal for Rfa1p phosphorylation. Because Rfa1p contains the major ssDNA binding activity of the RPA heterotrimer and is required for DNA replication, repair and recombination, it is possible that phosphorylation of this subunit is directly involved in modulating RPA activity during the checkpoint response.     

Replication protein A is sequentially phosphorylated during meiosis

Phosphorylation of the cellular single-stranded DNA-binding protein, replication protein A (RPA), occurs during normal mitotic cell cycle progression and also in response to genotoxic stress. In budding yeast, these reactions require the ATM homolog Mec1, a central regulator of the DNA replication and DNA damage checkpoint responses. We now demonstrate that the middle subunit of yeast RPA (Rfa2) becomes phosphorylated in two discrete steps during meiosis. Primary Rfa2 phosphorylation occurs early in meiotic progression and is independent of DNA replication, recombination and Mec1. In contrast, secondary Rfa2 phosphorylation is activated upon initiation of recombination and requires Mec1. While the primary Rfa2 phosphoisomer is detectable throughout most of meiosis, the secondary Rfa2 phosphoisomer is only transiently generated and begins to disappear soon after recombination is complete. Extensive secondary Rfa2 phosphorylation is observed in a recombination mutant defective for the pachytene checkpoint, indicating that Mec1-dependent Rfa2 phosphorylation does not function to maintain meiotic delay in response to DNA double-strand breaks. Our results suggest that Mec1-dependent RPA phosphorylation could be involved in regulating recombination rather than cell cycle or meiotic progression.     

Replication Protein A‐1 Has a Preference for the Telomeric G‐rich Sequence in Trypanosoma cruzi

Replication protein A (RPA), the major eukaryotic single‐stranded binding protein, is a heterotrimeric complex formed by RPA‐1, RPA‐2, and RPA‐3. RPA is a fundamental player in replication, repair, recombination, and checkpoint signaling. In addition, increasing evidences have been adding functions to RPA in telomere maintenance, such as interaction with telomerase to facilitate its activity and also involvement in telomere capping in some conditions. Trypanosoma cruzi, the etiological agent of Chagas disease is a protozoa parasite that appears early in the evolution of eukaryotes. Recently, we have showed that T. cruziRPA presents canonical functions being involved with DNA replication and DNA damage response. Here, we found by FISH/IF assays that T. cruziRPA localizes at telomeres even outside replication (S) phase. In vitro analysis showed that one telomeric repeat is sufficient to bind RPA‐1. Telomeric DNA induces different secondary structural modifications on RPA‐1 in comparison with other types of DNA. In addition, RPA‐1 presents a higher affinity for telomeric sequence compared to randomic sequence, suggesting that RPA may play specific roles in T. cruzi telomeric region.     

Therapeutic S and G2 checkpoint override causes centromere fragmentation in mitosis

In budding yeast, the meiosis-specific protein kinase Ime2 is required for normal meiotic progression.Current evidence suggests that Ime2 is functionally related to Cdc28, the major cyclin-dependent kinase in yeastthat is essential for both cell cycle and meiosis. We have previously reported that a natural target of Ime2 activityis replication protein A (RPA), the cellular single-stranded DNA-binding protein that performs critical functionsduring DNA replication, repair, and recombination. Ime2-dependent RPA phosphorylation first occursearly in meiosis and targets the middle subunit of the RPA heterotrimeric complex (Rfa2). We now demonstratethat Rfa2 serine 27 (S27) is required for Ime2-dependent Rfa2 phosphorylation in vivo. S27 is also required forRfa2 phosphorylation in vitro catalyzed by immunoprecipitated Ime2. In addition, Ime2 mediates in vitro phosphorylationof a short peptide containing Rfa2 amino acids 23 through 29, thereby providing evidence that S27itself is the phosphoacceptor. Phosphorylation site mapping supports this conclusion, as mass spectrometryanalysis has revealed that at least three residues within Rfa2 amino acids 2 through 35 become phosphorylatedspecifically during meiosis. Although S27 is embedded in a motif that is recognized by several protein kinases,this sequence is not a typical target of cyclin-dependent kinases. Therefore, the mechanism underlying Ime2substrate recognition could differ from that of Cdc28.     

