ABSTRACT
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.
Subject(s)
Cell Cycle Proteins/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Replication Protein A/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Amino Acid Sequence , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , DNA Damage/drug effects , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/genetics , Methyl Methanesulfonate/toxicity , Molecular Sequence Data , Plasmids/genetics , Plasmids/metabolism , Protein Interaction Domains and Motifs/drug effects , Protein Structure, Tertiary , Replication Protein A/chemistry , Replication Protein A/genetics , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Sequence Alignment , Two-Hybrid System TechniquesABSTRACT
In response to DNA damage, two general but fundamental processes occur in the cell: (1) a DNA lesion is recognized and repaired, and (2) concomitantly, the cell halts the cell cycle to provide a window of opportunity for repair to occur. An essential factor for a proper DNA-damage response is the heterotrimeric protein complex Replication Protein A (RPA). Of particular interest is hyperphosphorylation of the 32-kDa subunit, called RPA2, on its serine/threonine-rich amino (N) terminus following DNA damage in human cells. The unstructured N-terminus is often referred to as the phosphorylation domain and is conserved among eukaryotic RPA2 subunits, including Rfa2 in Saccharomyces cerevisiae. An aspartic acid/alanine-scanning and genetic interaction approach was utilized to delineate the importance of this domain in budding yeast. It was determined that the Rfa2 N-terminus is important for a proper DNA-damage response in yeast, although its phosphorylation is not required. Subregions of the Rfa2 N-terminus important for the DNA-damage response were also identified. Finally, an Rfa2 N-terminal hyperphosphorylation-mimetic mutant behaves similarly to another Rfa1 mutant (rfa1-t11) with respect to genetic interactions, DNA-damage sensitivity, and checkpoint adaptation. Our data indicate that post-translational modification of the Rfa2 N-terminus is not required for cells to deal with "repairable" DNA damage; however, post-translational modification of this domain might influence whether cells proceed into M-phase in the continued presence of unrepaired DNA lesions as a "last-resort" mechanism for cell survival.
Subject(s)
Cell Cycle Checkpoints , DNA Repair , Replication Protein A/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , DNA Damage , DNA, Fungal/metabolism , Phosphorylation , Protein Structure, Tertiary , Replication Protein A/chemistry , Replication Protein A/genetics , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
Maintenance of genome integrity is critical for proper cell growth. This occurs through accurate DNA replication and repair of DNA lesions. A key factor involved in both DNA replication and the DNA damage response is the heterotrimeric single-stranded DNA (ssDNA) binding complex Replication Protein A (RPA). Although the RPA complex appears to be structurally conserved throughout eukaryotes, the primary amino acid sequence of each subunit can vary considerably. Examination of sequence differences along with the functional interchangeability of orthologous RPA subunits or regions could provide insight into important regions and their functions. This might also allow for study in simpler systems. We determined that substitution of yeast Replication Factor A (RFA) with human RPA does not support yeast cell viability. Exchange of a single yeast RFA subunit with the corresponding human RPA subunit does not function due to lack of inter-species subunit interactions. Substitution of yeast Rfa2 with domains/regions of human Rpa2 important for Rpa2 function (i.e., the N-terminus and the loop 3-4 region) supports viability in yeast cells, and hybrid proteins containing human Rpa2 N-terminal phospho-mutations result in similar DNA damage phenotypes to analogous yeast Rfa2 N-terminal phospho-mutants. Finally, the human Rpa2 N-terminus (NT) fused to yeast Rfa2 is phosphorylated in a manner similar to human Rpa2 in human cells, indicating that conserved kinases recognize the human domain in yeast. The implication is that budding yeast represents a potential model system for studying not only human Rpa2 N-terminal phosphorylation, but also phosphorylation of Rpa2 N-termini from other eukaryotic organisms.
