scholarly journals Characterization of the Endonuclease and ATP-dependent Flap Endo/Exonuclease of Dna2

2011 ◽  
Vol 286 (27) ◽  
pp. 23763-23770 ◽  
Author(s):  
Barbara K. Fortini ◽  
Subhash Pokharel ◽  
Piotr Polaczek ◽  
Lata Balakrishnan ◽  
Robert A. Bambara ◽  
...  
Keyword(s):  
Protein A ◽  
Nuclease Activity ◽  
Strand Break ◽  
Detailed Characterization ◽  
Single Stranded Dna ◽  
Damage Repair ◽  
Dna Ends ◽  
Lagging Strand

Two processes, DNA replication and DNA damage repair, are key to maintaining genomic fidelity. The Dna2 enzyme lies at the heart of both of these processes, acting in conjunction with flap endonuclease 1 and replication protein A in DNA lagging strand replication and with BLM/Sgs1 and MRN/X in double strand break repair. In vitro, Dna2 helicase and flap endo/exonuclease activities require an unblocked 5′ single-stranded DNA end to unwind or cleave DNA. In this study we characterize a Dna2 nuclease activity that does not require, and in fact can create, 5′ single-stranded DNA ends. Both endonuclease and flap endo/exonuclease are abolished by the Dna2-K677R mutation, implicating the same active site in catalysis. In addition, we define a novel ATP-dependent flap endo/exonuclease activity, which is observed only in the presence of Mn2+. The endonuclease is blocked by ATP and is thus experimentally distinguishable from the flap endo/exonuclease function. Thus, Dna2 activities resemble those of RecB and AddAB nucleases even more closely than previously appreciated. This work has important implications for understanding the mechanism of action of Dna2 in multiprotein complexes, where dissection of enzymatic activities and cofactor requirements of individual components contributing to orderly and precise execution of multistep replication/repair processes depends on detailed characterization of each individual activity.

scholarly journals NBS1-CtIP–Mediated DNA End Resection Regulates cGAS Binding to Micronuclei

2020 ◽  
Author(s):  
Salim Abdisalaam ◽  
Shibani Mukherjee ◽  
Souparno Bhattacharya ◽  
Debapriya Sinha ◽  
Sharda Kumari ◽  
...  
Keyword(s):  
Critical Role ◽  
Protein A ◽  
Nijmegen Breakage Syndrome ◽  
Strand Break ◽  
Direct Role ◽  
Interacting Protein ◽  
Dna End Resection ◽  
End Resection ◽  
Dna Ends

AbstractCyclic GMP-AMP synthase (cGAS), an important component of immune signaling, is hyperactivated in cells defective for DNA damage response (DDR) signaling. However, a direct role for DDR factors in the regulation of cGAS functions is mostly unknown. Here, we provide novel evidence that Nijmegen breakage syndrome 1 (NBS1) protein, a well-studied DNA double-strand break (DSB) sensor, in coordination with ATM, a protein kinase, and CtBP-interacting protein (CtIP), a DNA end resection factor, functions as an upstream regulator of cGAS binding to micronuclei. Upon NBS1 binding to micronuclei via its fork-head–associated domain, it recruits ATM and CtIP via its N- and C-terminal domains, respectively. Subsequently, ATM stabilizes NBS1’s interaction with micronuclei, and CtIP converts DSB ends into single-strand DNA ends, and these two key events preclude cGAS from binding to micronuclei. Notably, we show that purified cGAS cannot form a complex with DNA substrates that mimic resected DNA ends in vitro. Thus, NBS1 together with its binding partners modify the chromatin architecture of the micronuclei and that plays a critical role in cGAS’s binding to micronuclei.

Functional characterization of replication protein A2 (RPA2) from Cryptosporidium parvum

Microbiology ◽  
2004 ◽  
Vol 150 (5) ◽  
pp. 1197-1205 ◽  
Author(s):  
Jason J. Millership ◽  
Xiaomin Cai ◽  
Guan Zhu
Keyword(s):  
Dna Binding ◽  
Cryptosporidium Parvum ◽  
Functional Characterization ◽  
Protein A ◽  
Replication Protein ◽  
Start Codon ◽  
Heterologous Proteins ◽  
Single Stranded Dna

