Subunits of human replication protein A are crosslinked by photoreactive primers synthesized by DNA polymerases (original) (raw)
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FEBS Letters, 1998
To analyze the influence of single-stranded template extension of DNA duplex on the conformation of human replication protein A (RPA) bound to DNA we have designed two template-primer systems differing by the size of the singlestranded template tail (9 and 19 nucleotides (nt)). Basesubstituted photoreactive dUTP analogs were used as substrates for elongation of radiolabeled template-primer by DNA polymerase L L in the absence or in the presence of RPA. Following UVcrosslinking it was demonstrated that the pattern of RPA subunit labeling and consequently RPA arrangement near the 3P-end of the primer is stronlgly dependent upon the length of the template extension.
Photoreactive DNA as a Tool to study Replication Protein A Functioning in DNA Replication and Repair
Photochemistry and Photobiology, 2020
Replication protein A (RPA), eukaryotic single-stranded DNA-binding protein, is a key player in multiple processes of DNA metabolism including DNA replication, recombination and DNA repair. Human RPA composed of subunits of 70-, 32-and 14-kDa binds ssDNA with high affinity and interacts specifically with multiple proteins. The RPA heterotrimer binds ssDNA in several modes, with occlusion lengths of 8-10, 13-22 and 30 nucleotides corresponding to global, transitional and elongated conformations of protein. Varying the structure of photoreactive DNA, the intermediates of different stages of DNA replication or DNA repair were designed and applied to identify positioning of the RPA subunits on the specific DNA structures. Using this approach, RPA interactions with various types of DNA structures attributed to replication and DNA repair intermediates were examined. This review is dedicated to blessed memory of Prof. Alain Favre who contributed to the development of photoreactive nucleotide derivatives and their application for the study of protein-nucleic acids interactions.
Polarity of human replication protein A binding to DNA
Nucleic Acids Research, 2001
Replication protein A (RPA), the nuclear singlestranded DNA binding protein is involved in DNA replication, nucleotide excision repair (NER) and homologous recombination. It is a stable heterotrimer consisting of subunits with molecular masses of 70, 32 and 14 kDa (p70, p32 and p14, respectively). Gapped DNA structures are common intermediates during DNA replication and NER. To analyze the interaction of RPA and its subunits with gapped DNA we designed structures containing 9 and 30 nucleotide gaps with a photoreactive arylazido group at the 3′end of the upstream oligonucleotide or at the 5′-end of the downstream oligonucleotide. UV crosslinking and subsequent analysis showed that the p70 subunit mainly interacts with the 5′-end of DNA irrespective of DNA structure, while the subunit orientation towards the 3′-end of DNA in the gap structures strongly depends on the gap size. The results are compared with the data obtained previously with the primer-template systems containing 5′-or 3′-protruding DNA strands. Our results suggest a model of polar RPA binding to the gapped DNA.
Nucleic Acids Research, 2004
The human nuclear single-stranded (ss) DNAbinding protein, replication protein A (RPA), is a heterotrimer consisting of three subunits: p70, p32 and p14. The protein±DNA interaction is mediated by several DNA-binding domains (DBDs): two major (A and B, also known as p70A and p70B) and several minor (C and D, also known as p70C and p32D, and, presumably, by p70N). Here, using crosslinking experiments, we investigated an interaction of RPA deletion mutants containing a subset of the DBDs with partial DNA duplexes containing 5¢protruding ssDNA tails of 10, 20 and 30 nt. The crosslinks were generated using either a`zerolength' photoreactive group (4-thio-2¢-deoxyuridine-5¢-monophosphate) embedded in the 3¢ end of the DNA primer, or a group connected to the 3¢ end by a lengthy linker (5-{N-[N-(4-azido-2,5-di¯uoro-3chloropyridine-6-yl)-3-aminopropionyl]-trans-3-aminopropenyl-1}-2¢-deoxyuridine-5¢-monophosphate). In the absence of two major DBDs, p70A and p70B, the RPA trimerization core (p70C´p32D´p14) was capable of correctly recognizing the primer± template junction and adopting an orientation similar to that in native RPA. Both p70C and p32D contributed to this recognition. However, the domain contribution differed depending on the size of the ssDNA. In contrast with the trimerization core, the RPA dimerization core (p32D´p14) was incapable of detectably recognizing the DNAjunction structures, suggesting an orchestrating role for p70C in this process.