In Vitro Analysis of the Role of Replication Protein A (RPA) and RPA Phosphorylation in ATR-mediated Checkpoint Signaling

Replication protein A (RPA) plays essential roles in DNA metabolism, including replication, checkpoint, and repair. Recently, we described an in vitro system in which the phosphorylation of human Chk1 kinase by ATR (ataxia telangiectasia mutated and Rad3-related) is dependent on RPA bound to single-stranded DNA. Here, we report that phosphorylation of other ATR targets, p53 and Rad17, has the same requirements and that RPA is also phosphorylated in this system. At high p53 or Rad17 concentrations, RPA phosphorylation is inhibited and, in this system, RPA with phosphomimetic mutations cannot support ATR kinase function, whereas a non-phosphorylatable RPA mutant exhibits full activity. Phosphorylation of these ATR substrates depends on the recruitment of ATR and the substrates by RPA to the RPA-ssDNA complex. Finally, mutant RPAs lacking checkpoint function exhibit essentially normal activity in nucleotide excision repair, revealing RPA separation of function for checkpoint and excision repair.     

DNA replication defects, spontaneous DNA damage, and ATM-dependent checkpoint activation in replication protein A-deficient cells

Replication protein A (RPA) is a heterotrimeric, single-stranded DNA-binding complex comprised of 70-kDa (RPA1), 32-kDa (RPA2), and 14-kDa (RPA3) subunits that is essential for DNA replication, recombination, and repair in eukaryotes. In addition, recent studies using vertebrate model systems have suggested an important role for RPA in the initiation of cell cycle checkpoints following exposure to DNA replication stress. Specifically, RPA has been implicated in the recruitment and activation of the ATM-Rad3-related protein kinase, ATR, which in conjunction with the related kinase, ATM (ataxia-telangiectasia-mutated), transmits checkpoint signals via the phosphorylation of downstream effectors. In this report, we have explored the effects of RPA insufficiency on DNA replication, cell survival, and ATM/ATR-dependent signal transduction in response to genotoxic stress. RNA interference-mediated suppression of RPA1 caused a slowing of S phase progression, G2/M cell cycle arrest, and apoptosis in HeLa cells. RPA-deficient cells demonstrated high levels of spontaneous DNA damage and constitutive activation of ATM, which was responsible for the terminal G2/M arrest phenotype. Surprisingly, we found that neither RPA1 nor RPA2 were essential for the hydroxyurea- or UV-induced phosphorylation of the ATR substrates CHK1 and CREB (cyclic AMP-response element-binding protein). These findings reveal that RPA is required for genomic stability and suggest that activation of ATR can occur through RPA-independent pathways.     

Sequential and synergistic modification of human RPA stimulates chromosomal DNA repair

The activity of human replication protein A (RPA) in DNA replication and repair is regulated by phosphorylation of the middle RPA2 subunit. It has previously been shown that up to nine different N-terminal residues are modified in vivo and in response to genotoxic stress. Using a novel antibody against phospho-Ser(29), a moiety formed by cyclin-Cdk, we observed that RPA2 was phosphorylated during mitosis in nonstressed cells. Robust phosphorylation of Ser(29) was also seen in interphase cells following treatment with the DNA-damaging agent camptothecin, a rare example of stress stimulating the modification of a repair factor by cyclin-Cdk. RPA2 phosphorylation is regulated both in cis and trans. Cis-phosphorylation follows a preferred pathway. (That is, the initial modification of Ser(33) by ATR stimulates subsequent phosphorylation of Cdk sites Ser(23) and Ser(29)). These events then facilitate modification of Thr(21) and extreme N-terminal sites Ser(4) and Ser(8), probably by DNA-PK. Our data also indicate that the phosphorylation of one RPA molecule can influence the phosphorylation of other RPA molecules in trans. Cells in which endogenous RPA2 was "replaced" with a double S23A/S29A-RPA2 mutant were seen to have an abnormal cell cycle distribution both in normal and in stressed cells. Such cells also showed aberrant DNA damage-dependent RPA foci and had persistent staining of gammaH2AX following DNA damage. Our data indicate that RPA phosphorylation facilitates chromosomal DNA repair. We postulate that the RPA phosphorylation pattern provides a means to regulate the DNA repair pathway utilized.     

Characterization of the Interaction between Rfa1 and Rad24 in Saccharomyces cerevisiae

