Fluorescent Single-Stranded DNA-Binding Proteins Enable In Vitro and In Vivo Studies

Abstract G-quadruplexes (G4s) are stable secondary structures that can lead to the stalling of replication forks and cause genomic instability. Pif1 is a 5′ to 3′ helicase, localized to both the mitochondria and nucleus that can unwind G4s in vitro and prevent fork stalling at G4 forming sequences in vivo. Using in vitro primer extension assays, we show that both G4s and stable hairpins form barriers to nuclear and mitochondrial DNA polymerases δ and γ, respectively. However, while single-stranded DNA binding proteins (SSBs) readily promote replication through hairpins, SSBs are only effective in promoting replication through weak G4s. Using a series of G4s with increasing stabilities, we reveal a threshold above which G4 through-replication is inhibited even with SSBs present, and Pif1 helicase is required. Because Pif1 moves along the template strand with a 5′-3′-directionality, head-on collisions between Pif1 and polymerase δ or γ result in the stimulation of their 3′-exonuclease activity. Both nuclear RPA and mitochondrial SSB play a protective role during DNA replication by preventing excessive DNA degradation caused by the helicase-polymerase conflict.

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Functional properties ofEscherichia coli single-stranded DNA binding proteins in vivo

Bulletin of Experimental Biology and Medicine ◽

10.1007/bf00840566 ◽

1987 ◽

Vol 103 (3) ◽

pp. 372-374

Author(s):

O. A. Aizenberg ◽

G. E. Fradkin

Keyword(s):

Dna Binding ◽

Functional Properties ◽

Binding Proteins ◽

Dna Binding Proteins ◽

Ofescherichia Coli ◽

Single Stranded Dna

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Transcriptional repression in Saccharomyces cerevisiae by a SIN3-LexA fusion protein

Molecular and Cellular Biology ◽

10.1128/mcb.13.3.1805-1814.1993 ◽

1993 ◽

Vol 13 (3) ◽

pp. 1805-1814

Author(s):

H Wang ◽

D J Stillman

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Binding ◽

Fusion Protein ◽

Protein Interactions ◽

Transcriptional Repression ◽

Binding Proteins ◽

Dna Binding Proteins ◽

Predicted Amino Acid Sequence

The yeast SIN3 gene (also known as SDI1, UME4, RPD1, and GAM2) has been identified as a transcriptional regulator. Previous work has led to the suggestion that SIN3 regulates transcription via interactions with DNA-binding proteins. Although the SIN3 protein is located in the nucleus, it does not bind directly to DNA in vitro. We have expressed a LexA-SIN3 fusion protein in Saccharomyces cerevisiae and show that this fusion protein represses transcription from heterologous promoters that contain lexA operators. The predicted amino acid sequence of the SIN3 protein contains four copies of a paired amphipathic helix (PAH) motif, similar to motifs found in HLH (helix-loop-helix) and TPR (tetratricopeptide repeat) proteins, and these motifs are proposed to be involved in protein-protein interactions. We have conducted a deletion analysis of the SIN3 gene and show that the PAH motifs are required for SIN3 activity. Additionally, the C-terminal region of the SIN3 protein is sufficient for repression activity in a LexA-SIN3 fusion, and deletion of a PAH motif in this region inactivates this repression activity. A model is presented in which SIN3 recognizes specific DNA-binding proteins in vivo in order to repress transcription.

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RFX1, a transactivator of hepatitis B virus enhancer I, belongs to a novel family of homodimeric and heterodimeric DNA-binding proteins

Molecular and Cellular Biology ◽

10.1128/mcb.14.2.1230-1244.1994 ◽

1994 ◽

Vol 14 (2) ◽

pp. 1230-1244

Author(s):

W Reith ◽

C Ucla ◽

E Barras ◽

A Gaud ◽

B Durand ◽

...

Keyword(s):

Hepatitis B Virus ◽

Dna Binding ◽

Hepatitis B ◽

Binding Proteins ◽

Dna Binding Proteins ◽

Mrna Levels ◽

B Virus ◽

Factor C

RFX1 is a transactivator of human hepatitis B virus enhancer I. We show here that RFX1 belongs to a previously unidentified family of DNA-binding proteins of which we have cloned three members, RFX1, RFX2, and RFX3, from humans and mice. Members of the RFX family constitute the nuclear complexes that have been referred to previously as enhancer factor C, EP, methylation-dependent DNA-binding protein, or rpL30 alpha. RFX proteins share five strongly conserved regions which include the two domains required for DNA binding and dimerization. They have very similar DNA-binding specificities and heterodimerize both in vitro and in vivo. mRNA levels for all three genes, particularly RFX2, are elevated in testis. In other cell lines and tissues, RFX mRNA levels are variable, particularly for RFX2 and RFX3. RFX proteins share several novel features, including new DNA-binding and dimerization motifs and a peculiar dependence on methylated CpG dinucleotides at certain sites.

