Secondary structure creates mismatched base pairs required for high-affinity binding of cAMP response element-binding protein to the human enkephalin enhancer.

Spiro, Craig; Richards, Jane P.; Чандрасекаран, С.; Brennan, Richard G.; McMurray, Cynthia T.

doi:10.1073/pnas.90.10.4606

Cited by 47 publications

(76 citation statements)

References 19 publications

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“…The existence of proteins whose interaction with DNA is based on conformation ± but not sequence-speci®c ± recognition has been documented (Bianchi et al, 1988;Herbert et al, 1993;Leith and Russel, 1993;Pearson et al, 1995;Solaro et al, 1995). Transcriptional regulation of many genes involves conformational changes in DNA (Delic et al, 1991;Hanke et al, 1995;Iyer and Struhl, 1995;Spiro et al, 1995;Michelotti et al, 1996), and binding of some transcription factors depends on the conformation of DNA within or adjacent to their binding sites (Grigoriev et al, 1992;Spiro et al, 1993;Hu et al, 1994;May®eld and Miller, 1994). At the level of the p53 protein, modi®cations of the p53 carboxy-terminal regulatory domain either by posttranslational modifications, alternative splicing (Kulesz-Martin et al, 1994;Flaman et al, 1996), or by binding of proteins mimicking the e ect of PAb421 binding would provide another level for determining the speci®city for transactivation of the ever growing list of potential p53 target genes.…”

Section: Discussionmentioning

confidence: 99%

DNA-conformation is an important determinant of sequence-specific DNA binding by tumor suppressor p53

1997

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Sequence-speci®c transactivation of target genes is one of the most important molecular properties of the tumor suppressor p53. Binding of p53 to its target DNAs is tightly regulated, with modi®cations in the carboxyterminal regulatory domain of the p53 protein playing an important role. In this study we examined the possible in¯uence of DNA structure on sequence-speci®c DNA binding by p53, by analysing its binding to p53 consensus elements adopting di erent conformations. We found that p53 has the ability to bind to consensus elements which are present in a double-helical form, as well as to consensus elements which are located within alternative non-B-DNA structures. The ability of a consensus element to adopt either one of these conformations is dependent on its sequence symmetry, and is strongly in¯uenced by its sequence environment. Our data suggest a model according to which the conformational status of the target DNA is an important determinant for sequence-speci®c DNA binding by p53. Modi®cations in the carboxy-terminal regulatory region of p53 possibly determine the preference of p53 for a given DNA conformation.

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Section: Discussionmentioning

confidence: 99%

DNA-conformation is an important determinant of sequence-specific DNA binding by tumor suppressor p53

1997

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“…Promoters (2,3), terminators (4), amplified genes (5), and origins of DNA replication from prokaryotes (6 -8), viruses (9), and eukaryotes (10) all contain IRs. Origins of DNA replication from higher eukaryotes, such as monkeys and humans (11)(12)(13)(14)(15), are also enriched in IRs.…”

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confidence: 99%

The Human Cruciform-binding Protein, CBP, Is Involved in DNA Replication and Associates in Vivo with Mammalian Replication Origins

