scholarly journals Structure-Based Mutational Analysis of the Bovine Papillomavirus E1 Helicase Domain Identifies Residues Involved in the Nonspecific DNA Binding Activity Required for Double Trimer Formation

2010 ◽  
Vol 84 (9) ◽  
pp. 4264-4276 ◽  
Author(s):  
Xiaofei Liu ◽  
Stephen Schuck ◽  
Arne Stenlund
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Mutational Analysis ◽  
Binding Activity ◽  
Helicase Domain ◽  
Bovine Papillomavirus ◽  
Dna Binding Activity ◽  
E1 Protein ◽  
Initiation Of Dna Replication ◽  
Dna Template

ABSTRACT The papillomavirus E1 protein is a multifunctional initiator protein responsible for preparing the viral DNA template for initiation of DNA replication. The E1 protein encodes two DNA binding activities that are required for initiation of DNA replication. A well-characterized sequence-specific DNA binding activity resides in the E1 DBD and is used to tether E1 to the papillomavirus ori. A non-sequence-specific DNA binding activity is also required for formation of the E1 double trimer (DT) complex, which is responsible for the local template melting that precedes loading of the E1 helicase. This DNA binding activity is very poorly understood. We use a structure-based mutagenesis approach to identify residues in the E1 helicase domain that are required for the non-sequence-specific DNA binding and DT formation. We found that three groups of residues are involved in nonspecific DNA binding: the E1 β-hairpin structure containing R505, K506, and H507; a hydrophobic loop containing F464; and a charged loop containing K461 together generate the binding surface involved in nonspecific DNA binding. These residues are well conserved in the T antigens from the polyomaviruses, indicating that the polyomaviruses share this nonspecific DNA binding activity.

scholarly journals Surface Mutagenesis of the Bovine Papillomavirus E1 DNA Binding Domain Reveals Residues Required for Multiple Functions Related to DNA Replication

2006 ◽  
Vol 80 (15) ◽  
pp. 7491-7499 ◽  
Author(s):  
Stephen Schuck ◽  
Arne Stenlund
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Binding Activity ◽  
Binding Domain ◽  
Bovine Papillomavirus ◽  
Dna Binding Domain ◽  
Multifunctional Protein ◽  
Dna Binding Activity ◽  
E1 Protein ◽  
Complex Functions

ABSTRACT The E1 protein from papillomaviruses is a multifunctional protein with complex functions required for the initiation of viral DNA replication. We have performed a surface mutagenesis of the well-characterized E1 DNA binding domain (DBD). We demonstrate that substitutions of multiple residues on the surface of the E1 DBD are defective for DNA replication without affecting the DNA binding activity of the protein. The defects of individual substitutions include failure to form the double trimer that melts the ori and failure to form the double hexamer that unwinds the ori. These results demonstrate that the DBD plays an essential role in multiple DNA replication-related processes apart from DNA binding.

scholarly journals Redox regulation of DNA binding activity of DREF (DNA replication-related element binding factor) in Drosophila

2004 ◽  
Vol 378 (3) ◽  
pp. 833-838 ◽  
Author(s):  
Tae-Yeong CHOI ◽  
S. Young PARK ◽  
Ho-Sung KANG ◽  
Jae-Hun CHEONG ◽  
Han-Do KIM ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Redox State ◽  
Redox Regulation ◽  
Binding Activity ◽  
Dna Binding Activity ◽  
Cell Extracts ◽  
Binding Factor ◽  
Related Element ◽  
Intracellular Redox

DREF [DRE (DNA replication-related element) binding factor] is an 80 kDa polypeptide homodimer which plays an important role in regulating cell proliferation-related genes. Both DNA binding and dimer formation activities are associated with residues 16–115 of the N-terminal region. However, the mechanisms by which DREF dimerization and DNA binding are regulated remain unknown. Here, we report that the DNA binding activity of DREF is regulated by a redox mechanism, and that the cysteine residues are involved in this regulation. Electrophoretic mobility shift analysis using Drosophila Kc cell extracts or recombinant DREF proteins indicated that the DNA binding domain is sufficient for redox regulation. Site-directed mutagenesis and transient transfection assays showed that Cys59 and/or Cys62 are critical both for DNA binding and for redox regulation, whereas Cys91 is dispensable. In addition, experiments using Kc cells indicated that the DNA binding activity and function of DREF are affected by the intracellular redox state. These findings give insight into the exact nature of DREF function in the regulation of target genes by the intracellular redox state.

scholarly journals Regulation of the DNA-binding and transcriptional activities of Xenopus laevis NFI-X by a novel C-terminal domain.