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Components of the ESCRT Pathway, DFG16, and YGR122w Are Required for Rim101 To Act as a Corepressor with Nrg1 at the Negative Regulatory Element of the DIT1 Gene of Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.25.15.6772-6788.2005 ◽

2005 ◽

Vol 25 (15) ◽

pp. 6772-6788 ◽

Cited By ~ 61

Author(s):

Karen Rothfels ◽

Jason C. Tanny ◽

Enikö Molnar ◽

Helena Friesen ◽

Cosimo Commisso ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Binding ◽

Binding Proteins ◽

Target Genes ◽

Regulatory Element ◽

Active Form ◽

Dna Binding Proteins ◽

Negative Regulatory Element

ABSTRACT The divergently transcribed DIT1 and DIT2 genes of Saccharomyces cerevisiae, which belong to the mid-late class of sporulation-specific genes, are subject to Ssn6-Tup1-mediated repression in mitotic cells. The Ssn6-Tup1 complex, which is required for repression of diverse sets of coordinately regulated genes, is known to be recruited to target genes by promoter-specific DNA-binding proteins. In this study, we show that a 42-bp negative regulatory element (NRE) present in the DIT1-DIT2 intergenic region consists of two distinct subsites and that a multimer of each subsite supports efficient Ssn6-Tup1-dependent repression of a CYC1-lacZ reporter gene. By genetic screening procedures, we identified DFG16, YGR122w, VPS36, and the DNA-binding proteins Rim101 and Nrg1 as potential mediators of NRE-directed repression. We show that Nrg1 and Rim101 bind simultaneously to adjacent target sites within the NRE in vitro and act as corepressors in vivo. We have found that the ability of Rim101 to be proteolytically processed to its active form and mediate NRE-directed repression not only depends on the previously characterized RIM signaling pathway but also requires Dfg16, Ygr122w, and components of the ESCRT trafficking pathway. Interestingly, Rim101 was processed in bro1 and doa4 strains but was unable to mediate efficient repression.

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RFX1, a transactivator of hepatitis B virus enhancer I, belongs to a novel family of homodimeric and heterodimeric DNA-binding proteins.

Molecular and Cellular Biology ◽

10.1128/mcb.14.2.1230 ◽

1994 ◽

Vol 14 (2) ◽

pp. 1230-1244 ◽

Cited By ~ 87

Author(s):

W Reith ◽

C Ucla ◽

E Barras ◽

A Gaud ◽

B Durand ◽

...

Keyword(s):

Hepatitis B Virus ◽

Dna Binding ◽

Hepatitis B ◽

Binding Proteins ◽

Dna Binding Proteins ◽

Mrna Levels ◽

B Virus ◽

Factor C

RFX1 is a transactivator of human hepatitis B virus enhancer I. We show here that RFX1 belongs to a previously unidentified family of DNA-binding proteins of which we have cloned three members, RFX1, RFX2, and RFX3, from humans and mice. Members of the RFX family constitute the nuclear complexes that have been referred to previously as enhancer factor C, EP, methylation-dependent DNA-binding protein, or rpL30 alpha. RFX proteins share five strongly conserved regions which include the two domains required for DNA binding and dimerization. They have very similar DNA-binding specificities and heterodimerize both in vitro and in vivo. mRNA levels for all three genes, particularly RFX2, are elevated in testis. In other cell lines and tissues, RFX mRNA levels are variable, particularly for RFX2 and RFX3. RFX proteins share several novel features, including new DNA-binding and dimerization motifs and a peculiar dependence on methylated CpG dinucleotides at certain sites.

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Bacillus subtilis single-stranded DNA-binding protein SsbA is phosphorylated at threonine 38 by the serine/threonine kinase YabT

Periodicum Biologorum ◽

10.18054/pb.v118i4.4572 ◽

2017 ◽

Vol 118 (4) ◽

Cited By ~ 2

Author(s):

Abderahmane Derouiche ◽

Dina Petranovic ◽

Boris Macek ◽

Ivan Mijakovic

Keyword(s):