Maintaining the integrity of the genome requires the high fidelity duplication of the genome and the ability of the cell to recognize and repair DNA lesions. The heterotrimeric single stranded DNA (ssDNA) binding complex Replication Protein A (RPA) is central to multiple DNA processes, which are coordinated by RPA through its ssDNA binding function and through multiple protein-protein interactions. Many RPA interacting proteins have been reported through large genetic and physical screens; however, the number of interactions that have been further characterized is limited. To gain a better understanding of how RPA functions in DNA replication, repair, and cell cycle regulation and to identify other potential functions of RPA, a yeast two hybrid screen was performed using the yeast 70 kDa subunit, Replication Factor A1 (Rfa1), as a bait protein. Analysis of 136 interaction candidates resulted in the identification of 37 potential interacting partners, including the cell cycle regulatory protein and DNA damage clamp loader Rad24. The Rfa1-Rad24 interaction is not dependent on ssDNA binding. However, this interaction appears affected by DNA damage. The regions of both Rfa1 and Rad24 important for this interaction were identified, and the region of Rad24 identified is distinct from the region reported to be important for its interaction with Rfc2 5. This suggests that Rad24-Rfc2-5 (Rad24-RFC) recruitment to DNA damage substrates by RPA occurs, at least partially, through an interaction between the N terminus of Rfa1 and the C terminus of Rad24. The predicted structure and location of the Rad24 C-terminus is consistent with a model in which RPA interacts with a damage substrate, loads Rad24-RFC at the 5’ junction, and then releases the Rad24-RFC complex to allow for proper loading and function of the DNA damage clamp.     

Replication-mediated DNA damage by camptothecin induces phosphorylation of RPA by DNA-dependent protein kinase and dissociates RPA:DNA-PK complexes

Replication protein A (RPA) is a DNA single-strand binding protein essential for DNA replication, recombination and repair. In human cells treated with the topoisomerase inhibitors camptothecin or etoposide (VP-16), we find that RPA2, the middle-sized subunit of RPA, becomes rapidly phosphorylated. This response appears to be due to DNA-dependent protein kinase (DNA-PK) and to be independent of p53 or the ataxia telangiectasia mutated (ATM) protein. RPA2 phosphorylation in response to camptothecin required ongoing DNA replication. Camptothecin itself partially inhibited DNA synthesis, and this inhibition followed the same kinetics as DNA-PK activation and RPA2 phosphorylation. DNA-PK activation and RPA2 phosphorylation were prevented by the cell-cycle checkpoint abrogator 7-hydroxystaurosporine (UCN-01), which markedly potentiates camptothecin cytotoxicity. The DNA-PK catalytic subunit (DNA-PKcs) was found to bind RPA which was replaced by the Ku autoantigen upon camptothecin treatment. DNA-PKcs interacted directly with RPA1 in vitro. We propose that the encounter of a replication fork with a topoisomerase-DNA cleavage complex could lead to a juxtaposition of replication fork-associated RPA and DNA double-strand end-associated DNA-PK, leading to RPA2 phosphorylation which may signal the presence of DNA damage to an S-phase checkpoint mechanism. Keywords: camptothecin/DNA damage/DNA-dependent protein kinase/RPA2 phosphorylation     

Initiation of DNA damage responses through XPG‐related nucleases

Lesion‐specific enzymes repair different forms of DNA damage, yet all lesions elicit the same checkpoint response. The common intermediate required to mount a checkpoint response is thought to be single‐stranded DNA (ssDNA), coated by replication protein A (RPA) and containing a primer‐template junction. To identify factors important for initiating the checkpoint response, we screened for genes that, when overexpressed, could amplify a checkpoint signal to a weak allele of chk1 in fission yeast. We identified Ast1, a novel member of the XPG‐related family of endo/exonucleases. Ast1 promotes checkpoint activation caused by the absence of the other XPG‐related nucleases, Exo1 and Rad2, the homologue of Fen1. Each nuclease is recruited to DSBs, and promotes the formation of ssDNA for checkpoint activation and recombinational repair. For Rad2 and Exo1, this is independent of their S‐phase role in Okazaki fragment processing. This XPG‐related pathway is distinct from MRN‐dependent responses, and each enzyme is critical for damage resistance in MRN mutants. Thus, multiple nucleases collaborate to initiate DNA damage responses, highlighting the importance of these responses to cellular fitness.     

Xenopus DNA2 is a helicase/nuclease that is found in complexes with replication proteins And-1/Ctf4 and Mcm10 and DSB response proteins Nbs1 and ATM

We have used the Xenopus laevis egg extract system to study the roles of vertebrate Dna2 in DNA replication and double-strand-break (DSB) repair. We first establish that Xenopus Dna2 is a helicase, as well as a nuclease. We further show that Dna2 is a nuclear protein that is actively recruited to DNA only after replication origin licensing. Dna2 co-localizes in foci with RPA and is found in a complex with replication fork components And-1 and Mcm10. Dna2 interacts with the DSB repair and checkpoint proteins Nbs1 and ATM. We also determine the order of arrival of ATM, MRN, Dna2, TopBP1, and RPA to duplex DNA ends and show that it is the same both in S phase and M phase extracts. Interestingly, Dna2 can bind to DNA ends independently of MRN, but efficient nucleolytic resection, as measured by RPA recruitment, requires both MRN and Dna2. The nuclease activity of Mre11 is required, since its inhibition delays both full Dna2 recruitment and resection. Dna2 depletion inhibits but does not block resection, and Chk1 and Chk2 induction occurs in the absence of Dna2.     

