TIBS 25 – MARCH 2000 REVIEWS 17 Lemon, B.J. and Peters, J.W. (1999) Binding of 21 Malki, S. et al. (1995) Characterization of an operon 25 Santangelo, J.D. et al. (1995) Characterization and exogenously added carbon monoxide at the active site encoding an NADP-reducing hydrogenase in Desulfovibrio expression of the hydrogenase-encoding gene from of the iron-only hydrogenase (CpI) from Clostridium fructosovorans. J. Bacteriol. 177, 2628–2636 Clostridium acetobutylicum P262. Microbiology 141, pasteurianum. Biochemistry 38, 12969–12973 22 Stokkermans, J. et al. (1989) hyd gamma, a gene from 171–180 18 Montet, Y. et al. (1997) Gas access to the active site Desulfovibrio vulgaris (Hildenborough) encodes a 26 Kraulis, P.J. (1991) MOLSCRIPT: a program to produce of Ni–Fe hydrogenases probed by X-ray crystallography polypeptide homologous to the periplasmic both detailed and schematic plots of protein structures. and molecular dynamics. Nat. Struct. Biol. 4, 523–526 hydrogenase. FEMS Microbiol. Lett. 49, 217–222 J. Appl. Crystallogr. 24, 946–950 19 Niu, S. et al. (1999) Theoretical characterization of the 23 Voordouw, G. et al. (1985) Cloning of the gene 27 Arnez, J.G. (1994) MINIMAGE: a program for plotting reaction intermediates in a model of the nickel–iron encoding the hydrogenase from Desulfovibrio vulgaris electron-density maps. J. Appl. Crystallogr. 27, hydrogenase of Desulfovibrio gigas. J. Am. Chem. Soc. (Hildenborough) and determination of the NH2-terminal 649–653 121, 4000–4007 sequence. Eur. J. Biochem. 148. 509–514 28 Merritt, E.A. and Bacon, D.J. (1997) Raster3D 20 Gorwa, M.F. et al. (1996) Molecular characterization 24 Voordouw, G. et al. (1989) Organization of the genes photorealistic molecular graphics. Methods Enzymol. and transcriptional analysis of the putative encoding [Fe] hydrogenase in Desulfovibrio vulgaris 277, 505–524 hydrogenase gene of Clostridium acetobutylicum ATCC subsp. oxamicus Monticello. J. Bacteriol. 171, 29 Nicholls, A. (1992) GRASP: Graphical Representation 824. J. Bacteriol. 178, 2668–2675 3881–3889 and Analysis of Surface Properties, Colombia University appears that nature created safety mech- anisms by employing various DNA pols for similar functional tasks. Translesion Eukaryotic DNA polymerases, a DNA synthesis, for example, requires at least DNA pols z and h, the former proba- growing family bly being responsible for error-prone translesion DNA synthesis and the latter performing error-free DNA translesion synthesis (reviewed in Ref. 3). In many cases, DNA pols have complex Ulrich Hübscher, Heinz-Peter Nasheuer and polypeptide structures because, in addi- tion to the polymerizing subunit (that Juhani E. Syväoja often contains a proofreading 39→59 ex- onuclease), they comprise other func- In eukaryotic cells, DNA polymerases are required to maintain the integrity tional subunits (Table 1). These functions of the genome during processes, such as DNA replication, various DNA include other enzymatic activities (e.g. repair events, translesion DNA synthesis, DNA recombination, and also in DNA primase) or allow the DNA poly- regulatory events, such as cell cycle control and DNA damage checkpoint merase to interact with other proteins, in- function. In the last two years, the number of known DNA polymerases has volved in check-point function, cell cycle increased to at least nine (called a, b, g, d, e, z, h, u and i), and yeast control, DNA replication or DNA repair. Saccharomyces cerevisiae contains REV1 deoxycytidyl transferase. The replicative DNA pols d and e, for ex- ample, are chaperoned by the accessory proteins replication factor C (RF-C), and ANY LIVING CELL and organism is faced discovery of DNA pol a in eukaryotic proliferating cell nuclear antigen (PCNA). with the tremendous task of keeping the cells in 1957, the number of DNA pols The interaction of these two accessory genome intact in order to develop in an identified has grown. In the early 1970s, proteins with the DNA pols permits a high organized manner, function in a complex DNA pol b and g were discovered leading speed of DNA synthesis coupled with a environment, divide at the right time and to the simple concept that DNA pol a is high accuracy4. die when it is appropriate. To achieve the enzyme involved in DNA replication, At the structural level, DNA pols ap- this, DNA synthesis is required to dupli- DNA pol b in DNA repair and DNA pol g in pear to possess a universal DNA poly- cate the genetic information prior to cell mitochondrial DNA replication. However, merase active site5. This is achieved by a division. DNA synthesis is also needed the