Replication protein A (RPA) is a heterotrimeric complex of single-stranded DNA-binding proteins that play multiple roles in eukaryotic DNA metabolism. The RPA complex is typically composed of heterologous proteins (termed RPA1, RPA2 and RPA3) in animals, plants and fungi, which possess different functions. Previously, two distinct, short-type RPA large subunits (CpRPA1 and CpRPA1B) from the apicomplexan parasite Cryptosporidium parvum were characterized. Here are reported the identification and characterization of a putative middle RPA subunit (CpRPA2) from this unicellular organism. Although the CpRPA2 gene encodes a predicted 40·1 kDa peptide, which is larger than other RPA2 subunits characterized to date, Western blot analysis of oocyst preparations detected a native CpRPA2 protein with a molecular mass of approximately 32 kDa, suggesting that CpRPA2 might undergo post-translational cleavage or the gene was translated at an alternative start codon. Immunofluorescence microscopy using a rabbit anti-CpRPA2 antibody revealed that CpRPA2 protein was mainly distributed in the cytosol (rather than the nuclei) of C. parvum sporozoites. Semi-quantitative RT-PCR data indicated that CpRPA2 was differentially expressed in a tissue culture model with highest expression in intracellular parasites infecting HCT-8 cells for 36 and 60 h. Sequence comparison suggests that RPA2 is a group of poorly conserved proteins. Nonetheless, functional analyses of recombinant proteins confirmed that CpRPA2 is a single-stranded DNA-binding protein and that it could serve as an in vitro phosphorylation target by a DNA-dependent protein kinase. The minimal length of poly(dT) required for CpRPA2 binding is 17 nucleotides, and the DNA-binding capability was inhibited by phosphorylation in vitro. These observations provide additional evidence on the divergence of RPA proteins between C. parvum and host, implying that the parasite DNA replication machinery could be explored as a chemotherapeutic target.

scholarly journals The Yeast Recombinational Repair Protein Rad59 Interacts With Rad52 and Stimulates Single-Strand Annealing

Genetics ◽  
2001 ◽  
Vol 159 (2) ◽  
pp. 515-525 ◽  
Author(s):  
Allison P Davis ◽  
Lorraine S Symington
Keyword(s):  
Protein A ◽  
Single Strand ◽  
Physical Interaction ◽  
Dna Double Strand Breaks ◽  
Single Stranded Dna ◽  
Single Strand Annealing ◽  
Strand Annealing ◽  
Recombination Pathway

Abstract The yeast RAD52 gene is essential for homology-dependent repair of DNA double-strand breaks. In vitro, Rad52 binds to single- and double-stranded DNA and promotes annealing of complementary single-stranded DNA. Genetic studies indicate that the Rad52 and Rad59 proteins act in the same recombination pathway either as a complex or through overlapping functions. Here we demonstrate physical interaction between Rad52 and Rad59 using the yeast two-hybrid system and co-immunoprecipitation from yeast extracts. Purified Rad59 efficiently anneals complementary oligonucleotides and is able to overcome the inhibition to annealing imposed by replication protein A (RPA). Although Rad59 has strand-annealing activity by itself in vitro, this activity is insufficient to promote strand annealing in vivo in the absence of Rad52. The rfa1-D288Y allele partially suppresses the in vivo strand-annealing defect of rad52 mutants, but this is independent of RAD59. These results suggest that in vivo Rad59 is unable to compete with RPA for single-stranded DNA and therefore is unable to promote single-strand annealing. Instead, Rad59 appears to augment the activity of Rad52 in strand annealing.

scholarly journals Cells Expressing Murine RAD52 Splice Variants Favor Sister Chromatid Repair

2006 ◽  
Vol 26 (10) ◽  
pp. 3752-3763 ◽  
Author(s):  
Peter H. Thorpe ◽  
Vanessa A. Marrero ◽  
Margaret H. Savitzky ◽  
Ivana Sunjevaric ◽  
Tom C. Freeman ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Sister Chromatid ◽  
Mammalian Cells ◽  
Splice Variants ◽  
Protein A ◽  
Single Stranded Dna ◽  
Culture Cells ◽  
Dominant Phenotype ◽  
Mouse Tissues

ABSTRACT The RAD52 gene is essential for homologous recombination in the yeast Saccharomyces cerevisiae. RAD52 is the archetype in an epistasis group of genes essential for DNA damage repair. By catalyzing the replacement of replication protein A with Rad51 on single-stranded DNA, Rad52 likely promotes strand invasion of a double-stranded DNA molecule by single-stranded DNA. Although the sequence and in vitro functions of mammalian RAD52 are conserved with those of yeast, one difference is the presence of introns and consequent splicing of the mammalian RAD52 pre-mRNA. We identified two novel splice variants from the RAD52 gene that are expressed in adult mouse tissues. Expression of these splice variants in tissue culture cells elevates the frequency of recombination that uses a sister chromatid template. To characterize this dominant phenotype further, the RAD52 gene from the yeast Saccharomyces cerevisiae was truncated to model the mammalian splice variants. The same dominant sister chromatid recombination phenotype seen in mammalian cells was also observed in yeast. Furthermore, repair from a homologous chromatid is reduced in yeast, implying that the choice of alternative repair pathways may be controlled by these variants. In addition, a dominant DNA repair defect induced by one of the variants in yeast is suppressed by overexpression of RAD51, suggesting that the Rad51-Rad52 interaction is impaired.