Subject(s)
DNA Replication , Replication Protein A/metabolism , Saccharomyces cerevisiae/metabolism , Blotting, Western , Cell Proliferation , Cells, Cultured , HeLa Cells , Humans , Mutation/genetics , Phosphorylation , Protein Structure, Tertiary , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Replication Protein A/genetics , Reverse Transcriptase Polymerase Chain Reaction , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Two-Hybrid System TechniquesABSTRACT
Replication protein A (RPA) is a heterotrimeric protein complex required for a large number of DNA metabolic processes, including DNA replication and repair. An alternative form of RPA (aRPA) has been described in which the RPA2 subunit (the 32-kDa subunit of RPA and product of the RPA2 gene) of canonical RPA is replaced by a homologous subunit, RPA4. The normal function of aRPA is not known; however, previous studies have shown that it does not support DNA replication in vitro or S-phase progression in vivo. In this work, we show that the RPA4 gene is expressed in normal human tissues and that its expression is decreased in cancerous tissues. To determine whether aRPA plays a role in cellular physiology, we investigated its role in DNA repair. aRPA interacted with both Rad52 and Rad51 and stimulated Rad51 strand exchange. We also showed that, by using a reconstituted reaction, aRPA can support the dual incision/excision reaction of nucleotide excision repair. aRPA is less efficient in nucleotide excision repair than canonical RPA, showing reduced interactions with the repair factor XPA and no stimulation of XPF-ERCC1 endonuclease activity. In contrast, aRPA exhibits higher affinity for damaged DNA than canonical RPA, which may explain its ability to substitute for RPA in the excision step of nucleotide excision repair. Our findings provide the first direct evidence for the function of aRPA in human DNA metabolism and support a model for aRPA functioning in chromosome maintenance functions in nonproliferating cells.
Subject(s)
DNA Repair/physiology , Replication Protein A/metabolism , DNA Repair/genetics , Enzyme-Linked Immunosorbent Assay , Humans , Immunoblotting , In Vitro Techniques , Polymerase Chain Reaction , Protein Binding/genetics , Protein Binding/physiology , Rad51 Recombinase/metabolism , Rad52 DNA Repair and Recombination Protein/metabolism , Replication Protein A/geneticsABSTRACT
Replication Protein A (RPA) is a single-stranded DNA-binding protein essential for DNA replication, repair, recombination and cell-cycle regulation. A human homolog of the RPA2 subunit, called RPA4, was previously identified and shown to be expressed in colon mucosal and placental cells; however, the function of RPA4 was not determined. To examine the function of RPA4 in human cells, we carried out knockdown and replacement studies to determine whether RPA4 can substitute for RPA2 in the cell. Unlike RPA2, exogenous RPA4 expression did not support chromosomal DNA replication and lead to cell-cycle arrest in G2/M. In addition, RPA4 localized to sites of DNA repair and reduced gamma-H2AX caused by RPA2 depletion. These studies suggest that RPA4 cannot support cell proliferation but can support processes that maintain the genomic integrity of the cell.
Subject(s)
Cell Cycle , DNA Replication , DNA-Binding Proteins/metabolism , Amino Acid Sequence , Apoptosis , DNA Repair , DNA-Binding Proteins/chemistry , Genomics , HeLa Cells , Humans , Molecular Sequence Data , Phenotype , Protein Subunits/metabolism , Replication Protein A/antagonists & inhibitors , Replication Protein A/metabolism , Sequence Homology, Amino AcidABSTRACT
Meiotic recombination in Saccharomyces cerevisiae is initiated by the creation of DNA double strand breaks (DSBs), an event requiring 10 recombination initiation proteins. Published data indicate that these 10 proteins form three main interaction subgroups [(Spo11-Rec102-Rec104-Ski8), (Rec114-Rec107-Mei4), and (Mre11-Rad50-Xrs2)], but certain components from each subgroup may also interact. Although several of the protein-protein interactions have been defined, the mechanism for DSB formation has been challenging to define. Using a variation of the approach pioneered by others, we have tethered 8 of the 10 initiation proteins to a recombination coldspot and discovered that in addition to Spo11, 6 others (Rec102, Rec104, Ski8, Rec114, Rec107, and Mei4) promote DSB formation at the coldspot, albeit with different frequencies. Of the 8 proteins tested, only Mre11 was unable to cause DSBs even though it binds to UAS(GAL) at GAL2. Our results suggest there may be several ways that the recombination initiation proteins can associate to form a functional initiation complex that can create DSBs.
