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Regulation of Nearest-Neighbor Cooperative Binding of E. coli SSB Protein to DNA.

ABSTRACT: Escherichia coli single-strand (ss) DNA-binding protein (SSB) is an essential protein that binds ssDNA intermediates formed during genome maintenance. SSB homotetramers bind ssDNA in several modes differing in occluded site size and cooperativity. The 35-site-size ((SSB)35) mode favored at low [NaCl] and high SSB/DNA ratios displays high "unlimited" nearest-neighbor cooperativity (?35), forming long protein clusters, whereas the 65-site-size ((SSB)65) mode in which ssDNA wraps completely around the tetramer is favored at higher [NaCl] (>200 mM) and displays "limited" cooperativity (?65), forming only dimers of tetramers. In addition, a non-nearest-neighbor high cooperativity can also occur in the (SSB)65 mode on long ssDNA even at physiological salt concentrations in the presence of glutamate and requires its intrinsically disordered C-terminal linker (IDL) region. However, whether cooperativity exists between the different modes and the role of the IDL in nearest-neighbor cooperativity has not been probed. Here, we combine sedimentation velocity and fluorescence titration studies to examine nearest-neighbor cooperativity in each binding mode and between binding modes using (dT)70 and (dT)140. We find that the (SSB)35 mode always shows extremely high "unlimited" cooperativity that requires the IDL. At high salt, wild-type SSB and a variant without the IDL, SSB-?L, bind in the (SSB)65 mode but show little cooperativity, although cooperativity increases at lower [NaCl] for wild-type SSB. We also find significant intermode nearest-neighbor cooperativity (?65/35), with ?65 ? ?65/35

SUBMITTER: Kozlov AG

PROVIDER: S-EPMC6895745 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Regulation of Nearest-Neighbor Cooperative Binding of E. coli SSB Protein to DNA.

Kozlov Alexander G AG Shinn Min Kyung MK Lohman Timothy M TM

Biophysical journal 20191028 11

Escherichia coli single-strand (ss) DNA-binding protein (SSB) is an essential protein that binds ssDNA intermediates formed during genome maintenance. SSB homotetramers bind ssDNA in several modes differing in occluded site size and cooperativity. The 35-site-size ((SSB)35) mode favored at low [NaCl] and high SSB/DNA ratios displays high "unlimited" nearest-neighbor cooperativity (ω35), forming long protein clusters, whereas the 65-site-size ((SSB)65) mode in whi ...[more]

PMID: 31708161

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Project description:BackgroundThe enumeration of tetrameric and other sequence motifs that are positively or negatively correlated with in vivo antisense DNA effects has been a useful addition to the arsenal of information needed to predict effective targets for antisense DNA control of gene expression. Such retrospective information derived from in vivo cellular experiments characterizes aspects of the sequence dependence of antisense inhibition that are not predicted by nearest-neighbor (NN) thermodynamic parameters derived from in vitro experiments. However, quantitation of the antisense contributions of motifs is problematic, since individual motifs are not isolated from the effects of neighboring nucleotides, and motifs may be overlapping. These problems are circumvented by a next-nearest-neighbor (NNN) analysis of antisense DNA effects in which the overlapping nature of nearest-neighbors is taken into account.ResultsNext-nearest-neighbor triplet combinations of nucleotides are the simplest that include overlapping sequence effects and therefore can encompass interactions beyond those of nearest neighbors. We used singular value decomposition (SVD) to fit experimental data from our laboratory in which phosphorothioate-modified antisense DNAs (S-DNAs) 20 nucleotides long were used to inhibit cellular protein expression in 112 experiments involving four gene targets and two cell lines. Data were fitted using a NNN model, neglecting end effects, to derive NNN inhibition parameters that could be combined to give parameters for a set of 49 sequences that represents the inhibitory effects of all possible overlapping triplet interactions in the cellular targets of these antisense S-DNAs. We also show that parameters to describe subsets of the data, such as the mRNAs being targeted and the cell lines used, can be included in such a derivation. While NNN triplet parameters provided an adequate model to fit our data, NN doublet parameters did not.ConclusionsThe methodology presented illustrates how NNN antisense inhibitory information can be derived from in vivo cellular experiments. Subsequent calculations of the antisense inhibitory parameters for any mRNA target sequence automatically take into account the effects of all possible overlapping combinations of nearest-neighbors in the sequence. This procedure is more robust than the tallying of tetrameric motifs that have positive or negative antisense effects. The specific parameters derived in this work are limited in their applicability by the relatively small database of experiments that was used in their derivation.

Dataset Information

Regulation of Nearest-Neighbor Cooperative Binding of E. coli SSB Protein to DNA.

Publications

Regulation of Nearest-Neighbor Cooperative Binding of E. coli SSB Protein to DNA.

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