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Structure and Function of Nucleases in DNA Repair: Shape, Grip and Blade of the DNA Scissors

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Structure and Function of Nucleases in DNA Repair: Shape, Grip and Blade of the DNA Scissors Oncogene (2002) 21, 9022 – 9032 ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc Structure and function of nucleases in DNA repair: shape, grip and blade of the DNA scissors Tatsuya Nishino1 and Kosuke Morikawa*,1 1Department of Structural Biology, Biomolecular Engineering Research Institute (BERI), 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan DNA nucleases catalyze the cleavage of phosphodiester mismatched nucleotides. They also recognize the bonds. These enzymes play crucial roles in various DNA replication or recombination intermediates to facilitate repair processes, which involve DNA replication, base the following reaction steps through the cleavage of excision repair, nucleotide excision repair, mismatch DNA strands (Table 1). repair, and double strand break repair. In recent years, Nucleases can be regarded as molecular scissors, new nucleases involved in various DNA repair processes which cleave phosphodiester bonds between the sugars have been reported, including the Mus81 : Mms4 (Eme1) and the phosphate moieties of DNA. They contain complex, which functions during the meiotic phase and conserved minimal motifs, which usually consist of the Artemis : DNA-PK complex, which processes a V(D)J acidic and basic residues forming the active site. recombination intermediate. Defects of these nucleases These active site residues coordinate catalytically cause genetic instability or severe immunodeficiency. essential divalent cations, such as magnesium, Thus, structural biology on various nuclease actions is calcium, manganese or zinc, as a cofactor. However, essential for the elucidation of the molecular mechanism the requirements for actual cleavage, such as the types of complex DNA repair machinery. Three-dimensional and the numbers of metals, are very complicated, but structural information of nucleases is also rapidly are not common among the nucleases. It appears that accumulating, thus providing important insights into the the major role of the metals is to stabilize inter- molecular architectures, as well as the DNA recognition mediates, thereby facilitating the phosphoryl transfer and cleavage mechanisms. This review focuses on the reactions. Cleavage reactions occur either at the end three-dimensional structure-function relationships of or within DNA, and thus DNA nucleases are nucleases crucial for DNA repair processes. categorized as exonucleases and endonucleases, respec- Oncogene (2002) 21, 9022 – 9032. doi:10.1038/sj.onc. tively (Figure 1). Exonucleases can be further 1206135 classified as 5’ end processing or 3’ end processing enzymes, according to their polarity of consecutive Keywords: DNA repair; nuclease; metal-dependent cleavage. cleavage; protein-DNA interaction; structure-function This review describes the three-dimensional (3D) relationships structural views of the actions of various nucleases involved in many DNA repair pathways. The rapidly accumulating genomic, biochemical and structural data have allowed us to classify various nucleases into Introduction folding families. In general, the nucleases involved in DNA repair recognize the damaged moiety through the Quality control of genetic material is a function remarkably large deformation of DNA duplexes, and conserved in all living organisms. DNA suffers from thus in terms of their DNA recognition mode, they many environmental stresses, including attacks by apparently differ from the sequence-specific endonu- reactive oxygen species, radiation, UV light, and cleases, such as the restriction enzymes. The active sites carcinogens, which modify the DNA. In addition, of DNA repair nucleases have some similarity with there are intrinsic errors and unusual structures, which other nucleases, including the metal-coordinating are formed during replication or recombination, and residues; however, they also display pronounced they must be corrected by the various repair protein diversity. machineries to avoid alterations of the base sequences or entanglement of the DNA. These DNA repair Nucleases in various categories of DNA repair proteins may function independently, but in many cases, they form complexes to perform more efficient Replication repair reactions. In the repair complexes, nucleases play important roles in eliminating the damaged or DNA polymerase replicates a new strand of DNA, the sequence of which is complementary to the template DNA. Most DNA polymerases in prokaryotes and *Correspondence: K Morikawa; E-mail: [email protected] eukaryotes are composed of two different enzymes, a Structure and function of DNA repair nuclease T Nishino and K Morikawa 9023 Table 1 Nucleases involved in DNA repair