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In molecular biology, endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain (namely DNA or RNA). Some, such as deoxyribonuclease I, cut DNA relatively nonspecifically (without regard to sequence), while many, typically called restriction endonucleases or restriction enzymes, cleave only at very specific nucleotide sequences. Endonucleases differ from exonucleases, which cleave the ends of recognition sequences instead of the middle (endo) portion. Some enzymes known as "exo-endonucleases", however, are not limited to either nuclease function, displaying qualities that are both endo- and exo-like. Evidence suggests that endonuclease activity experiences a lag compared to exonuclease activity. Restriction enzymes are endonucleases from eubacteria and archaea that recognize a specific DNA sequence. The nucleotide sequence recognized for cleavage by a restriction enzyme is called the restriction site. Typically, a restriction site will be a palindromic sequence about four to six nucleotides long. Most restriction endonucleases cleave the DNA strand unevenly, leaving complementary single-stranded ends. These ends can reconnect through hybridization and are termed "sticky ends". Once paired, the phosphodiester bonds of the fragments can be joined by DNA ligase. There are hundreds of restriction endonucleases known, each attacking a different restriction site. The DNA fragments cleaved by the same endonuclease can be joined regardless of the origin of the DNA. Such DNA is called recombinant DNA; DNA formed by the joining of genes into new combinations. Restriction endonucleases (restriction enzymes) are divided into three categories, Type I, Type II, and Type III, according to their mechanism of action. These enzymes are often used in genetic engineering to make recombinant DNA for introduction into bacterial, plant, or animal cells, as well as in synthetic biology. One of the more famous endonucleases is Cas9. Ultimately, there are three categories of restriction endonucleases that relatively contribute to the cleavage of specific sequences.

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Combining RNAi and in vivo confocal microscopy analysis of the photoconvertible fluorescent protein Dendra2 to study a DNA repair protein

Matteo Di Piazza

Clinical approaches for tumor treatment often rely on combination therapy where a DNA damaging agent is used in combination with a DNA repair protein inhibitor. For this reason, great efforts have been made during the last decade to identify inhibitors of DNA repair proteins or, alternatively, small molecules that specifically alter protein stability or trafficking. Unfortunately, when studying these drug candidates, classical biochemical approaches are prone to artifacts. The apurinic/apyrimidinic endonuclease (APE1) protein is an essential component of the base excision repair (BER) pathway that is responsible for repairing DNA damage caused by oxidative and alkylating agents. In this work, we combined conditional gene expression knockdown of APE1 protein by RNA interference (RNAi) technology with re-expression of an ectopic recombinant form of APE1 fused with the photoconvertible fluorescent protein (PCFP) Dendra2. Dendra2 did not alter the subcellular localization or endonuclease activity of APEI. We calculated APE1 half-life and compared these results with the classical biochemical approach, which is based on cycloheximide (CHX) treatment. In conclusion, we combined RNAi and in vivo confocal microscopy to study a DNA repair protein demonstrating the feasibility and the advantage of this approach for the study of the cellular dynamic of a DNA repair protein

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Endonucléase

Une endonucléase est une nucléase, qui coupe un acide nucléique en fragments plus courts. Les endonucléases sont capables de couper au milieu de la chaîne, par opposition aux exonucléases qui n'attaquent que les nucléotides situés aux extrémités des fragments. Certaines endonucléases dites endonucléases de restriction sont des enzymes de restriction qui coupent un brin d'ADN bicaténaire en des endroits caractérisés par une séquence spécifique de nucléotides : le site de restriction. On parle de digestion de l'ADN.

Recombinaison homologue

thumb | 275px | alt=Schéma du chromosome 1 après recombinaison homologue | Figure 1. La recombinaison homologue peut produire de nouvelles combinaisons d'allèles entre les chromosomes parentaux, notamment lors de la méiose.La recombinaison homologue est un type de recombinaison génétique où les séquences de nucléotides sont échangées entre des molécules d'ADN identiques (homologues) ou similaires (Figure 1). Au sens large, la recombinaison homologue est un mécanisme ubiquitaire de réparation des cassures double-brins de l'ADN.

Nucléase

A nuclease (also archaically known as nucleodepolymerase or polynucleotidase) is an enzyme capable of cleaving the phosphodiester bonds between nucleotides of nucleic acids. Nucleases variously effect single and double stranded breaks in their target molecules. In living organisms, they are essential machinery for many aspects of DNA repair. Defects in certain nucleases can cause genetic instability or immunodeficiency. Nucleases are also extensively used in molecular cloning.

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