Subject(s)
DNA Breaks, Double-Stranded , Recombination, Genetic , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , DNA-Binding Proteins/genetics , Diploidy , Monosaccharide Transport Proteins/genetics , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Transcription Factors/geneticsABSTRACT
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.
Subject(s)
DNA Replication/physiology , DNA, Viral/biosynthesis , Multiprotein Complexes/metabolism , Replication Protein A/metabolism , Simian virus 40/physiology , Virus Replication/physiology , DNA, Viral/chemistry , HeLa Cells , Humans , Multiprotein Complexes/chemistry , Protein Structure, Tertiary/physiology , Replication Protein A/chemistry , Simian virus 40/chemistryABSTRACT
In eukaryotes, the single strand DNA (ssDNA)-binding protein, replication protein A (RPA), is essential for DNA replication, repair, and recombination. RPA is composed of the following three subunits: RPA1, RPA2, and RPA3. The RPA1 subunit contains four structurally related domains and is responsible for high affinity ssDNA binding. This study uses a depletion/replacement strategy in human cells to reveal the contributions of each domain to RPA cellular functions. Mutations that substantially decrease ssDNA binding activity do not necessarily disrupt cellular RPA function. Conversely, mutations that only slightly affect ssDNA binding can dramatically affect cellular function. The N terminus of RPA1 is not necessary for DNA replication in the cell; however, this region is important for the cellular response to DNA damage. Highly conserved aromatic residues in the high affinity ssDNA-binding domains are essential for DNA repair and cell cycle progression. Our findings suggest that as long as a threshold of RPA-ssDNA binding activity is met, DNA replication can occur and that an RPA activity separate from ssDNA binding is essential for function in DNA repair.
Subject(s)
Cell Cycle/physiology , DNA Repair/physiology , DNA Replication/physiology , DNA, Single-Stranded/metabolism , Replication Protein A/metabolism , DNA, Single-Stranded/genetics , HeLa Cells , Humans , Mutation , Protein Structure, Tertiary/physiology , Protein Subunits/genetics , Protein Subunits/metabolism , Replication Protein A/geneticsABSTRACT
The activation of a telomere maintenance mechanism is required for cancer development in humans. While most tumors achieve this by expressing the enzyme telomerase, a fraction (5-15%) employs a recombination-based mechanism termed alternative lengthening of telomeres (ALT). Here we show that loss of the single-stranded DNA-binding protein replication protein A (RPA) in human ALT cells, but not in telomerase-positive cells, causes increased exposure of single-stranded G-rich telomeric DNA, cell cycle arrest in G2/M phase, accumulation of single-stranded telomeric DNA within ALT-associated PML bodies (APBs), and formation of telomeric aggregates at the ends of metaphase chromosomes. This study demonstrates differences between ALT cells and telomerase-positive cells in the requirement for RPA in telomere processing and implicates the ALT mechanism in tumor cells as a possible therapeutic target.
Subject(s)
DNA, Single-Stranded/metabolism , Neoplasms/genetics , Replication Protein A/physiology , Telomere/metabolism , Cell Cycle , Cell Line, Transformed , Cell Line, Tumor , Humans , RNA Interference , Replication Protein A/antagonists & inhibitors , Telomere/chemistrySubject(s)
Telomere/metabolism , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/metabolism , Protein Binding , Protein Folding , Replication Protein A/chemistry , Replication Protein A/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Telomere-Binding Proteins/chemistry , Telomere-Binding Proteins/metabolismABSTRACT
Replication protein A (RPA) is a heterotrimeric, single-stranded DNA-binding protein. RPA is conserved in all eukaryotes and is essential for DNA replication, DNA repair, and recombination. RPA also plays a role in coordinating DNA metabolism and the cellular response to DNA damage. Assays have been established for many of these reactions. This chapter provides an overview of the methods used for analyzing RPA-DNA interactions, RPA-protein interactions, and functional activities of RPA. Methods are also discussed for visualizing RPA in the cell and analyzing the effects of RPA function on cell cycle progression in mammalian cells.