1995 ◽  
Vol 15 (10) ◽  
pp. 5552-5562 ◽  
Author(s):  
E Roulet ◽  
M T Armentero ◽  
G Krey ◽  
B Corthésy ◽  
C Dreyer ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Xenopus Laevis ◽  
Transcriptional Activation ◽  
Binding Activity ◽  
New Members ◽  
Binding Domains ◽  
Dna Binding Activity

The nuclear factor I (NFI) family consists of sequence-specific DNA-binding proteins that activate both transcription and adenovirus DNA replication. We have characterized three new members of the NFI family that belong to the Xenopus laevis NFI-X subtype and differ in their C-termini. We show that these polypeptides can activate transcription in HeLa and Drosophila Schneider line 2 cells, using an activation domain that is subdivided into adjacent variable and subtype-specific domains each having independent activation properties in chimeric proteins. Together, these two domains constitute the full NFI-X transactivation potential. In addition, we find that the X. laevis NFI-X proteins are capable of activating adenovirus DNA replication through their conserved N-terminal DNA-binding domains. Surprisingly, their in vitro DNA-binding activities are specifically inhibited by a novel repressor domain contained within the C-terminal part, while the dimerization and replication functions per se are not affected. However, inhibition of DNA-binding activity in vitro is relieved within the cell, as transcriptional activation occurs irrespective of the presence of the repressor domain. Moreover, the region comprising the repressor domain participates in transactivation. Mechanisms that may allow the relief of DNA-binding inhibition in vivo and trigger transcriptional activation are discussed.

scholarly journals Mutational analysis of the DNA-binding domain of the CYS3 regulatory protein of Neurospora crassa.

1991 ◽  
Vol 11 (9) ◽  
pp. 4356-4362 ◽  
Author(s):  
M N Kanaan ◽  
G A Marzluf
Keyword(s):  
Dna Binding ◽  
Neurospora Crassa ◽  
Mutational Analysis ◽  
Regulatory Gene ◽  
Regulatory Protein ◽  
Binding Activity ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Dna Binding Domain ◽  
Dna Binding Activity

cys-3, the major sulfur regulatory gene of Neurospora crassa, activates the expression of a set of unlinked structural genes which encode sulfur catabolic-related enzymes during conditions of sulfur limitation. The cys-3 gene encodes a regulatory protein of 236 amino acid residues with a leucine zipper and an upstream basic region (the b-zip region) which together may constitute a DNA-binding domain. The b-zip region was expressed in Escherichia coli to examine its DNA-binding activity. The b-zip domain protein binds to the promoter region of the cys-3 gene itself and of cys-14, the sulfate permease II structural gene. A series of CYS3 mutant proteins obtained by site-directed mutagenesis were expressed and tested for function, dimer formation, and DNA-binding activity. The results demonstrate that the b-zip region of cys-3 is critical for both its function in vivo and specific DNA-binding in vitro.

scholarly journals Role of Single-Stranded DNA Binding Activity of T Antigen in Simian Virus 40 DNA Replication

2001 ◽  
Vol 75 (6) ◽  
pp. 2839-2847 ◽  
Author(s):  
Chunxiao Wu ◽  
Rupa Roy ◽  
Daniel T. Simmons
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Single Point ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Binding Activity ◽  
T Antigen ◽  
Amino Acid Residues ◽  
Single Stranded Dna ◽  
Dna Binding Activity

ABSTRACT We have previously mapped the single-stranded DNA binding domain of large T antigen to amino acid residues 259 to 627. By using internal deletion mutants, we show that this domain most likely begins after residue 301 and that the region between residues 501 and 550 is not required. To study the function of this binding activity, a series of single-point substitutions were introduced in this domain, and the mutants were tested for their ability to support simian virus 40 (SV40) replication and to bind to single-stranded DNA. Two replication-defective mutants (429DA and 460EA) were grossly impaired in single-stranded DNA binding. These two mutants were further tested for other biochemical activities needed for viral DNA replication. They bound to origin DNA and formed double hexamers in the presence of ATP. Their ability to unwind origin DNA and a helicase substrate was severely reduced, although they still had ATPase activity. These results suggest that the single-stranded DNA binding activity is involved in DNA unwinding. The two mutants were also very defective in structural distortion of origin DNA, making it likely that single-stranded DNA binding is also required for this process. These data show that single-stranded DNA binding is needed for at least two steps during SV40 DNA replication.

