scholarly journals Development of a single-stranded DNA-binding protein fluorescent fusion toolbox

2020 ◽  
Vol 48 (11) ◽  
pp. 6053-6067
Author(s):  
Katarzyna Dubiel ◽  
Camille Henry ◽  
Lisanne M Spenkelink ◽  
Alexander G Kozlov ◽  
Elizabeth A Wood ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Dna Binding ◽  
Single Molecule ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Single Stranded Dna ◽  
Intrinsically Disordered ◽  
C Terminus ◽  
Sites Of Action

Abstract Bacterial single-stranded DNA-binding proteins (SSBs) bind single-stranded DNA and help to recruit heterologous proteins to their sites of action. SSBs perform these essential functions through a modular structural architecture: the N-terminal domain comprises a DNA binding/tetramerization element whereas the C-terminus forms an intrinsically disordered linker (IDL) capped by a protein-interacting SSB-Ct motif. Here we examine the activities of SSB-IDL fusion proteins in which fluorescent domains are inserted within the IDL of Escherichia coli SSB. The SSB-IDL fusions maintain DNA and protein binding activities in vitro, although cooperative DNA binding is impaired. In contrast, an SSB variant with a fluorescent protein attached directly to the C-terminus that is similar to fusions used in previous studies displayed dysfunctional protein interaction activity. The SSB-IDL fusions are readily visualized in single-molecule DNA replication reactions. Escherichia coli strains in which wildtype SSB is replaced by SSB-IDL fusions are viable and display normal growth rates and fitness. The SSB-IDL fusions form detectible SSB foci in cells with frequencies mirroring previously examined fluorescent DNA replication fusion proteins. Cells expressing SSB-IDL fusions are sensitized to some DNA damaging agents. The results highlight the utility of SSB-IDL fusions for biochemical and cellular studies of genome maintenance reactions.

scholarly journals E. coli primase and DNA polymerase III holoenzyme are able to bind concurrently to a primed template during DNA replication

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Andrea Bogutzki ◽  
Natalie Naue ◽  
Lidia Litz ◽  
Andreas Pich ◽  
Ute Curth
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Protein Interactions ◽  
Dna Polymerase Iii ◽  
Protein Protein Interactions ◽  
Single Stranded Dna ◽  
E Coli ◽  
Polymerase Iii ◽  
C Terminus ◽  
Pol Iii

Abstract During DNA replication in E. coli, a switch between DnaG primase and DNA polymerase III holoenzyme (pol III) activities has to occur every time when the synthesis of a new Okazaki fragment starts. As both primase and the χ subunit of pol III interact with the highly conserved C-terminus of single-stranded DNA-binding protein (SSB), it had been proposed that the binding of both proteins to SSB is mutually exclusive. Using a replication system containing the origin of replication of the single-stranded DNA phage G4 (G4ori) saturated with SSB, we tested whether DnaG and pol III can bind concurrently to the primed template. We found that the addition of pol III does not lead to a displacement of primase, but to the formation of higher complexes. Even pol III-mediated primer elongation by one or several DNA nucleotides does not result in the dissociation of DnaG. About 10 nucleotides have to be added in order to displace one of the two primase molecules bound to SSB-saturated G4ori. The concurrent binding of primase and pol III is highly plausible, since even the SSB tetramer situated directly next to the 3′-terminus of the primer provides four C-termini for protein-protein interactions.

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.

Properties of the high-affinity single-stranded DNA binding state of the Escherichia coli recA protein

Biochemistry ◽  
1988 ◽  
Vol 27 (4) ◽  
pp. 1205-1212 ◽  
Author(s):  
Joseph P. Menetski ◽  
Abraham Varghese ◽  
Stephen C. Kowalczykowski
Keyword(s):  
Escherichia Coli ◽  
Dna Binding ◽  
Reca Protein ◽  
Single Stranded Dna ◽  
High Affinity

scholarly journals F sex factor of Escherichia coli K-12 codes for a single-stranded DNA binding protein.

1983 ◽  
Vol 80 (14) ◽  
pp. 4422-4426 ◽  
Author(s):  
A. L. Kolodkin ◽  
M. A. Capage ◽  
E. I. Golub ◽  
K. B. Low
Keyword(s):  
Escherichia Coli ◽  
Dna Binding ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Single Stranded Dna ◽  
K 12

scholarly journals Single-molecule DREEM imaging reveals DNA wrapping around human mitochondrial single-stranded DNA binding protein

2018 ◽  
Vol 46 (21) ◽  
pp. 11287-11302 ◽  
Author(s):  
Parminder Kaur ◽  
Matthew J Longley ◽  
Hai Pan ◽  
Hong Wang ◽  
William C Copeland
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Single Stranded Dna ◽  
Dna Wrapping
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