scholarly journals A New Direct Single-Molecule Observation Method for DNA Synthesis Reaction Using Fluorescent Replication Protein A

Sensors ◽  
2014 ◽  
Vol 14 (3) ◽  
pp. 5174-5182 ◽  
Author(s):  
Shunsuke Takahashi ◽  
Shohei Kawasaki ◽  
Hidefumi Miyata ◽  
Hirofumi Kurita ◽  
Takeshi Mizuno ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Single Molecule ◽  
Protein A ◽  
Replication Protein A ◽  
Replication Protein ◽  
Synthesis Reaction ◽  
Observation Method

scholarly journals The Schizosaccharomyces pombe rad11+ gene encodes the large subunit of replication protein A.

1997 ◽  
Vol 17 (5) ◽  
pp. 2381-2390 ◽  
Author(s):  
A E Parker ◽  
R K Clyne ◽  
A M Carr ◽  
T J Kelly
Keyword(s):  
Dna Repair ◽  
Dna Synthesis ◽  
Schizosaccharomyces Pombe ◽  
Excision Repair ◽  
Protein A ◽  
Simian Virus 40 ◽  
Replication Protein A ◽  
S Phase ◽  
Replication Protein

Replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein present in all eukaryotes. In vitro studies have implicated RPA in simian virus 40 DNA synthesis and nucleotide excision repair, but little direct information is available about the in vivo roles of the protein. We report here the cloning of the largest subunit of RPA (rpa1+) from the fission yeast Schizosaccharomyces pombe. The rpa1+ gene is essential for viability and is expressed specifically at S phase of the cell cycle. Genetic analysis revealed that rpa1+ is the locus of the S. pombe radiation-sensitive mutation rad11. The rad11 allele exhibits pleiotropic effects consistent with an in vivo role for RPA in both DNA repair and DNA synthesis. The mutant is sensitive to both UV and ionizing radiation but is not defective in the DNA damage-dependent checkpoint, consistent with the hypothesis that RPA is part of the enzymatic machinery of DNA repair. When incubated in hydroxyurea, rad11 cells initially arrest with a 1C DNA content but then lose viability coincident with reentry into S phase, suggesting that DNA synthesis is aberrant under these conditions. A significant fraction of the mutant cells subsequently undergo inappropriate mitosis in the presence of hydroxyurea, indicating that RPA also plays a role in the checkpoint mechanism that monitors the completion of S phase. We propose that RPA is required to maintain the integrity of replication complexes when DNA replication is blocked. We further suggest that the rad11 mutation leads to the premature breakdown of such complexes, thereby preventing recovery from the hydroxyurea arrest and eliminating a signal recognized by the S-phase checkpoint mechanism.

scholarly journals Concentration-Dependent Exchange of Replication Protein A on Single-Stranded DNA Revealed by Single-Molecule Imaging

PLoS ONE ◽  
2014 ◽  
Vol 9 (2) ◽  
pp. e87922 ◽  
Author(s):  
Bryan Gibb ◽  
Ling F. Ye ◽  
Stephanie C. Gergoudis ◽  
YoungHo Kwon ◽  
Hengyao Niu ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein A ◽  
Replication Protein A ◽  
Replication Protein ◽  
Single Molecule Imaging ◽  
Single Stranded Dna

Direct Observation Method of Individual Single-Stranded DNA Molecules Using Fluorescent Replication Protein A

2011 ◽  
Vol 21 (3) ◽  
pp. 1189-1194 ◽  
Author(s):  
Masahiko Oshige ◽  
Shohei Kawasaki ◽  
Hiroki Takano ◽  
Kouji Yamaguchi ◽  
Hirofumi Kurita ◽  
...  
Keyword(s):  
Direct Observation ◽  
Protein A ◽  
Replication Protein A ◽  
Replication Protein ◽  
Dna Molecules ◽  
Single Stranded Dna ◽  
Observation Method ◽  
Direct Observation Method

scholarly journals Human RPA activates BLM’s bidirectional DNA unwinding from a nick

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Zhenheng Qin ◽  
Lulu Bi ◽  
Xi-Miao Hou ◽  
Siqi Zhang ◽  
Xia Zhang ◽  
...  
Keyword(s):  
Dna Repair ◽  
Dna Replication ◽  
Single Molecule ◽  
Genome Stability ◽  
Protein A ◽  
Replication Protein A ◽  
Replication Protein ◽  
High Abundance ◽  
Dna Unwinding ◽  
Dna Replication And Repair