Bacillus Subtilis ◽

Dna Binding ◽

Tyrosine Phosphorylation ◽

Binding Proteins ◽

Phosphorylation Site ◽

Dna Binding Proteins ◽

Mass Spectrometry Analysis ◽

Single Stranded Dna ◽

Arginine Residues

Background and purpose: Single-stranded DNA-binding proteins participate in all stages of DNA metabolism that involve single-stranded DNA, from replication, recombination, repair of DNA damage, to natural competence in species such as Bacillus subtilis. B. subtilis single-stranded DNA-binding proteins have previously been found to be phosphorylated on tyrosine and arginine residues. While tyrosine phosphorylation was shown to enhance the DNA-binding properties of SsbA, arginine phosphorylation was not functionally characterized.Materials and methods: We used mass spectrometry analysis to detect phosphorylation of SsbA purified from B. subtilis cells. The detected phosphorylation site was assessed for its influence on DNA-binding in vitro, using electrophoretic mobility shift assays. The ability of B. subtilis serine/threonine kinases to phosphorylate SsbA was assessed using in vitro phosphorylation assays.Results: In addition to the known tyrosine phosphorylation of SsbA on tyrosine 82, we identified a new phosphorylation site: threonine 38. The in vitro assays demonstrated that SsbA is preferentially phosphorylated by the B. subtilis Hanks-type kinase YabT, and phosphorylation of threonine 38 leads to enhanced cooperative binding to DNA.Conclusions: Our findings contribute to the emerging picture that bacterial proteins, exemplified here by SsbA, undergo phosphorylation at multiple residues. This results in a complex regulation of cellular functions, and suggests that the complexity of the bacterial cellular regulation may be underestimated.

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Zinc finger protein ZNF384 is an adaptor of Ku to DNA during classical non-homologous end-joining

Nature Communications ◽

10.1038/s41467-021-26691-0 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Jenny Kaur Singh ◽

Rebecca Smith ◽

Magdalena B. Rother ◽

Anton J. L. de Groot ◽

Wouter W. Wiegant ◽

...

Keyword(s):

Dna Binding ◽

Zinc Finger ◽

Binding Proteins ◽

Zinc Finger Protein ◽

Dna Binding Proteins ◽

End Joining ◽

Dsb Repair ◽

Non Homologous End Joining

AbstractDNA double-strand breaks (DSBs) are among the most deleterious types of DNA damage as they can lead to mutations and chromosomal rearrangements, which underlie cancer development. Classical non-homologous end-joining (cNHEJ) is the dominant pathway for DSB repair in human cells, involving the DNA-binding proteins XRCC6 (Ku70) and XRCC5 (Ku80). Other DNA-binding proteins such as Zinc Finger (ZnF) domain-containing proteins have also been implicated in DNA repair, but their role in cNHEJ remained elusive. Here we show that ZNF384, a member of the C2H2 family of ZnF proteins, binds DNA ends in vitro and is recruited to DSBs in vivo. ZNF384 recruitment requires the poly(ADP-ribosyl) polymerase 1 (PARP1)-dependent expansion of damaged chromatin, followed by binding of its C2H2 motifs to the exposed DNA. Moreover, ZNF384 interacts with Ku70/Ku80 via its N-terminus, thereby promoting Ku70/Ku80 assembly and the accrual of downstream cNHEJ factors, including APLF and XRCC4/LIG4, for efficient repair at DSBs. Altogether, our data suggest that ZNF384 acts as a ‘Ku-adaptor’ that binds damaged DNA and Ku70/Ku80 to facilitate the build-up of a cNHEJ repairosome, highlighting a role for ZNF384 in DSB repair and genome maintenance.

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Reovirus Protein ςNS Binds in Multiple Copies to Single-Stranded RNA and Shares Properties with Single-Stranded DNA Binding Proteins

Journal of Virology ◽

10.1128/jvi.74.13.5939-5948.2000 ◽

2000 ◽

Vol 74 (13) ◽

pp. 5939-5948 ◽

Cited By ~ 40

Author(s):

Anne Lynn Gillian ◽

Stephen C. Schmechel ◽

Jonathan Livny ◽

Leslie A. Schiff ◽

Max L. Nibert

Keyword(s):

Dna Binding ◽

Nucleic Acids ◽

Binding Proteins ◽

Atp Hydrolysis ◽

Nonstructural Protein ◽

Dna Binding Proteins ◽

Mobility Shift ◽

Single Stranded Dna ◽

Gel Mobility Shift

ABSTRACT Reovirus nonstructural protein ςNS interacts with reovirus plus-strand RNAs in infected cells, but little is known about the nature of those interactions or their roles in viral replication. In this study, a recombinant form of ςNS was analyzed for in vitro binding to nucleic acids using gel mobility shift assays. Multiple units of ςNS bound to single-stranded RNA molecules with positive cooperativity and with each unit covering about 25 nucleotides at saturation. The ςNS protein did not bind preferentially to reovirus RNA over nonreovirus RNA in competition experiments but did bind preferentially to single-stranded over double-stranded nucleic acids and with a slight preference for RNA over DNA. In addition, ςNS bound to single-stranded RNA to which a 19-base DNA oligonucleotide was hybridized at either end or near the middle. When present in saturative amounts, ςNS displaced this oligonucleotide from the partial duplex. The strand displacement activity did not require ATP hydrolysis and was inhibited by MgCl2, distinguishing it from a classical ATP-dependent helicase. These properties of ςNS are similar to those of single-stranded DNA binding proteins that are known to participate in genomic DNA replication, suggesting a related role for ςNS in replication of the reovirus RNA genome.

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