Physical and Functional Interaction of Mitochondrial Single-Stranded DNA-Binding Protein and the Catalytic Subunit of DNA Polymerase Gamma

The maintenance of the mitochondrial genome depends on a suite of nucleus-encoded proteins, among which the catalytic subunit of the mitochondrial replicative DNA polymerase, Pol γα, plays a pivotal role. Mutations in the Pol γα-encoding gene, POLG, are a major cause of human mitochondrial disorders. Here we present a study of direct and functional interactions of Pol γα with the mitochondrial single-stranded DNA-binding protein (mtSSB). mtSSB coordinates the activity of the enzymes at the DNA replication fork. However, the mechanism of this functional relationship is elusive, and no direct interactions between the replicative factors have been identified to date. This contrasts strikingly with the extensive interactomes of SSB proteins identified in other homologous replication systems. Here we show for the first time that mtSSB binds Pol γα directly, in a DNA-independent manner. This interaction is strengthened in the absence of the loop 2.3 structure in mtSSB, and is abolished upon preincubation with Pol γβ. Together, our findings suggest that the interaction between mtSSB and polymerase gamma holoenzyme (Pol γ) involves a balance between attractive and repulsive affinities, which have distinct effects on DNA synthesis and exonucleolysis.

Sub-millisecond conformational transitions of short single-stranded DNA lattices by photon correlation single-molecule FRET

Thermally-driven conformational fluctuations (or 'breathing') of DNA plays important roles in the function and regulation of the 'macromolecular machinery of genome expression.' Fluctuations in double-stranded (ds) DNA are involved in the transient exposure of pathways to protein binding sites within the DNA framework, leading to the binding of functional and regulatory proteins to single-stranded (ss) DNA templates. These interactions often require that the ssDNA sequences, as well as the proteins involved, assume transient conformations critical for successful binding. Here we use microsecond-resolved single-molecule F&oumlrster Resonance Energy Transfer (smFRET) experiments to investigate the backbone fluctuations of short (ss) oligo- oligo(dT)n templates within DNA constructs that can also serve as models for ss-dsDNA junctions. Such junctions, as well as the attached ssDNA sequences, are involved in the binding of ssDNA binding (ssb) proteins that control and integrate the mechanisms of DNA replication complexes. We have used these data to determine multi-order time-correlation functions (TCFs) and probability distribution functions (PDFs) that characterize the kinetic and thermodynamic behavior of the system. We find that the oligo(dT)n tails of ss-dsDNA constructs inter-convert, on sub-millisecond time-scales, between three macrostates with distinctly different end-to-end distances. These are: (i) a 'compact' macrostate that represents the dominant species at equilibrium; (ii) a 'partially extended' macrostate that exists as a minority species; and (iii) a 'highly extended' macrostate that is present in trace amounts. We propose a model for ssDNA secondary structure that advances our understanding of how spontaneously formed nucleic acid conformations may facilitate the activities of ssDNA associating proteins.

The Characteristics of New SSB Proteins from Metagenomic Libraries and Their Use in Biotech Applications

Single-stranded DNA binding proteins (SSBs) bind to single-stranded DNA in a sequence-independent manner to prevent formation of secondary structures and protect DNA from nuclease degradation. These ubiquitous proteins are present in prokaryotes, eukaryotes, and viruses, and play a pivotal role in the following major cellular processes: replication, recombination, and repair of genetic material. In DNA replication, SSB proteins specifically stimulate DNA polymerase, increase fidelity of DNA synthesis, assist the advance of DNA polymerase, and organize and stabilize replication forks. Here, we present our characterization of four SSB proteins of different origins. One of them was isolated from Clostridium sp. phage phiCP130 (SSB C1: 124 aa, Mr = 13,905). Three others (SSB M2: 136 aa, Mr = 15,009; SSB M3: 144 aa, Mr = 16,106; and SSB M5: 138 aa, Mr = 15,851) were isolated from metagenomics libraries. They show high similarity to SSB proteins from Caldanaerovirga acetigigens, Caldanaerobius fijiensis, and Fervidobacterium gondwanense. The recombinant proteins were overproduced in E. coli Rosetta (pRARE), except for SSB M5, which was overproduced in E. coli BL21. Proteins were purified using a metal-affinity chromatography as His-tagged fusion proteins. Electrophoretic mobility shift assay was used to examine their DNA binding activity with fluorescein-labeled oligonucleotide (dT40) used as a substrate. Thermal stability analysis revealed that they are stable at elevated temperatures, with the exception of SSB protein C1, which loses its activity above 65 °C. The other proteins are active at high temperatures, SSB M3 up to 85 °C, while SSB M2 and SSB M5 are active up to 98.7 °C. The subunit structure of proteins was analyzed by gel filtration on Superdex 75 column (AKTA). This allowed us to conclude that in solution, the analyzed proteins exist in oligomeric form, a feature which is characteristic of other SSB proteins. Purified SSB proteins were tested to improve specificity of PCR-based DNA amplification.

