Sequence-Specific Binding to Telomeric DNA Is Not a Conserved Property of the Cdc13 DNA Binding Domain

Biochemistry ◽  
2011 ◽  
Vol 50 (29) ◽  
pp. 6289-6291 ◽  
Author(s):  
Edward K. Mandell ◽  
Amy D. Gelinas ◽  
Deborah S. Wuttke ◽  
Victoria Lundblad
Keyword(s):  
Dna Binding ◽  
Specific Binding ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Telomeric Dna

scholarly journals Radiation-induced oxidative damage to the DNA-binding domain of the lactose repressor

2007 ◽  
Vol 403 (3) ◽  
pp. 463-472 ◽  
Author(s):  
Nathalie Gillard ◽  
Stephane Goffinont ◽  
Corinne Buré ◽  
Marie Davidkova ◽  
Jean-Claude Maurizot ◽  
...  
Keyword(s):  
Dna Binding ◽  
Conformational Changes ◽  
Specific Binding ◽  
Classical Model ◽  
Binding Domain ◽  
Oh Radicals ◽  
Water Radiolysis ◽  
Dna Binding Domain ◽  
Fluorescence Measurements ◽  
Radiation Induced

Understanding the cellular effects of radiation-induced oxidation requires the unravelling of key molecular events, particularly damage to proteins with important cellular functions. The Escherichia coli lactose operon is a classical model of gene regulation systems. Its functional mechanism involves the specific binding of a protein, the repressor, to a specific DNA sequence, the operator. We have shown previously that upon irradiation with γ-rays in solution, the repressor loses its ability to bind the operator. Water radiolysis generates hydroxyl radicals (OH· radicals) which attack the protein. Damage of the repressor DNA-binding domain, called the headpiece, is most likely to be responsible of this loss of function. Using CD, fluorescence spectroscopy and a combination of proteolytic cleavage with MS, we have examined the state of the irradiated headpiece. CD measurements revealed a dose-dependent conformational change involving metastable intermediate states. Fluorescence measurements showed a gradual degradation of tyrosine residues. MS was used to count the number of oxidations in different regions of the headpiece and to narrow down the parts of the sequence bearing oxidized residues. By calculating the relative probabilities of reaction of each amino acid with OH· radicals, we can predict the most probable oxidation targets. By comparing the experimental results with the predictions we conclude that Tyr7, Tyr12, Tyr17, Met42 and Tyr47 are the most likely hotspots of oxidation. The loss of repressor function is thus correlated with chemical modifications and conformational changes of the headpiece.

scholarly journals The Crystal Structure of the DNA-Binding Domain of Yeast RAP1 in Complex with Telomeric DNA

Cell ◽  
1996 ◽  
Vol 85 (1) ◽  
pp. 125-136 ◽  
Author(s):  
Peter König ◽  
Rafael Giraldo ◽  
Lynda Chapman ◽  
Daniela Rhodes
Keyword(s):  
Crystal Structure ◽  
Dna Binding ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Telomeric Dna

scholarly journals Specific Binding of Integrase to the Origin of Transfer (oriT) of the Conjugative Transposon Tn916

2001 ◽  
Vol 183 (9) ◽  
pp. 2947-2951 ◽  
Author(s):  
Douglas Hinerfeld ◽  
Gordon Churchward
Keyword(s):  
Dna Binding ◽  
Specific Binding ◽  
Binding Domain ◽  
Conjugal Transfer ◽  
Dna Binding Domain ◽  
Conjugative Transposon ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
A Site ◽  
Integrase Protein

ABSTRACT Purified integrase protein (Int) of the conjugative transposon Tn916 was shown, using nuclease protection experiments, to bind specifically to a site within the origin of conjugal transfer of the transposon, oriT. A sequence similar to the ends of the transposon that are bound by the C-terminal DNA-binding domain of Int was present in the protected region. However, Int binding tooriT required both the N- and C-terminal DNA-binding domains of Int, and the pattern of nuclease protection differed from that observed when Int binds to the transposon ends and flanking DNA. Binding of Int to oriT may be part of a mechanism to prevent premature conjugal transfer of Tn916 prior to excision from the donor DNA.