Novac

Álvarez

Pearson

et al. 2002

Journal of Biological Chemistry

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We previously identified and purified from human (HeLa) cells a 66-kDa cruciform-binding protein, CBP, with binding specificity for cruciform DNA regardless of its sequence. DNA cruciforms have been implicated in the regulation of initiation of DNA replication. CBP is a member of the 14-3-3 family of proteins, which are conserved regulatory molecules expressed in all eukaryotes. Here, the in vivo association of CBP/14-3-3 with mammalian origins of DNA replication was analyzed by studying its association with the monkey replication origins ors8 and ors12, as assayed by a chromatin immunoprecipitation assay and quantitative PCR analysis. The association of the 14-3-3␤, -⑀, -␥, andisoforms with these origins was found to be ϳ9-fold higher, compared with other portions of the genome, in logarithmically growing cells. In addition, the association of these isoforms with ors8 and ors12 was also analyzed as a function of the cell cycle. Higher binding of 14-3-3␤, -⑀, -␥, and -isoforms with ors8 and ors12 was found at the G 1 /S border, by comparison with other stages of the cell cycle. The CBP/14-3-3 cruciform binding activity was also found to be maximal at the G 1 /S boundary. The involvement of 14-3-3 in mammalian DNA replication was analyzed by studying the effect of anti-14-3-3␤, -⑀, -␥, and -antibodies in the in vitro replication of p186, a plasmid containing the minimal replication origin of ors8. Anti-14-3-3⑀, -␥, and -antibodies alone or in combination inhibited p186 replication by ϳ50 -80%, while anti-14-3-3␤ antibodies had a lesser effect (ϳ25-50%). All of the antibodies tested were also able to interfere with CBP binding to cruciform DNA. The results indicate that CBP/14-3-3 is an origin-binding protein, acting at the initiation step of DNA replication by binding to cruciform-containing molecules, and dissociates after origin firing.Inverted repeat sequences (IRs) 1 are a common feature of prokaryotic and eukaryotic regulatory regions, and their distribution is nonrandom and clustered (1). Promoters (2, 3), terminators (4), amplified genes (5), and origins of DNA replication from prokaryotes (6 -8), viruses (9), and eukaryotes (10) We and others have previously demonstrated the involvement of cruciform structures in the initiation of mammalian DNA replication (20, 23-25) (reviewed in Ref. 16) We previously identified and isolated from human cells (HeLa) a cruciform-specific binding protein, CBP (26), of 66 kDa with binding specificity for cruciform-containing DNA (26). CBP binds at the base of four-way junctions (27), interacting with them in a different manner from other proteins known to bind such junctions (26,27).CBP is a member of the 14-3-3 protein family, a highly conserved family (28) of proteins through plants, invertebrates, and higher eukaryotes (reviewed in Ref. 29). 14-3-3 proteins are multifunctional, involved in diverse cellular processes, such as neurotransmitter biosynthesis, signal transduction pathways, and cell cycle control (reviewed in Ref. 30). 14-3-3-associated proteins include rec...

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“…We know that these non-specific electrostatic interactions are occurring primarily within the CREB bZIP domain, since the absorbance and elution profile from the size-exclusion column of CREB 254-327 is virtually identical to that of full-length CREB. Furthermore, CREB has been shown to bind with high affinity to hairpin binding sites [20], which may account for the tight association of the bZIP domain with contaminating tRNA. These observations raise the question as to how universal contaminating nucleic acid may be in purified preparations of proteins that have DNA binding activity.…”

Section: Discussionmentioning

confidence: 99%

Purification of CREB to apparent homogeneity: Removal of truncation products and contaminating nucleic acid

Lopez¹,

Mick²,

Nyborg³

2007

Protein Expression and Purification

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The cAMP response element binding protein (CREB) is a mammalian transcription factor which regulates the expression of many cellular genes. CREB is commonly expressed in E. coli and purified by heat-extraction followed by affinity chromatography. We have discovered that although this purification yields a reasonably pure product which is active in DNA-binding and functional assays, it contains a large amount of nucleic acid as well as CREB truncation products and other polypeptides. Consequently, this CREB is inadequate for use in biophysical studies including crystallography, and spectroscopic analysis such as analytical ultracentrifugation, FRET, and circular dichroism. We revised the purification protocol to incorporate expression in the Rosetta™ host strain, nuclease treatment, and denaturing/high salt size-exclusion chromatography. We typically obtain 10 mg of CREB per liter of culture media that is 99% homogenous, free of nucleic acid, and amenable to biophysical studies. Comparison of CREB from the original and revised protocols shows similar affinities for the cAMP response element (CRE) but small differences in their secondary structures when assayed by limited proteolysis and circular dichroism.

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Secondary structure creates mismatched base pairs required for high-affinity binding of cAMP response element-binding protein to the human enkephalin enhancer.

Cited by 47 publications

References 19 publications

DNA-conformation is an important determinant of sequence-specific DNA binding by tumor suppressor p53

DNA-conformation is an important determinant of sequence-specific DNA binding by tumor suppressor p53

The Human Cruciform-binding Protein, CBP, Is Involved in DNA Replication and Associates in Vivo with Mammalian Replication Origins

Purification of CREB to apparent homogeneity: Removal of truncation products and contaminating nucleic acid

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