SSB protein

The low torsion constant at a = —0.025 is very similar to that observed in a supercoiled pBR322 that was partially relaxed by saturation binding of Escherichia coli single-strand binding (ssb) protein, and which persisted for over a month.(56) It is also similar to that recently inferred from an in vivo assay based on variation in repression efficiency with size of a putative DNA loop.(234) Indeed, it appears that anomalously low torsion constants may be universally encountered in the course of either partial or complete relaxation of supercoiled DNAs, regardless of whether the superhelix density is reduced by action of topoisomerase I, binding of ssb protein, binding of intercalated... 

Fig. 9 Electropherograms showing 3 nM fluorescently labeled 11-mer in the absence (A) and in the presence (B) of 0.7 /j,M SSB protein in the running buffer. The conditions used were as follows separation capillary, 35 cm, 20-/xm i.d. running buffer, 25 mM disodium tetraborate (pH 9.1) separation voltage, 25 kV excitation wavelength, 488 nm emission wavelength, 515 nm and temperature, 25 (1°C. Approximately 1 nL of sample solution was injected electrokinetically. The asterisk indicates the migration time of the solvent, The traces Iv and Ih, corresponding to vertically and horizontally polarized fluorescence intensities, respectively, are shown separated for clarity. (From Ref. 48.)...

Fig. 10 Binding curves obtained by measuring the electrophoretic mobility ( ) and fluorescence anisotropy (o) of F-ll-mer at varying concentrations of SSB protein in the running buffer. Conditions were the same as for Figure 9. (From Ref. 48.)...

Two other protein complexes also function in eukaryotic DNA replication. RPA (replication protein A) is a eukaryotic single-stranded DNA-binding protein, equivalent in function to the E. coli SSB protein. RFC (replication/actor (J) is a clamp loader for PCNA and facilitates the assembly of active replication complexes. The subunits of the RFC complex have significant sequence similarity to the subunits of the bacterial clamploading (y) complex. 

The E. coli single-strand binding protein, another helix-destabilizing protein that is usually called simply SSB, is a tetramer of 18.5-kDa subunits.265,3,2,313 It is essential to DNA replication. About 35 nucleotides may bind to each tetramer.314 The situation is not as clear in eukaryotes where DNA is largely coiled around histones in the nucleosomes. Several singlestrand binding proteins have been identified,315 but the need for SSB proteins in eukaryotic nuclear replication is uncertain.316 A human mitochondrial SSB resembles that of E. coli.317... 

The SSPsubfamily of Hsp70 in S. cerevisiaehas also recently been implicated in [PSI metabolism (Chemoff et al, 1999). This group consists of two members, Ssbl and Ssb2, which have identical amino acid sequences except at four positions (Boorstein et al., 1994). SSB proteins are not... 

Helicases (Figure 24.28)- SSB proteins do not actively denature DNA and cannot actively unwind duplex DNA strands. Nevertheless, unwinding of this kind is essential if single-strand templates are to be exposed for polymerase action. The helicase proteins... 

Tyrosine residues can also be used to describe the structoe and the dynamics of a protein. Single stranded DNA binding protein modulates DNA replication and repair, transcription, translation, regulation and recombination. However, while most of SSB proteins activate DNA replication, e.g. Escherichia coli SSB and 029 SSB, some SSBs such as those of filamentous phages Ml 3, are inhibitory. 

In vivo, elongation process of DNA replication is dependent on the presence of a virally-encoded SSB protein, 029 SSB. 029 SSB binds to ssDNA with relatively low affinity ( Ka = 10 M ) covering 3-4 nt per monomer, probably as a single array of protein units along the DNA. 

Suppose that a bacterial mutant is found to replicate its DNA at a very low rate. Upon analysis, it is found to have normal levels of activity of DNA polymerases 1 and 111, DNA gyrase, and DNA ligase. It also makes normal amounts of the wild-types of dnaA, dnaB, dnaC, and SSB proteins. The sequence of the oriC region of its chromosome is found to be wild type. What defect might account for the abnormally low rate of DNA replication in this mutant Explain briefly. 

Measurement of Temperature Inside the Capillary The use of NECEEM for measuring temperature inside the capillary has been demonstrated experimentally by measuring koff as a function of temperature for the interaction between SSB protein and fluorescently labeled ssDNA (Berezovski and Krylov, 2004). The calibration curve was built with a verified Beckman MDQ CE instrument, which uses a liquid heat exchanger to remove excess heat efficiently from the capillary. This calibration curve was then used to measure the temperature inside the capillary in a custom-built instrument with the capillary exposed to the ambient atmosphere at 20°C. It was found that the temperature inside the capillary was 15°C higher than the ambient temperature. As affinity interactions are very sensitive to temperature, the unjustified assumption that the capillary temperature... 

Fig. 8. Basic requirements for Pol V-mediated translesion synthesis in vitro sorting out the published literature. The groups of Dr. Goodman (Goodman, 2002), Dr. Livneh (Livneh, 2001), and ourselves (Fujii et ai, 2004) have published work on the reconstitution of lesion bypass using Pol V and accessory factors. As outlined in this figure, the three studies differ in (i) the nature of the Pol V preparation and (ii) the structure of the primer template. The length of the single-stranded region downstream from the lesion site that appears to be a critical parameter (see text) is indicated for each substrate. Except for RecA protein that was found to be essential in all three studies, differences with respect to the requirements of SSB protein, ATP or ATP S, and the f3 clamp have been reported as discussed in the text and summarized in this figure.

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