‘Drc’, a structurally novel ssDNA-binding transcription regulator of N4-related bacterial viruses

Bacterial viruses encode a vast number of ORFan genes that lack similarity to any other known proteins. Here, we present a 2.20 Å crystal structure of N4-related Pseudomonas virus LUZ7 ORFan gp14, and elucidate its function. We demonstrate that gp14, termed here as Drc (ssDNA-binding RNA Polymerase Cofactor), preferentially binds single-stranded DNA, yet contains a structural fold distinct from other ssDNA-binding proteins (SSBs). By comparison with other SSB folds and creation of truncation and amino acid substitution mutants, we provide the first evidence for the binding mechanism of this unique fold. From a biological perspective, Drc interacts with the phage-encoded RNA Polymerase complex (RNAPII), implying a functional role as an SSB required for the transition from early to middle gene transcription during phage infection. Similar to the coliphage N4 gp2 protein, Drc likely binds locally unwound middle promoters and recruits the phage RNA polymerase. However, unlike gp2, Drc does not seem to need an additional cofactor for promoter melting. A comparison among N4-related phage genera highlights the evolutionary diversity of SSB proteins in an otherwise conserved transcription regulation mechanism.

Variations in amino acid composition in bacterial single stranded DNA–binding proteins correlate with GC content

Background and purposeSSB proteins are essential for the maintenance of the genome in all domains of life. Most bacterial SSBs are active as homotetramers. Each monomer comprises N-terminal domain (OB-fold) which is responsible for ssDNA binding and a disordered C-terminal domain (Ct) with a conserved acidic tail responsible for protein interactions.The variations in these essential proteins prompted us to conduct in silico analyses of the aa composition and properties of two distinct SSB domains in relation to bacterial GC content.Materials and methodsSSB sequences were collected from genomes covering a wide range of GC content from 14 bacterial phyla. The maximum-likelihood (ML) trees were constructed for SSB sequences and corresponding 16S rRNA genes. The aa contents of OB folds and Ct domains were subsequently analysed. ResultsWe showed that SSB proteins followed predicted amino acid (aa) composition as a function of genomic GC content. However, two distinct domains of SSB exhibit significant differences to the expected aa composition. Variations in aa proportion were more prominent in Ct domains. Elevated accumulation of Gly (up to 60 %) and Pro (up to 24 %), significant drop in the proportion of basic Lys and reduction in hydrophobic Leu, Ile and Val were identified in Ct domains of SSBs from high GC genomes. Consequently, this influences the biochemical properties of Ct domains.ConclusionsBased on this comparative study of SSBs we conclude that genomic GC content and two distinct domains with different functional roles participate in shaping aa composition of SSB proteins.

Chemo-mechanical pushing of proteins along single-stranded DNA

Single-stranded (ss)DNA binding (SSB) proteins bind with high affinity to ssDNA generated during DNA replication, recombination, and repair; however, these SSBs must eventually be displaced from or reorganized along the ssDNA. One potential mechanism for reorganization is for an ssDNA translocase (ATP-dependent motor) to push the SSB along ssDNA. Here we use single molecule total internal reflection fluorescence microscopy to detect such pushing events. When Cy5-labeled Escherichia coli (Ec) SSB is bound to surface-immobilized 3′-Cy3–labeled ssDNA, a fluctuating FRET signal is observed, consistent with random diffusion of SSB along the ssDNA. Addition of Saccharomyces cerevisiae Pif1, a 5′ to 3′ ssDNA translocase, results in the appearance of isolated, irregularly spaced saw-tooth FRET spikes only in the presence of ATP. These FRET spikes result from translocase-induced directional (5′ to 3′) pushing of the SSB toward the 3′ ssDNA end, followed by displacement of the SSB from the DNA end. Similar ATP-dependent pushing events, but in the opposite (3′ to 5′) direction, are observed with EcRep and EcUvrD (both 3′ to 5′ ssDNA translocases). Simulations indicate that these events reflect active pushing by the translocase. The ability of translocases to chemo-mechanically push heterologous SSB proteins along ssDNA provides a potential mechanism for reorganization and clearance of tightly bound SSBs from ssDNA.