The internal dynamics of gene 32 protein-DNA complexes studied by quasi-elastic light scattering

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The complex formed between adenovirus DNA-binding protein (AdDBP) and poly(rA) was investigated with circular dichroism spectroscopy. The binding process was studied at a variety of salt concentrations, and the titration curves were analyzed according to the contiguous cooperative binding model given by McGhee and von Hippel [McGhee, J.D., & von Hippel, P.H. (1974) J. Mol. Biol. 86, 469-489]. The cooperativity factor omega of the binding process is low (omega approximately 20-30) and independent of the salt concentration. This in contrast to the binding constant K for which a moderately strong salt dependence is observed: delta log (K omega)/delta log [NaCl] = -3.1. The size of the binding site was consistently calculated to be about 13. We also studied the C-terminal 39-kDa fragment which is sufficient for DNA replication in vitro. Complex formation between this fragment of AdDBP and poly(rA) appeared to be characterized by spectroscopic and binding properties similar to those of the intact protein. Only, the binding constant in 50 mM NaCl is a factor of 5 lower.

Section: Resultssupporting

confidence: 71%

Section: Methodsmentioning

confidence: 99%

Complex formation between the adenovirus DNA-binding protein and single-stranded poly(rA). Cooperativity and salt dependence

Kuil

Amerongen²,

Vliet³

et al. 1989

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“…nm(Kuil et al, 1988(Kuil et al, ,1990). An increase of the base-base distance to 0.47 nm was observed by Delius using electron microscopy for GP32 binding to fd DNA(Delius et al, 1972).…”

mentioning

confidence: 99%

Study of the binding of single-stranded DNA-binding protein to DNA and poly(rA) using electric field induced birefringence and circular dichroism spectroscopy

Kuil¹,

Holmlund²,

Vlaanderen³

et al. 1990

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Binding of the single-stranded DNA-binding protein (SSB) of Escherichia coli to single-stranded (ss) polynucleotides produces characteristic changes in the absorbance (OD) and circular dichroism (CD) spectra of the polynucleotides. By use of these techniques, complexes of SSB protein and poly(rA) were shown to display two of the binding modes reported by Lohman and Overman [Lohman, T.M., & Overman, L. (1985) J. Biol. Chem. 260, 3594-3603]. The circular dichroism spectra of the "low salt" (10 mM NaCl) and "high salt" (greater than 50 mM NaCl) binding mode are similar in shape, but not in intensity. SSB binding to poly(rA) yields a complexed CD spectrum that shares several characteristics with the spectra obtained for the binding of AdDBP, GP32, and gene V protein to poly(rA). We therefore propose that the local structure of the SSB-poly(rA) complex is comparable to the structures proposed for the complexes of these three-stranded DNA-binding proteins with DNA (and RNA) and independent of the SSB-binding mode. Electric field induced birefringence experiments were used to show that the projected base-base distance of the complex is about 0.23 nm, in agreement with electron microscopy results. Nevertheless, the local distance between the successive bases in the complex will be quite large, due to the coiling of the DNA around the SSB tetramer, thus partly explaining the observed CD changes induced upon complexation with single-stranded DNA and RNA.

“…There has been much discussion about the size of the binding site («) and values obtained range from 5 to 11 nucleotides per protein, but it has been argued that inactivation of a fraction of the protein has caused the lower values (Bobst et al, 1982;Scheerhagen et al, 1986b). Minimizing the contribution of inactive protein during the determination of the site size led to values for n close to 10 (Bobst et al, 1982;Scheerhagen et al, 1986b;Kuil et al, 1988). In addition, Watanabe (1989) reported that in the analysis of a titration experiment the cooperativity can easily be overestimated, which could explain the lower values of n. Reanalysis of titration curves yielded a value of = 9 for the binding of GP32 to poly(rA), in agreement with our recent results (Kuil et al, 1989).…”

mentioning

confidence: 99%

“…Since at that time the interpretation of the hydrodynamic measurements in terms of flexibility seemed difficult, Scheerhagen chose to model the complex as a regular superhelix, where the local complex axis at every position in the complex has the same orientation with respect to the overall axis (superhelix axis). Recently, hydrodynamic studies (Kuil et al, 1988(Kuil et al, , 1990 were undertaken with larger fragments of single-stranded DNA with the aim of obtaining more specific data about the flexibility of the GP32-single- stranded DNA complex. It appeared that the flexibility of the complex is comparable to and probably even higher than that of double-stranded DNA.…”

mentioning

confidence: 99%

Structure calculations for single-stranded DNA complexed with the single-stranded DNA binding protein GP32 of bacteriophage T4: a remarkable DNA structure

Amerongen¹,

Kuil²,

Scheerhagen³

et al. 1990

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In this study it is established by calculation which regular conformations single-stranded DNA and RNA can adopt in the complex with the single-stranded DNA binding protein GP32 of bacteriophage T4. In order to do so, information from previous experiments about base orientations and the length and diameter of the complexes is used together with knowledge about bond lengths and valence angles between chemical bonds. It turns out that there is only a limited set of similar conformations which are in agreement with experimental data. The arrangement of neighboring bases is such that there is ample space for aromatic residues of the protein to partly intercalate between the bases, which is in agreement with a previously proposed model for the binding domain of the protein [Prigodich, R. V., Shamoo, Y., Williams, K. R., Chase, J. W., Konigsberg, W. H., & Coleman, J. E. (1986) Biochemistry 25, 3666-3671]. Both C2'endo and C3'endo sugar conformations lead to calculated DNA conformations that are consistent with experimental data. The orientation of the O2' atoms of the sugars in RNA can explain why the binding affinity of GP32 for polyribonucleotides is lower than for polydeoxyribonucleotides.