Prokaryote/ Bacteriophage Archaea Yeast Mammals Replication Proofreading PolI, II PolB, D Pold, e, g Pold, e, g DnaQ Okazaki fragment processing RNaseH RNaseHII RNaseH RNaseH FEN1 FEN1 Dna2 Replication fork cleavage Hef Mus81 Mus81 (+Mms4[Eme1])a,b Wrn Base excision Repair EndoV APN1 Abasic site processing EndolIV HAP1[APE,APEX]b ExoIII Mismatchrepair MutH Nucleotide excision repair 5’ processing UvrC(+UvrB)a Rad1(+Rad10)a XPF(+ERCC1)a 3’ processing UvrC Rad2 XPG Short patch repair Vsr Double strand break repair End processing RecB(+RecCD)a Dna2 SbcD(+SbcC)a Mre11(+Rad50)a Mre11(+Rad50)a Mre11(+Rad50)a RecJ ExoVII[RecE]b ExoI[SbcB]b Artemis(+DNA-PK)a Holliday junction resolvase RuvC Ccell[Ydc2]b RusA Hjc T4 endoVII T7 endoI aProteins in parenthesis form a complex. bProteins in brackets are a homolog or alternative name of the protein Most of the Okazaki fragments are eliminated by RNaseH, enzyme ubiquitously present in all living organisms. RNaseH produces nicks in the RNA region of Okazaki fragments (Figure 2). In eukaryotes and in archaea, FEN1 endonucleases also participate in the removal of Okazaki fragments (reviewed in Lieber, 1997). FEN1 is a multi-functional enzyme. In addition to the 5’ to 3’ exonuclease activity to remove the Okazaki fragments, the enzyme can also generate an incision at the junction point of a 5’ flap DNA Figure 1 Schematic diagram of the nuclease activity. The two structure. This latter activity is required to eliminate strands of DNA are schematically drawn. The cleavage made by the nuclease is represented by arrowhead non-homologous tails in base excision repair and in recombination intermediates. The replication process is stalled by various modes of DNA damage. Upon the halt of fork progression, polymerase and an exonuclease, encoded within the the DNA polymerase and other protein complexes same polypeptide, but sometimes they are formed by abandon the replication fork. The remaining fork must different subunits. The exonuclease degrades misincor- be processed by various fork-specific protein machi- porated DNA strand in the 3’ to 5’ direction (Figure 2) neries. The most notable protein among them is (reviewed in Shevelev and Hubscher, 2002). Deletion Mus81, which was recently found as a new fork/ of these proofreading nucleases results in lethal or junction specific endonuclease (Boddy et al., 2000, strong mutator phenotypes in bacteria (Fijalkowska 2001; Interthal and Heyer, 2000; Kaliraman et al., and Schaaper, 1996) and in yeast (Morrison et al., 2001; Mullen et al., 2001). Genetic and biochemical 1993), and causes cancer in mice (Goldsby et al., analyses have revealed that this endonuclease is 2001). completely conserved in eukaryotes, while its homolog The removal of Okazaki fragments is another has been found in archaea. The loss of Mus81 in yeast important process in replication. This DNA : RNA causes UV or methylation damage sensitivity (Interthal hybrid is required to initialize DNA polymerization, and Heyer, 2000) and defects in sporulation (Mullen et but once the replication starts, it is rapidly degraded. al., 2001). Oncogene Structure and function of DNA repair nuclease T Nishino and K Morikawa 9024 Base Excision Repair Figure 2 Nuclease associated DNA repair pathways. The substrate DNAs are drawn schematically and the arrowheads denote nuclease cleavage. RNA regions are drawn in bold line responsible for mismatches in certain sequences Base excision repair (reviewed in Modrich and Lahue, 1996; Yang, 2000; Abasic sites within DNA duplexes are frequently Tsutakawa and Morikawa, 2001). In the MutSLH produced by the actions of various DNA glycosylases system, the MutS protein recognizes and binds involved in the base excision repair pathway, in mismatched base moieties of DNA. MutL mediates addition to the spontaneous hydrolysis of bases. These the interaction between the MutS and MutH proteins. apyrimidine or apurine (AP) sites are removed by AP MutH recognizes a hemimethylated GATC sequence, endonucleases which cleave the phosphdiester bond and cleaves next to the G of the non-methylated strand next to an abasic site (Figure 2) (reviewed in Mol et (Figure 2). The cleavage activity of MutH is enhanced al., 2000a). E. coli cells contain two AP endonucleases: by the MutL protein, although its mechanism remains endonuclease IV (endoIV) and exonuclease III unclear. Vsr is a mismatch-specific endonuclease (exoIII). Interestingly, these two enzymes show no involved in very short patch repair, and recognizes a sequence similarity to each other; although their AP TG mismatch at the specific sequence CT(A/T)GG, endonuclease activities are quite similar. In eukaryotes, where the mismatch occurs at the second thymine, there seems to be a single, major AP endonuclease upon spontaneous deamination. Vsr makes an incision working in each organism. APN1, the yeast homolog next to the mismatched
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