MEC1-dependent phosphorylation of yeast RPA1 in vitro

Replication protein A (RPA) is a conserved single-stranded DNA (ssDNA) binding protein with well-characterized roles in DNA metabolism. RPA is phosphorylated in response to genotoxic stress and is required for efficient checkpoint function, although these aspects of RPA function are not well understood. We have investigated the association between RPA and the checkpoint kinase Mec1 in yeast. RPA and Mec1 were found to be physically associated during unperturbed cell growth and in response to DNA damage. Using a Mec1 immunoprecipitate (IP)-kinase assay, we show that the two large subunits, RPA1 and RPA2, are good substrates for Mec1 kinase. The major phosphorylation site of RPA1 was further investigated as it was found to be localized to its amino terminus (RPA1N), which is a non-ssDNA binding domain implicated in regulatory function. This phosphorylation site mapped to serine 178 and phosphorylation-defective mutant protein, expressed from rfa1-S178A, showed reduced physical interaction with Mec1. Phenotypic analysis in vivo revealed that the rfa1-S178A mutation affected the kinetics of RPA1 and Rad53 phosphorylation but did not otherwise affect the checkpoint response. We suggest that phosphorylation of RPA1N by Mec1 may function together with other checkpoint events to regulate the checkpoint response.     

Functional compartmentalization of Rad9 and Hus1 reveals diverse assembly of the 9‐1‐1 complex components during the DNA damage response in Leishmania

The Rad9‐Rad1‐Hus1 (9‐1‐1) complex is a key component in the coordination of DNA damage sensing, cell cycle progression and DNA repair pathways in eukaryotic cells. This PCNA‐related trimer is loaded onto RPA‐coated single stranded DNA and interacts with ATR kinase to mediate effective checkpoint signaling to halt the cell cycle and to promote DNA repair. Beyond these core activities, mounting evidence suggests that a broader range of functions can be provided by 9‐1‐1 structural diversification. The protozoan parasite Leishmania is an early‐branching eukaryote with a remarkably plastic genome, which hints at peculiar genome maintenance mechanisms. Here, we investigated the existence of homologs of the 9‐1‐1 complex subunits in L. major and found that LmRad9 and LmRad1 associate with chromatin in response to replication stress and form a complex in vivo with LmHus1. Similar to LmHus1, LmRad9 participates in telomere homeostasis and in the response to both replication stress and double strand breaks. However, LmRad9 and LmHus1‐deficient cells present markedly opposite phenotypes, which suggest their functional compartmentalization. We show that some of the cellular pool of LmRad9 forms an alternative complex and that some of LmHus1 exists as a monomer. We propose that the diverse assembly of the Leishmania 9‐1‐1 subunits mediates functional compartmentalization, which has a direct impact on the response to genotoxic stress.     

Replication-mediated disassociation of replication protein A-XPA complex upon DNA damage: implications for RPA handing off

RPA (replication protein A), the eukaryotic ssDNA (single-stranded DNA)-binding protein, participates in most cellular processes in response to genotoxic insults, such as NER (nucleotide excision repair), DNA, DSB (double-strand break) repair and activation of cell cycle checkpoint signalling. RPA interacts with XPA (xeroderma pigmentosum A) and functions in early stage of NER. We have shown that in cells the RPA-XPA complex disassociated upon exposure of cells to high dose of UV irradiation. The dissociation required replication stress and was partially attributed to tRPA hyperphosphorylation. Treatment of cells with CPT (camptothecin) and HU (hydroxyurea), which cause DSB DNA damage and replication fork collapse respectively and also leads to the disruption of RPA-XPA complex. Purified RPA and XPA were unable to form complex in vitro in the presence of ssDNA. We propose that the competition-based RPA switch among different DNA metabolic pathways regulates the dissociation of RPA with XPA in cells after DNA damage. The biological significances of RPA-XPA complex disruption in relation with checkpoint activation, DSB repair and RPA hyperphosphorylation are discussed.     