Cooperative DNA binding of the human HoxB5 (Hox-2.1) protein is under redox regulation in vitro

1993 ◽  
Vol 13 (8) ◽  
pp. 4609-4617
Author(s):  
C K Galang ◽  
C A Hauser
Keyword(s):  
Dna Binding ◽  
Redox Regulation ◽  
Mutational Analysis ◽  
Cysteine Residue ◽  
Binding Activity ◽  
Nf Kappa B ◽  
Amino Terminal ◽  
Dna Binding Activity ◽  
Specific Oxidation

The human HoxB5 (Hox-2.1) gene product is a sequence-specific DNA binding protein. Cooperative interactions stabilize in vitro DNA binding of the HoxB5 protein to tandem binding sites by at least 100-fold relative to binding to a single site. The HoxB5 homeodomain is sufficient for sequence-specific DNA binding but not for cooperative DNA binding. Here we report that the additional protein sequence required for cooperativity is a small domain adjacent to the homeodomain on the amino-terminal side. We further show that cooperative DNA binding is under redox regulation. The HoxB5 protein binds to DNA in vitro both when oxidized or reduced but binds cooperatively only when oxidized. Mutational analysis has revealed that the cysteine residue in the turn between homeodomain helices 2 and 3 is necessary for cooperative binding and redox regulation. The enhanced DNA binding of oxidized HoxB5 protein is the opposite of the redox regulation reported for other mammalian transcription factors such as Fos, Jun, USF, NF-kappa B, c-Myb, and v-Rel, in which oxidation of cysteine residues inhibits DNA binding. Thus, specific oxidation of nuclear proteins is a potential regulatory mechanism that can act to either decrease or increase their DNA binding activity.

scholarly journals Cell-Type-Dependent Activity of the Ubiquitous Transcription Factor USF in Cellular Proliferation and Transcriptional Activation

1999 ◽  
Vol 19 (2) ◽  
pp. 1508-1517 ◽  
Author(s):  
Yibing Qyang ◽  
Xu Luo ◽  
Tao Lu ◽  
Preeti M. Ismail ◽  
Dmitry Krylov ◽  
...  
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Transcriptional Activity ◽  
Cellular Proliferation ◽  
Mutational Analysis ◽  
Growth Control ◽  
Binding Activity ◽  
Dual Function ◽  
Osteosarcoma Cell ◽  
Dna Binding Activity

ABSTRACT USF1 and USF2 are basic helix-loop-helix transcription factors implicated in the control of cellular proliferation. In HeLa cells, the USF proteins are transcriptionally active and their overexpression causes marked growth inhibition. In contrast, USF overexpression had essentially no effect on the proliferation of the Saos-2 osteosarcoma cell line. USF1 and USF2 also lacked transcriptional activity in Saos-2 cells when assayed by transient cotransfection with USF-dependent reporter genes. Yet, there was no difference in the expression, subcellular localization, or DNA-binding activity of the USF proteins in HeLa and Saos-2 cells. Furthermore, Gal4-USF1 and Gal4-USF2 fusion proteins activated transcription similarly in both cell lines. Mutational analysis and domain swapping experiments revealed that the small, highly conserved USF-specific region (USR) was responsible for the inactivity of USF in Saos-2 cells. In HeLa, the USR serves a dual function. It acts as an autonomous transcriptional activation domain at promoters containing an initiator element and also induces a conformational change that is required for USF activity at promoters lacking an initiator. Taken together, these results suggest a model in which the transcriptional activity of the USF proteins, and consequently their antiproliferative activity, is tightly controlled by interaction with a specialized coactivator that recognizes the conserved USR domain and, in contrast to USF, is not ubiquitous. The activity of USF is therefore context dependent, and evidence for USF DNA-binding activity in particular cells is insufficient to indicate USF function in transcriptional activation and growth control.

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