Mutational Analyses of Aquifex pyrophilus DNA Ligase Define Essential Domains for Self-Adenylation and DNA Binding Activity

2001 ◽  
Vol 388 (2) ◽  
pp. 253-260 ◽  
Author(s):  
Jae-Hwan Lim ◽  
Juhyun Choi ◽  
Wookhyun Kim ◽  
Byung-Yoon Ahn ◽  
Ye Sun Han
Keyword(s):  
Dna Binding ◽  
Binding Activity ◽  
Dna Ligase ◽  
Dna Binding Activity ◽  
Aquifex Pyrophilus

scholarly journals Cloning and Functional Characterization of an NAD+-Dependent DNA Ligase from Staphylococcus aureus

2001 ◽  
Vol 183 (10) ◽  
pp. 3016-3024 ◽  
Author(s):  
Frank S. Kaczmarek ◽  
Richard P. Zaniewski ◽  
Thomas D. Gootz ◽  
Dennis E. Danley ◽  
Mahmoud N. Mansour ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Amino Acid ◽  
Dna Binding ◽  
Functional Characterization ◽  
Limited Proteolysis ◽  
Binding Activity ◽  
Dna Ligase ◽  
E Coli ◽  
Dna Binding Activity ◽  
C Terminus

ABSTRACT A Staphylococcus aureus mutant conditionally defective in DNA ligase was identified by isolation of complementing plasmid clones that encode the S. aureus ligA gene. Orthologues of the putative S. aureus NAD+-dependent DNA ligase could be identified in the genomes of Bacillus stearothermophilus and other gram-positive bacteria and confirmed the presence of four conserved amino acid motifs, including motif I, KXDG with lysine 112, which is believed to be the proposed site of adenylation. DNA sequence comparison of the ligA genes from wild type and temperature-sensitive S. aureus strain NT64 identified a single base alteration that is predicted to result in the amino acid substitution E46G. The S. aureus ligA gene was cloned and overexpressed in Escherichia coli, and the enzyme was purified to near homogeneity. NAD+-dependent DNA ligase activity was demonstrated with the purified enzyme by measuring ligation of 32P-labeled 30-mer and 29-mer oligonucleotides annealed to a complementary strand of DNA. Limited proteolysis of purified S. aureus DNA ligase by thermolysin produced products with apparent molecular masses of 40, 22, and 21 kDa. The fragments were purified and characterized by N-terminal sequencing and mass analysis. The N-terminal fragment (40 kDa) was found to be fully adenylated. A fragment from residues 1 to 315 was expressed as a His-tagged fusion in E. coli and purified for functional analysis. Following deadenylation with nicotinamide mononucleotide, the purified fragment could self-adenylate but lacked detectable DNA binding activity. The 21- and 22-kDa C-terminal fragments, which lacked the last 76 amino acids of the DNA ligase, had no adenylation activity or DNA binding activity. The intact 30-kDa C terminus of the S. aureus LigA protein expressed in E. coli did demonstrate DNA binding activity. These observations suggest that, as in the case with the NAD+-dependent DNA ligase fromB. stearothermophilus, two independent functional domains exist in S. aureus DNA ligase, consisting of separate adenylation and DNA binding activities. They also demonstrate a role for the extreme C terminus of the ligase in DNA binding. As there is much evidence to suggest that DNA ligase is essential for bacterial survival, its discovery in the important human pathogen S. aureus indicates its potential as a broad-spectrum antibacterial target for the identification of novel antibiotics.

scholarly journals Structure of the p53/RNA polymerase II assembly

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Shu-Hao Liou ◽  
Sameer K. Singh ◽  
Robert H. Singer ◽  
Robert A. Coleman ◽  
Wei-Li Liu
Keyword(s):  
Dna Binding ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Conformational Changes ◽  
P53 Protein ◽  
Binding Activity ◽  
Transactivation Domain ◽  
Pol Ii ◽  
Dna Binding Activity ◽  
Regulated Gene Expression

