Binding of the dimeric Deinococcus radiodurans single-stranded DNA binding protein to single-stranded DNA
Alexander G Kozlov1, Julie M Eggington, Michael M Cox
1Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63110, USA.
Biochemistry
|August 28, 2010
Summary
Deinococcus radiodurans single-stranded DNA binding protein (DrSSB) exhibits ssDNA binding modes similar to EcoSSB, with quantitative differences in site size and binding enthalpy. These findings highlight conserved SSB protein functions in DNA processes.
Area of Science:
- Molecular Biology
- Biochemistry
Background:
- Deinococcus radiodurans single-stranded (ss) DNA binding protein (DrSSB) is crucial for DNA repair, replication, and recombination in a radiation-resistant bacterium.
- DrSSB shares structural similarities with Escherichia coli SSB (EcoSSB), possessing four DNA binding domains (OB-folds) despite functioning as a homodimer.
Purpose of the Study:
- To compare the equilibrium binding of DrSSB to ssDNA with that of EcoSSB.
- To investigate the ssDNA binding modes and characteristics of DrSSB.
Main Methods:
- Equilibrium binding studies using poly(dT) and phage M13 ssDNA.
- Electrophoretic mobility shift assays.
- Isothermal titration calorimetry (ITC) to determine binding stoichiometry and enthalpy.
Main Results:
- DrSSB exhibits a transition in occluded ssDNA site size (∼45 to 50-55 nucleotides) with varying salt concentrations, similar to EcoSSB but with a less pronounced difference.
- Stable, highly cooperative complexes of DrSSB with M13 ssDNA were observed under low-salt conditions.
- ITC revealed stoichiometric binding of DrSSB to oligo(dT)s with a ΔH(obs) of approximately -94 ± 4 kcal/mol, and binding enthalpy showed significant salt concentration sensitivity.
Conclusions:
- DrSSB and EcoSSB share common ssDNA binding features, suggesting conserved mechanisms for SSB protein versatility in vivo.
- Quantitative differences in ssDNA binding modes and enthalpy exist between DrSSB and EcoSSB, indicating distinct pathways of ssDNA wrapping.
Related Concept Videos
01:03Single-Strand DNA Binding Proteins
For successful DNA replication, the unwinding of double-stranded DNA must be accompanied by stabilization and protection of the separated single strands of the DNA. This crucial task is performed by single-strand DNA-binding (SSB) proteins. They bind to the DNA in a sequence-independent manner, which means that the nitrogenous bases of the DNA need not be present in a specific order for binding of SSB proteins to it. The binding of SSB proteins straightens single-stranded DNA (ssDNA) and makes...
02:13Cooperative Binding of Transcription Regulators
Transcriptional regulators bind to specific cis-regulatory sequences in the DNA to regulate gene transcription. These cis-regulatory sequences are very short, usually less than ten nucleotide pairs in length. The short length means that there is a high probability of the exact same sequence randomly occurring throughout the genome. Since regulators can also bind to groups of similar sequences, this further increases the chances of random binding. Transcriptional regulators form dimers that...
03:01The Replisome
DNA replication is carried out by a large complex of proteins that act in a coordinated matter to achieve high-fidelity DNA replication. Together this complex is known as the DNA replication machinery or the replisome.
The synthesis of the leading and lagging strands is a highly coordinated process. To explain this, the “Trombone model” was proposed by Bruce Alberts in 1980. The DNA loop formation starts when a primer is synthesized on the parent lagging strand. The loop grows with the...
The synthesis of the leading and lagging strands is a highly coordinated process. To explain this, the “Trombone model” was proposed by Bruce Alberts in 1980. The DNA loop formation starts when a primer is synthesized on the parent lagging strand. The loop grows with the...
00:55DNA Helicases
DNA unwinding helicase enzymes are a type of motor protein. Motor proteins can translocate along filaments or polymers using energy generated from ATP hydrolysis. Helicases are involved in all the important cellular processes where DNA unwinding is required, such as DNA replication, repair, recombination, and transcription. They are present in all living organisms, but vary in their structure, function, and mechanism of action. For example, in prokaryotes, DnaB helicase binds and translocates...
02:31Homologous Recombination
The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
02:04Fixing Double-strand Breaks
The double-stranded structure of DNA has two major advantages. First, it serves as a safe repository of genetic information where one strand serves as the back-up in case the other strand is damaged. Second, the double-helical structure can be wrapped around proteins called histones to form nucleosomes, which can then be tightly wound to form chromosomes. This way, DNA chains up to 2 inches long can be contained within microscopic structures in a cell. A double-stranded break not only damages...