The karyopherin Kap95 and the C-termini of Rfa1, Rfa2, and Rfa3 are necessary for efficient nuclear import of functional RPA complex proteins in Saccharomyces cerevisiae

Nuclear protein import in eukaryotic cells is mediated by karyopherin proteins, which bind to specific nuclear localization signals on substrate proteins and transport them across the nuclear envelope and into the nucleus. Replication protein A (RPA) is a nuclear protein comprised of three subunits (termed Rfa1, Rfa2, and Rfa3 in Saccharomyces cerevisiae) that binds single-stranded DNA and is essential for DNA replication, recombination, and repair. RPA associates with two different karyopherins in yeast, Kap95, and Msn5/Kap142. However, it is unclear which of these karyopherins is responsible for RPA nuclear import. We have generated GFP fusion proteins with each of the RPA subunits and demonstrate that these Rfa-GFP chimeras are functional in yeast cells. The intracellular localization of the RPA proteins in live cells is similar in wild-type and msn5Δ deletion strains but becomes primarily cytoplasmic in cells lacking functional Kap95. Truncating the C-terminus of any of the RPA subunits results in mislocalization of the proteins to the cytoplasm and a loss of protein-protein interactions between the subunits. Our data indicate that Kap95 is likely the primary karyopherin responsible for RPA nuclear import in yeast and that the C-terminal regions of Rfa1, Rfa2, and Rfa3 are essential for efficient nucleocytoplasmic transport of each RPA subunit.     

Distinct roles for DNA-PK,ATM and ATR in RPA phosphorylation and checkpoint activation in response to replication stress

DNA damage encountered by DNA replication forks poses risks of genome destabilization, a precursor to carcinogenesis. Damage checkpoint systems cause cell cycle arrest, promote repair and induce programed cell death when damage is severe. Checkpoints are critical parts of the DNA damage response network that act to suppress cancer. DNA damage and perturbation of replication machinery causes replication stress, characterized by accumulation of single-stranded DNA bound by replication protein A (RPA), which triggers activation of ataxia telangiectasia and Rad3 related (ATR) and phosphorylation of the RPA32, subunit of RPA, leading to Chk1 activation and arrest. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) [a kinase related to ataxia telangiectasia mutated (ATM) and ATR] has well characterized roles in DNA double-strand break repair, but poorly understood roles in replication stress-induced RPA phosphorylation. We show that DNA-PKcs mutant cells fail to arrest replication following stress, and mutations in RPA32 phosphorylation sites targeted by DNA-PKcs increase the proportion of cells in mitosis, impair ATR signaling to Chk1 and confer a G2/M arrest defect. Inhibition of ATR and DNA-PK (but not ATM), mimic the defects observed in cells expressing mutant RPA32. Cells expressing mutant RPA32 or DNA-PKcs show sustained H2AX phosphorylation in response to replication stress that persists in cells entering mitosis, indicating inappropriate mitotic entry with unrepaired damage.     

Cdk5 promotes DNA replication stress checkpoint activation through RPA-32 phosphorylation,and impacts on metastasis free survival in breast cancer patients

Cyclin dependent kinase 5 (Cdk5) is a determinant of PARP inhibitor and ionizing radiation (IR) sensitivity. Here we show that Cdk5-depleted (Cdk5-shRNA) HeLa cells show higher sensitivity to S-phase irradiation, chronic hydroxyurea exposure, and 5-fluorouracil and 6-thioguanine treatment, with hydroxyurea and IR sensitivity also seen in Cdk5-depleted U2OS cells. As Cdk5 is not directly implicated in DNA strand break repair we investigated in detail its proposed role in the intra-S checkpoint activation. While Cdk5-shRNA HeLa cells showed altered basal S-phase dynamics with slower replication velocity and fewer active origins per DNA megabase, checkpoint activation was impaired after a hydroxyurea block. Cdk5 depletion was associated with reduced priming phosphorylations of RPA32 serines 29 and 33 and SMC1-Serine 966 phosphorylation, lower levels of RPA serine 4 and 8 phosphorylation and DNA damage measured using the alkaline Comet assay, gamma-H2AX signal intensity, RPA and Rad51 foci, and sister chromatid exchanges resulting in impaired intra-S checkpoint activation and subsequently higher numbers of chromatin bridges. In vitro kinase assays coupled with mass spectrometry demonstrated that Cdk5 can carry out the RPA32 priming phosphorylations on serines 23, 29, and 33 necessary for this checkpoint activation. In addition we found an association between lower Cdk5 levels and longer metastasis free survival in breast cancer patients and survival in Cdk5-depleted breast tumor cells after treatment with IR and a PARP inhibitor. Taken together, these results show that Cdk5 is necessary for basal replication and replication stress checkpoint activation and highlight clinical opportunities to enhance tumor cell killing.