Nuclease-induced stepwise photodropping (NISP) to precisely investigate single-stranded DNA degradation behaviors of exonucleases and endonucleases.

Here, we employed a fluorescence-based single molecule method called nuclease-induced stepwise photodropping (NISP) to measure in real time the DNA degradation mediated by mitochondrial genome maintenance exonuclease 1 (MGME1), a bidirectional single-stranded DNA (ssDNA)-specific exonuclease. The method detects a stepwise decrease in fluorescence signals from Cy3 fluorophores labeled on an immobilized DNA substrate. Using NISP, we successfully determined the DNA degradation rates of 6.

Structural basis of how MGME1 processes DNA 5' ends to maintain mitochondrial genome integrity.

Mitochondrial genome maintenance exonuclease 1 (MGME1) helps to ensure mitochondrial DNA (mtDNA) integrity by serving as an ancillary 5'-exonuclease for DNA polymerase γ. Curiously, MGME1 exhibits unique bidirectionality in vitro, being capable of degrading DNA from either the 5' or 3' end. The structural basis of this bidirectionally and, particularly, how it processes DNA from the 5' end to assist in mtDNA maintenance remain unclear.

Structures, Mechanisms, and Functions of His-Me Finger Nucleases.

His-Me finger (also called HNH or ββα-me) nucleases, are a large superfamily of nucleases that share limited sequence homology, but all members carry a highly similar catalytic motif exhibiting a ββα topology. This review represents a structural comparison of His-Me finger nucleases, summarizing their substrate-binding and recognition strategies, mechanisms of enzymatic hydrolysis, cellular functions, and the various means of activity regulation. His-Me finger nucleases usually function as monomers, making a single nick in nucleic acids to degrade foreign or host genomes, or as homodimers that introduce double-stranded DNA breaks for DNA restriction, integration, recombination, and repair.

A unique exonuclease ExoG cleaves between RNA and DNA in mitochondrial DNA replication.

Replication of sufficient mitochondrial DNA (mtDNA) is essential for maintaining mitochondrial functions in mammalian cells. During mtDNA replication, RNA primers must be removed before the nascent circular DNA strands rejoin. This process involves mitochondrial RNase H1, which removes most of the RNA primers but leaves two ribonucleotides attached to the 5' end of nascent DNA.

Producing irreversible topoisomerase II-mediated DNA breaks by site-specific Pt(II)-methionine coordination chemistry.

Human type II topoisomerase (Top2) isoforms, hTop2α and hTop2β, are targeted by some of the most successful anticancer drugs. These drugs induce Top2-mediated DNA cleavage to trigger cell-death pathways. The potency of these drugs correlates positively with their efficacy in stabilizing the enzyme-mediated DNA breaks.

Structural basis for specific ligation of the peroxisome proliferator-activated receptor δ.

The peroxisome proliferator-activated receptor (PPAR) family comprises three subtypes: PPARα, PPARγ, and PPARδ. PPARδ transcriptionally modulates lipid metabolism and the control of energy homeostasis; therefore, PPARδ agonists are promising agents for treating a variety of metabolic disorders. In the present study, we develop a panel of rationally designed PPARδ agonists.

Crystal structure of endonuclease G in complex with DNA reveals how it nonspecifically degrades DNA as a homodimer.

Endonuclease G (EndoG) is an evolutionarily conserved mitochondrial protein in eukaryotes that digests nucleus chromosomal DNA during apoptosis and paternal mitochondrial DNA during embryogenesis. Under oxidative stress, homodimeric EndoG becomes oxidized and converts to monomers with diminished nuclease activity. However, it remains unclear why EndoG has to function as a homodimer in DNA degradation.

On the structural basis and design guidelines for type II topoisomerase-targeting anticancer drugs.

Type II topoisomerases (Top2s) alter DNA topology via the formation of an enzyme-DNA adduct termed cleavage complex, which harbors a transient double-strand break in one DNA to allow the passage of another. Agents targeting human Top2s are clinically active anticancer drugs whose trapping of Top2-mediated DNA breakage effectively induces genome fragmentation and cell death. To understand the structural basis of this drug action, we previously determined the structure of human Top2 β-isoform forming a cleavage complex with the drug etoposide and DNA, and described the insertion of drug into DNA cleavage site and drug-induced decoupling of catalytic groups.

New insights into DNA-binding by type IIA topoisomerases.

Type IIA topoisomerases catalyze the passage of two DNA duplexes across each other to resolve the entanglements and coiling of cellular DNA. The ability of these enzymes to interact simultaneously but differentially with two DNA segments is central to their DNA-manipulating functions: one duplex DNA is bound and cleaved to produce a transient double-strand break through which another DNA segment can be transported. Recent structural analyses have revealed in atomic detail how type IIA enzymes contact DNA and how the enzyme-DNA interactions may be exploited by drugs to achieve therapeutic purposes.

Structural basis of type II topoisomerase inhibition by the anticancer drug etoposide.

Type II topoisomerases (TOP2s) resolve the topological problems of DNA by transiently cleaving both strands of a DNA duplex to form a cleavage complex through which another DNA segment can be transported. Several widely prescribed anticancer drugs increase the population of TOP2 cleavage complex, which leads to TOP2-mediated chromosome DNA breakage and death of cancer cells. We present the crystal structure of a large fragment of human TOP2β complexed to DNA and to the anticancer drug etoposide to reveal structural details of drug-induced stabilization of a cleavage complex.