Nucleic Acids Research, 2008
Replication Protein A is a single-stranded (ss) DNAbinding protein that is highly conserved in eukaryotes and plays essential roles in many aspects of nucleic acid metabolism, including replication, recombination, DNA repair and telomere maintenance. It is a heterotrimeric complex consisting of three subunits: RPA1, RPA2 and RPA3. It possesses four DNA-binding domains (DBD), DBD-A, DBD-B and DBD-C in RPA1 and DBD-D in RPA2, and it binds ssDNA via a multistep pathway. Unlike the RPA1 and RPA2 subunits, no ssDNA-RPA3 interaction has as yet been observed although RPA3 contains a structural motif found in the other DBDs. We show here using 4-thiothymine residues as photoaffinity probe that RPA3 interacts directly with ssDNA on the 3'-side on a 31 nt ssDNA.
Localization of the large subunit of replication factor C near the 5? end of DNA primers
Journal of Molecular Recognition, 2001
Replication factor C (RFC) is a heteropentameric sliding clamp loader protein essential for processive synthesis of DNA by eukaryotic DNA polymerases d and e. To study the interaction of RFC with 3' and 5' ends of the DNA primer, we have developed chemical photocrosslinking assay using a synthetic DNA gap and DNA primer-template structures. We have found that the radioactively labeled primers containing a photoreactive group at their 5' end could crosslink with the largest RFC subunit (RFC140) on primertemplates and DNA gap structures, but that 3' end photoreactive primers could only crosslink with RFC140 within the DNA gap structure. Addition of replication protein A (RPA) to the reaction mixture resulted in the crosslinking of RPA subunits and inhibited crosslinking of RFC140 using 3' but not 5' photoreactive primers present at the gap. The results suggest specific contacts between RFC140 and the 5' end of the DNA primer. Together with previous data, these experiments allow us to propose a model for the DNA polymerase switch during eukaryotic DNA replication.
Journal of Biological Chemistry, 2004
Interactions between the minor groove of the DNA and DNA polymerases appear to play a major role in the catalysis and fidelity of DNA replication. In particular, Arg 668 of Escherichia coli DNA polymerase I (Klenow fragment) makes a critical contact with the N-3-position of guanine at the primer terminus. We investigated the interaction between Arg 668 and the ring oxygen of the incoming deoxynucleotide triphosphate (dNTP) using a combination of site-specific mutagenesis of the protein and atomic substitution of the DNA and dNTP. Hydrogen bonds from Arg 668 were probed with the site-specific mutant R668A. Hydrogen bonds from the DNA were probed with oligodeoxynucleotides containing either guanine or 3-deazaguanine (3DG) at the primer terminus. Hydrogen bonds from the incoming dNTP were probed with (1R,3R,4R)-1-[3-hydroxy-4-(triphosphorylmethyl)cyclopent-1-yl]uracil (dcUTP), an analog of dUTP in which the ring oxygen of the deoxyribose moiety was replaced by a methylene group. We found that the pre-steady-state parameter k pol was decreased 1,600 to 2,000-fold with each of the single substitutions. When the substitutions were combined, there was no additional decrease (R668A and 3DG), a 5-fold decrease (3DG and dcUTP), and a 50-fold decrease (R668A and dcUTP) in k pol. These results are consistent with a hydrogenbonding fork from Arg 668 to the primer terminus and incoming dNTP. These interactions may play an important role in fidelity as well as catalysis of DNA replication.
Interactions of human replication protein A with single-stranded DNA adducts
Biochemical Journal, 2005
Human RPA (replication protein A), a single-stranded DNA-binding protein, is required for many cellular pathways including DNA repair, recombination and replication. However, the role of RPA in nucleotide excision repair remains elusive. In the present study, we have systematically examined the binding of RPA to a battery of well-defined ssDNA (single-stranded DNA) substrates using fluorescence spectroscopy. These substrates contain adducts of (6-4) photoproducts, N-acetyl-2-aminofluorene-, 1-aminopyrene-, BPDE (benzo[a]pyrene diol epoxide)- and fluorescein that are different in many aspects such as molecular structure and size, DNA disruption mode (e.g. base stacking or non-stacking), as well as chemical properties. Our results showed that RPA has a lower binding affinity for damaged ssDNA than for non-damaged ssDNA and that the affinity of RPA for damaged ssDNA depends on the type of adduct. Interestingly, the bulkier lesions have a greater effect. With a fluorescent base-stacking...