scholarly journals Functional Dissection of Human and Mouse POT1 Proteins

2008 ◽  
Vol 29 (2) ◽  
pp. 471-482 ◽  
Author(s):  
Wilhelm Palm ◽  
Dirk Hockemeyer ◽  
Tatsuya Kibe ◽  
Titia de Lange
Keyword(s):  
Dna Damage ◽  
Dna Binding ◽  
Dna Damage Response ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Telomeric Dna ◽  
C Terminus ◽  
Damage Response ◽  
End Resection

ABSTRACT The single-stranded telomeric DNA binding protein POT1 protects mammalian chromosome ends from the ATR-dependent DNA damage response, regulates telomerase-mediated telomere extension, and limits 5′-end resection at telomere termini. Whereas most mammals have a single POT1 gene, mice have two POT1 proteins that are functionally distinct. POT1a represses the DNA damage response, and POT1b controls 5′-end resection. In contrast, as we report here, POT1a and POT1b do not differ in their ability to repress telomere recombination. By swapping domains, we show that the DNA binding domain of POT1a specifies its ability to repress the DNA damage response. However, no differences were detected in the in vitro DNA binding features of POT1a and POT1b. In contrast to the repression of ATR signaling by POT1a, the ability of POT1b to control 5′-end resection was found to require two regions in the C terminus, one corresponding to the TPP1 binding domain and a second representing a new domain located between amino acids (aa) 300 and 350. Interestingly, the DNA binding domain of human POT1 can replace that of POT1a to repress ATR signaling, and the POT1b region from aa 300 to 350 required for the regulation of the telomere terminus is functionally conserved in human POT1. Thus, human POT1 combines the features of POT1a and POT1b.

scholarly journals Structure of the DNA-binding domain of human telomeric protein, TRF1 and its interaction with telomeric DNA

2001 ◽  
Vol 1 (1) ◽  
pp. 273-274 ◽  
Author(s):  
T. Nishikawa ◽  
H. Okamura ◽  
A. Nagadoi ◽  
P. Koig ◽  
D. Rhodes ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Telomeric Dna ◽  
Telomeric Protein

UV photoaffinity labeling of Tn3 transposase–DNA complexes: identification of DNA binding domains

1996 ◽  
Vol 42 (1) ◽  
pp. 46-59
Author(s):  
Geoffrey S. Gottlieb ◽  
Michael A. Fennewald
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Specific Binding ◽  
Binding Domain ◽  
Target Site ◽  
Site Directed Mutagenesis ◽  
Cross Linking ◽  
Dna Binding Domain ◽  
Transposition Frequency ◽  
Terminal Domain

The prokaryotic transposon Tn3 requires the transposase protein, as well as the cis-acting terminal inverted repeats (IRs), for transposition. The first step in the transposition process requires transposase binding to the IRs, as well as target site selection for element insertion. The primary aim of this study is to define the relationship between the structure of Tru3 transposase and its DNA binding functions. We have defined, by UV cross-linking, two broad regions of transposase that interact with DNA: a 70-kDa N-terminal domain and a 30-kDa C-terminal domain. The 70-kDa N-terminal domain encompasses the IR sequence-specific binding domain, as well as a nonspecific DNA binding domain that has been previously described. We have also defined, by UV cross-linking, a region in the nonspecific DNA binding domain centered at amino acids 376 and 381 that is in contact with DNA. We have used site-directed mutagenesis of amino acids 376 and 381 to help delineate the function of this region of the transposase protein. Mutations in this region reduce transposition frequency to 30–40% of the wild type. These mutations reduce nonspecific DNA binding three- to four-fold but do not appear to affect specific binding to the IR. Transposition immunity is unaffected by mutations in the nonspecific DNA binding domain. This suggests that this region may be involved in target site selection.Key words: transposon, Tn3, DNA–protein cross-linking, UV cross-linking, transposase.

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