Genetic Analysis of Yeast RPA1 Reveals Its Multiple Functions in DNA Metabolism

Genetics ◽  
1998 ◽  
Vol 148 (3) ◽  
pp. 989-1005 ◽  
Author(s):  
Keiko Umezu ◽  
Neal Sugawara ◽  
Clark Chen ◽  
James E Haber ◽  
Richard D Kolodner
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Protein A ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Physical Analysis ◽  
Single Stranded Dna ◽  
Temperature Sensitive Mutants

Abstract Replication protein A (RPA) is a single-stranded DNA-binding protein identified as an essential factor for SV40 DNA replication in vitro. To understand the in vivo functions of RPA, we mutagenized the Saccharomyces cerevisiae RFA1 gene and identified 19 ultraviolet light (UV) irradiation- and methyl methane sulfonate (MMS)-sensitive mutants and 5 temperature-sensitive mutants. The UV- and MMS-sensitive mutants showed up to 104 to 105 times increased sensitivity to these agents. Some of the UV- and MMS-sensitive mutants were killed by an HO-induced double-strand break at MAT. Physical analysis of recombination in one UV- and MMS-sensitive rfa1 mutant demonstrated that it was defective for mating type switching and single-strand annealing recombination. Two temperature-sensitive mutants were characterized in detail, and at the restrictive temperature were found to have an arrest phenotype and DNA content indicative of incomplete DNA replication. DNA sequence analysis indicated that most of the mutations altered amino acids that were conserved between yeast, human, and Xenopus RPA1. Taken together, we conclude that RPA1 has multiple roles in vivo and functions in DNA replication, repair, and recombination, like the single-stranded DNA-binding proteins of bacteria and phages.

Differential Regulation of Articular Cartilage Biomechanical and Biochemical Properties by IGF-1 and TGF-β1 During In Vitro Growth

2009 ◽  
Author(s):  
Michael E. Stender ◽  
Christian R. Flores ◽  
Kristin J. Dills ◽  
Gregory M. Williams ◽  
Kevin M. Stewart ◽  
...  
Keyword(s):  
Articular Cartilage ◽  
Growth Models ◽  
Biochemical Properties ◽  
Low Friction ◽  
Cartilage Growth ◽  
Detailed Characterization ◽  
Naturally Occurring ◽  
Tgf Β1

Articular cartilage (AC) is a load bearing material that provides a low friction wear resistant interface in synovial joints. Naturally-occurring and stimulated intrinsic repair of damaged AC is ineffective. Thus, there is a desire to engineer effective replacement tissue that could be used for AC repair. Previous studies [1] have shown that culture of immature cartilage with medium including TGF-β1 will result in a more mature tissue than culture with IGF-1. Detailed characterization of tissue mechanical properties would be helpful for development of cartilage growth models [2].

scholarly journals Purification and In Vitro Characterization of the Serratia marcescens NucC Protein, a Zinc-Binding Transcription Factor Homologous to P2 Ogr

2003 ◽  
Vol 185 (6) ◽  
pp. 1808-1816 ◽  
Author(s):  
Victor McAlister ◽  
Chao Zou ◽  
Robert H. Winslow ◽  
Gail E. Christie
Keyword(s):  
Rna Polymerase ◽  
Serratia Marcescens ◽  
Specific Binding ◽  
Protein A ◽  
Upstream Region ◽  
Late Gene Expression ◽  
In Vitro Characterization ◽  
Template Dna

ABSTRACT NucC is structurally and functionally homologous to a family of prokaryotic zinc finger transcription factors required for late gene expression in P2- and P4-related bacteriophages. Characterization of these proteins in vitro has been hampered by their relative insolubility and tendency to aggregate. We report here the successful purification of soluble, active, wild-type NucC protein. Purified NucC exhibits site-specific binding to a conserved DNA sequence that is located upstream of NucC-dependent Serratia marcescens promoters and the late promoters of P2-related phages. This sequence is sufficient for binding of NucC in vitro. NucC binding to the S. marcescens nuclease promoter P nucA and to the sequence upstream of the P2 late promoter P F is accompanied by DNA bending. NucC protects about 25 nucleotides of the P F upstream region from DNase I digestion, and RNA polymerase protects the promoter region only in the presence of NucC. Template DNA, RNA polymerase holoenzyme, and purified NucC are the only macromolecular components required for transcription from P F in vitro.

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