Subject(s)
Replication Protein A/physiology , Chromatography, Affinity , DNA Repair , DNA Replication , Enzyme-Linked Immunosorbent Assay , Fluorescence Polarization , Fluorescent Antibody Technique, Indirect , HeLa Cells , Humans , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Replication Protein A/isolation & purification , Surface Plasmon ResonanceABSTRACT
Two of the unique events that occur in meiosis are high levels of genetic recombination and the reductional division. Our previous work demonstrated that the REC102, REC104, REC114, and RAD50 genes, required to initiate meiotic recombination in Saccharomyces cerevisiae, are needed for the proper timing of the first meiotic (MI) division. If these genes are absent, the MI division actually begins at an earlier time. This paper demonstrates that the meiotic recombination genes MER2/REC107, SPO11, and MRE2 and the synaptonemal complex genes HOP1 and RED1 are also required for the normal delay of the MI division. A rec103/ski8 mutant starts the MI division at the same time as in wild-type cells. Our data indicate no obvious correlation between the timing of premeiotic S phase and the timing of the first division in Rec- mutants. Cells with rec102 or rec104 mutations form MI spindles before wild-type cells, suggesting that the initiation signal acts prior to spindle formation. Neither RAD9 nor RAD24 is needed to transduce the signal, which delays the first division. The timing of the MI division in RAD24 mutants indicates that the pachytene checkpoint is not active in Rec+ cells and suggests that the coordination between recombination and the MI division in wild-type cells may occur primarily due to the initiation signal. Finally, at least one of the targets of the recombination initiation signal is the NDT80 gene, a transcriptional regulator of middle meiotic gene expression required for the first division.
Subject(s)
Meiosis/genetics , Recombination, Genetic , Saccharomyces cerevisiae/physiology , Signal Transduction/physiology , Spindle Apparatus/metabolism , DNA-Binding Proteins/metabolism , Endodeoxyribonucleases , Esterases/metabolism , Mutation , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Signal Transduction/geneticsABSTRACT
Meiotic recombination is not random along chromosomes; rather, there are preferred regions for initiation called hotspots. Although the general properties of meiotic hotspots are known, the requirements at the DNA sequence level for the determination of hotspot activity are still unclear. The sequence of six known hotspots in Saccharomyces cerevisiae was compared to identify a common homology region (CoHR). They reported that the locations of CoHR sequences correspond to mapped double-strand break (DSB) sites along three chromosomes (I, III, VI). We report here that a deletion of CoHR at HIS2, a hotspot used to identify the motif, has no significant effect on recombination. In the absence of CoHR, DSB formation occurs at a high frequency and at the same sequences as in wild-type strains. In cases where the deletion of sequences containing the CoHR motif has been shown to reduce recombination, we propose that it may be a reflection of the location of the deletion, rather than the loss of CoHR, per se.
Subject(s)
Chromosome Breakage , Chromosomes, Fungal , DNA, Fungal/genetics , Meiosis/genetics , Saccharomyces cerevisiae/genetics , Amino Acid Motifs , Base Sequence , Consensus Sequence , Molecular Sequence Data , Recombination, Genetic , Sequence Deletion , Sequence Homology, Nucleic AcidABSTRACT
This study addresses three questions about the properties of recombination hotspots in Saccharomyces cerevisiae: How much DNA is required for double-strand-break (DSB) site recognition? Do naturally occurring DSB sites compete with each other in meiotic recombination? What role does the sequence located at the sites of DSBs play? In S. cerevisiae, the HIS2 meiotic recombination hotspot displays a high level of gene conversion, a 3'-to-5' conversion gradient, and two DSB sites located approximately 550 bp apart. Previous studies of hotspots, including HIS2, suggest that global chromosome structure plays a significant role in recombination activity, raising the question of how much DNA is sufficient for hotspot activity. We find that 11.5 kbp of the HIS2 region is sufficient to partially restore gene conversion and both DSBs when moved to another yeast chromosome. Using a variety of different constructs, studies of hotspots have indicated that DSB sites compete with one another for DSB formation. The two naturally occurring DSBs at HIS2 afforded us the opportunity to examine whether or not competition occurs between these native DSB sites. Small deletions of DNA at each DSB site affect only that site; analyses of these deletions show no competition occurring in cis or in trans, indicating that DSB formation at each site at HIS2 is independent. These small deletions significantly affect the frequency of DSB formation at the sites, indicating that the DNA sequence located at a DSB site can play an important role in recombination initiation.