AbstractThe tumor suppressor p53 protein activates expression of a vast gene network in response to stress stimuli for cellular integrity. The molecular mechanism underlying how p53 targets RNA polymerase II (Pol II) to regulate transcription remains unclear. To elucidate the p53/Pol II interaction, we have determined a 4.6 Å resolution structure of the human p53/Pol II assembly via single particle cryo-electron microscopy. Our structure reveals that p53’s DNA binding domain targets the upstream DNA binding site within Pol II. This association introduces conformational changes of the Pol II clamp into a further-closed state. A cavity was identified between p53 and Pol II that could possibly host DNA. The transactivation domain of p53 binds the surface of Pol II’s jaw that contacts downstream DNA. These findings suggest that p53’s functional domains directly regulate DNA binding activity of Pol II to mediate transcription, thereby providing insights into p53-regulated gene expression.

scholarly journals Abundance Changes of the Response Regulator RcaC Require Specific Aspartate and Histidine Residues and Are Necessary for Normal Light Color Responsiveness

2008 ◽  
Vol 190 (21) ◽  
pp. 7241-7250 ◽  
Author(s):  
Lina Li ◽  
David M. Kehoe
Keyword(s):  
Dna Binding ◽  
Dynamic Range ◽  
Response Regulator ◽  
Red Light ◽  
Green Light ◽  
Binding Activity ◽  
Transcriptional Responses ◽  
Dna Binding Activity ◽  
Normal Light ◽  
Light Color

ABSTRACT RcaC is a large, complex response regulator that controls transcriptional responses to changes in ambient light color in the cyanobacterium Fremyella diplosiphon. The regulation of RcaC activity has been shown previously to require aspartate 51 and histidine 316, which appear to be phosphorylation sites that control the DNA binding activity of RcaC. All available data suggest that during growth in red light, RcaC is phosphorylated and has relatively high DNA binding activity, while during growth in green light RcaC is not phosphorylated and has less DNA binding activity. RcaC has also been found to be approximately sixfold more abundant in red light than in green light. Here we demonstrate that the light-controlled abundance changes of RcaC are necessary, but not sufficient, to direct normal light color responses. RcaC abundance changes are regulated at both the RNA and protein levels. The RcaC protein is significantly less stable in green light than in red light, suggesting that the abundance of this response regulator is controlled at least in part by light color-dependent proteolysis. We provide evidence that the regulation of RcaC abundance does not depend on any RcaC-controlled process but rather depends on the presence of the aspartate 51 and histidine 316 residues that have previously been shown to control the activity of this protein. We propose that the combination of RcaC abundance changes and modification of RcaC by phosphorylation may be necessary to provide the dynamic range required for transcriptional control of RcaC-regulated genes.

Differential use of SCL/TAL-1 DNA-binding domain in developmental hematopoiesis

Blood ◽  
2008 ◽  
Vol 112 (4) ◽  
pp. 1056-1067 ◽  
Author(s):  
Mira T. Kassouf ◽  
Hedia Chagraoui ◽  
Paresh Vyas ◽  
Catherine Porcher
Keyword(s):  
Dna Binding ◽  
Molecular Mechanisms ◽  
Binding Activity ◽  
Hematopoietic Stem ◽  
Helix Loop Helix ◽  
Experimental Systems ◽  
Dna Binding Activity ◽  
Independent Functions

Abstract Dissecting the molecular mechanisms used by developmental regulators is essential to understand tissue specification/differentiation. SCL/TAL-1 is a basic helix-loop-helix transcription factor absolutely critical for hematopoietic stem/progenitor cell specification and lineage maturation. Using in vitro and forced expression experimental systems, we previously suggested that SCL might have DNA-binding–independent functions. Here, to assess the requirements for SCL DNA-binding activity in vivo, we examined hematopoietic development in mice carrying a germline DNA-binding mutation. Remarkably, in contrast to complete absence of hematopoiesis and early lethality in scl-null embryos, specification of hematopoietic cells occurred in homozygous mutant embryos, indicating that direct DNA binding is dispensable for this process. Lethality was forestalled to later in development, although some mice survived to adulthood. Anemia was documented throughout development and in adulthood. Cellular and molecular studies showed requirements for SCL direct DNA binding in red cell maturation and indicated that scl expression is positively autoregulated in terminally differentiating erythroid cells. Thus, different mechanisms of SCL's action predominate depending on the developmental/cellular context: indirect DNA binding activities and/or sequestration of other nuclear regulators are sufficient in specification processes, whereas direct DNA binding functions with transcriptional autoregulation are critically required in terminal maturation processes.

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