BLM is a multifunctional helicase that plays critical roles in maintaining genome stability. It processes distinct DNA substrates, but not nicked DNA, during many steps in DNA replication and repair. However, how BLM prepares itself for diverse functions remains elusive. Here, using a combined single-molecule approach, we find that a high abundance of BLMs can indeed unidirectionally unwind dsDNA from a nick when an external destabilizing force is applied. Strikingly, human replication protein A (hRPA) not only ensures that limited quantities of BLMs processively unwind nicked dsDNA under a reduced force but also permits the translocation of BLMs on both intact and nicked ssDNAs, resulting in a bidirectional unwinding mode. This activation necessitates BLM targeting on the nick and the presence of free hRPAs in solution whereas direct interactions between them are dispensable. Our findings present novel DNA unwinding activities of BLM that potentially facilitate its function switching in DNA repair.

scholarly journals Concentration-Dependent Exchange of Replication Protein a on Single-Stranded DNA Revealed by Single-Molecule Imaging

2014 ◽  
Vol 106 (2) ◽  
pp. 274a ◽  
Author(s):  
Bryan Gibb ◽  
Ling F. Ye ◽  
Stephanie C. Gergoudis ◽  
YoungHo Kwon ◽  
Hengyao Niu ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein A ◽  
Replication Protein A ◽  
Replication Protein ◽  
Single Molecule Imaging ◽  
Single Stranded Dna

scholarly journals SV40 T antigen interactions with ssDNA and replication protein A: a regulatory role of T antigen monomers in lagging strand DNA replication

2020 ◽  
Vol 48 (7) ◽  
pp. 3657-3677 ◽  
Author(s):  
Nichodemus O Onwubiko ◽  
Angela Borst ◽  
Suraya A Diaz ◽  
Katharina Passkowski ◽  
Felicia Scheffel ◽  
...  
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Dissociation Constants ◽  
Protein A ◽  
Replication Protein A ◽  
T Antigen ◽  
Replication Initiation ◽  
Replication Protein ◽  
Central Process ◽  
Lagging Strand

Abstract DNA replication is a central process in all living organisms. Polyomavirus DNA replication serves as a model system for eukaryotic DNA replication and has considerably contributed to our understanding of basic replication mechanisms. However, the details of the involved processes are still unclear, in particular regarding lagging strand synthesis. To delineate the complex mechanism of coordination of various cellular proteins binding simultaneously or consecutively to DNA to initiate replication, we investigated single-stranded DNA (ssDNA) interactions by the SV40 large T antigen (Tag). Using single molecule imaging by atomic force microscopy (AFM) combined with biochemical and spectroscopic analyses we reveal independent activity of monomeric and oligomeric Tag in high affinity binding to ssDNA. Depending on ssDNA length, we obtain dissociation constants for Tag-ssDNA interactions (KD values of 10–30 nM) that are in the same order of magnitude as ssDNA binding by human replication protein A (RPA). Furthermore, we observe the formation of RPA-Tag-ssDNA complexes containing hexameric as well as monomeric Tag forms. Importantly, our data clearly show stimulation of primase function in lagging strand Okazaki fragment synthesis by monomeric Tag whereas hexameric Tag inhibits the reaction, redefining DNA replication initiation on the lagging strand.

scholarly journals Identification of nuclear pre-replication centers poised for DNA synthesis in Xenopus egg extracts: immunolocalization study of replication protein A.

1992 ◽  
Vol 119 (1) ◽  
pp. 1-15 ◽  
Author(s):  
Y Adachi ◽  
U K Laemmli
Keyword(s):  
Dna Synthesis ◽  
Protein A ◽  
Replication Protein A ◽  
T Antigen ◽  
Replication Protein ◽  
Mitotic Chromosomes ◽  
Membrane Breakdown ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

We demonstrate by immunofluorescence that a 70-kD protein (P70) purified from Xenopus egg extracts is associated with subnuclear foci (about 200) which we propose to be an assembly of DNA pre-replication centers (preRCs). A cDNA encoding this protein reveals that P70 is the Xenopus homologue of replication protein A (RPA also called RF-A). RPA is know to be a cellular, three-subunit single-stranded DNA binding protein, which assists T-antigen in the assembly of the pre-priming complex in the SV40 replication system. The punctated preRCs exist transiently; they form post-mitotically during the period of nuclear membrane breakdown and disappear during ongoing DNA replication. P70 is homogeneously associated with chromatin at the later stages of the S-phase and is displaced from chromatin post replication, so that P70 cannot be detected on mitotic chromosomes. Double-immunofluorescence studies using biotin-dUTP demonstrate that initiation of DNA synthesis is confined to preRCs, resulting in the punctated replication pattern observed previously by others. PreRCs form efficiently on decondensed chromatin in membrane-free egg extracts if ATP and divalent cations are present. Our results suggest that preRCs are composed of an assembly of a large number of pre-initiation replication complexes poised for initiation at discreet subnuclear regions prior to nuclear reconstruction and initiation of DNA synthesis.

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