Current Organic Chemistry - Volume 6, Issue 9, 2002

Highly fluorescent nucleotide base analogs provide sensitive probes for studying the structure, dynamics and interactions of nucleic acids. These analogs can be incorporated site-specifically into oligonucleotides through standard automated synthetic methods, allowing them to serve as sensitive probes of changes in the microenvironment of bases that may result from variation in buffer condition, ion concentration, temperature or molecular interactions. Global conformational changes in nucleic acids and nucleic acid complexes can also be detected using fluorescent base analogs. A significant strength of fluorescent base analogs is their similarity in molecular constitution and chemical properties to natural bases. In contrast to bulkier chromophores, incorporation of nucleotide base analogs into oligonucleotides can normally be accomplished without introducing significant structural or chemical changes that might alter the measurement. Here, we review the characteristics of currently available fluorescent nucleotide base analogs that have made them useful probes in fluorescence studies of nucleic acids and nucleic acid complexes. A range of applications, that include measurement of fluorescence emission quenching, spectral shifting, depolarization (anisotropy), fluorescence lifetime and fluorescence resonance energy transfer, will be presented to demonstrate the broad utility of fluorescence base analog probes in characterizing the steady-state equilibrium properties and real-time kinetics in nucleic acid systems.

Proteins that bind and stabilize single-stranded DNA are critical for proper DNA metabolism. In eukaryotes, the major single-stranded DNA-binding protein is replication protein A (RPA), an evolutionarily conserved heterotrimeric complex required for DNA replication, repair, and recombination. While much of the early work on RPA established its role in DNA replication, a great deal of attention is now being paid to the specific mechanisms by which RPA operates in recombination. As described in this review, significant insight has been gained from studies employing proteins purified from both yeast and human cells. Of particular importance, these analyses have revealed that RPA is centrally involved in the initiation of homologous recombination. Research into recombination and its influence by RPA is especially relevant to our understanding of disease development, as inappropriate chromosomal rearrangement is known to be associated with a number of human disorders.

DNA topology and topological changes are important in DNA packaging, replication, transcription, and recombination. The importance of topology to biology results from the ability of topological changes to manifest as geometric changes in twist, writhe, or both, and therefore to potentiate the formation of wrapped, looped, or melted structures that are high-energy intermediates in DNA transactions. The energetics of topological change and its partitioning into twist and writhe depend on the size and shape of the topological domain. The remodeling of chromatin structure and stability by ATPase motor proteins and by histone acetylases is accompanied by topological changes. Recent results on the mechanisms of remodeling and the challenges in interpretation of topology-based experiments are discussed. Topology can also be manipulated as an experimental variable to serve as a reporter of conformation or as a means of introducing strain into a system. Examples are given from work on DNA looping. Topological change can be transmitted along DNA (action at a distance) even when there is no permanent change in linking number. Dynamic supercoiling can be introduced by tracking proteins such as RNA polymerase (the twindomain model). The dissipation of dynamic supercoiling may be slowed by hydrodynamic resistance to axial rotation conferred by large or bent DNA, or by nucleosomes and other proteins on the DNA. Topoisomerases reduce transcription-induced supercoiling and control the steady-state levels of DNA topological strain. The roles of dynamic supercoiling in transcription initiation and gene regulation at the c-Myc promoter and at several E. coli promoters are discussed.

DNA ligases play an essential role in DNA replication, repair, and recombination and are classified as ATP-dependent or NAD-dependent based on their adenylation cofactor requirement. ATP-dependent ligases have been found in bacteriophages, viruses, archaea, eukaryotes and bacteria. NAD-dependent ligases exist in eubacteria and entomopoxviruses. Both types of ligases share a catalytic core which consists of conserved motifs found in nucleotidyl transferase superfamily. These motifs are essential for adenylation cofactor binding, metal ion coordination, and ligation chemistry, as validated by X-ray crystal structure determination and mutational analyses. Viral ATP ligases are generally small in size and have been used as models for elucidating the catalytic mechanism. More complex genomes, such as yeast and humans, encode multiple ATP ligases with additional non-catalytically essential domains at N-terminal and / or C-teminal regions of the catalytic core. Some of these additional domains are involved in protein-protein interactions, which target different ATP ligases to specific loci to fulfill specific cellular replication and repair functions. As a consequence of multiple protein interactions, ligase activity may be enhanced to complete a specific strand joining reaction. In humans, three ligase genes encoding four ligases (ligase I, ligase IIIα, ligase IIIβ, and ligase IV) are required for replication, recombination, repair, and nonhomologous end-joining (NHEJ). In addition to ligation activities, other enzymatic activities such as AP lyase activity have been detected in ATP ligases, implicating an expanded role of ligases in DNA repair. The discovery of ATP ligases in eubacteria indicates their ubiquity in living organisms. NAD ligases are composed of a nucleotidyl transferase catalytic core and additional C-terminal domains. The discovery of NAD ligases in entomopox virus genomes changes the notion that NAD ligases are only associated with eubacteria. While NAD and ATP ligases complement each other in rescuing growth defects, specific cellular functions such as DNA repair appear to require proteinprotein interactions specified by domains associated with NAD or ATP ligases. A ligase-DNA complex structure is needed to better understand the recognition and catalytic mechanism during DNA strand joining. The complex protein-protein interactions have to be elucidated to define the cellular functions. Newly identified putative ATP ligases in bacteria may open a new dimension in DNA ligation.