discovery of DNA pol d and e in mam- two-metal-ion-catalysed mechanism and during DNA repair processes, including malian cells during the 1980s compli- guarantees the incorporation of the cor- DNA recombination and bypassing le- cated this interpretation. It also sug- rectly base-paired deoxyribonucleoside sions when the DNA has been damaged gested that a particular DNA pol might triphosphate (according to the Watson– (translesion DNA synthesis). DNA syn- have more than one functional task Crick base pairing rule A–T and G–C) thesis is performed by enzymes, called in a cell, and that a particular DNA syn- onto a growing template. However, DNA DNA polymerases (DNA pol)1. Since the thetic event can require more than one pols do differ in various aspects of their DNA pol (reviewed in Ref. 2). Genetic structural architecture5, as a result of U. Hübscher is at the Institute of Veterinary studies performed with budding yeast the many possible interactions of DNA Biochemistry, University of Zürich-Irchel, Saccharomyces cerevisiae, for example, pols with other proteins and enzymes. Winterthurerstrasse 190, CH-8057 Zürich, showed that the three DNA pols a, d and Thus, the active site of the DNA pol is Switzerland; H-P. Nasheuer is at the Institute e share the task of replicating the cellular very conserved in evolution, whereas of Molecular Biotechnology, Dept of genome, and that DNA repair events such the structure of the surface of the mol- Biochemistry, Beutenbergstrasse 11, as base excision repair might require not ecules might differ considerably. D-07745 Jena, Germany; and J.E. Syväoja is only DNA pol b but also DNA pol d or at the Biocenter Oulu and Dept of e a Biochemistry, University of Oulu, FIN-90570 DNA pol , or both, especially in long- Functional roles of DNA pol Oulu, Finland, and Dept of Biology, University patch base excision repair. Because both DNA polymerase a–primase (DNA pol a– of Joensuu, FIN-80100 Joensuu, Finland. replication and repair are of primary prim) has an important role in DNA Email: [email protected] importance for cells and organisms, it replication (Fig. 1, reviewed in Ref. 6). 0968 – 0004/00/$ – See front matter © 2000, Elsevier Science Ltd. All rights reserved. PII: S0968-0004(99)01523-6 143 REVIEWS TIBS 25 – MARCH 2000 A direct role of DNA pol a–prim in Table 1. The eukaryotic DNA polymerases: polypeptide compositions, enzymatic activities and functionsa DNA repair and DNA recombination is still under discussion2. Recent findings Pol Subunitsb Enzymatic activity Functional taskc suggested that all replicative DNA pols, (M 3 103) a, d and e, are needed for double- stranded break repair in yeast by ho- a 165 (167) DNA pol Replication: initiation; repair: DSBR; telomer 67 (79/86) length regulation; cell cycle regulation mologous recombination with DNA pol 58 (58) a–prim probably initiating DNA repli- 48 (48) Primase cation on the lagging strand9. b 39 (68) DNA pol, 59 phosphatase Repair: BER, DSBR; meiosis Functional roles of DNA pol b g 125 (143) DNA pol, 39→59 exonuclease Mitochondrial DNA replication Pol b is a single polypeptide of 39 kDa 35 comprising 335 amino acid residues (re- d 125 (125) DNA pol, 39→59 exonuclease Replication: leading strand, lagging strand; viewed in Ref. 10). It consists of two do- 66 (55) repair: MMR, DSBR, BER, NER; translesion mains connected by a protease- 50 (40) DNA synthesis; cell cycle regulation sensitive hinge region. The 8-kDa N- d (22) terminal domain carries dRpase (to re- e 261 (256) DNA pol, 39→59 exonuclease Replication: lagging strand; repair: DSBR, move the 59-deoxyribose phosphate) 59 (79) BER, NER; cell cycle regulation and template-binding functions, whereas (34) the 31-kDa domain carries the DNA pol (29) activity (Table 1). The 3-D structure of DNA pol b has been determined, and the z 353 (173) DNA pol Error-prone translesion DNA synthesis roles of critical amino acid residues in (29) the catalytic center have been studied5. h 78 (70) DNA pol (Rad30) Error-free translesion DNA synthesis The 39OH primer terminus is perfectly positioned in the active site to conduct a u 198 ? DNA pol, helicase? Repair of interstrand crosslinks nucleophilic attack on the a-phosphorus i ? ? DNA pol (Rad 30B) Error-prone translesion DNA synthesis of the incoming, base-pairing deoxyribonucleoside-59triphosphate. This REV1 (116) Deoxycytidyl transferase Abasic site synthesise mechanism gurantees the proper chain elongation according to the base pair aFor references, see text bMammalian cells (yeast Saccharomyces cerevisiae) rule A–T and G–C. The 3-D structure of cDSBR, double-stranded break repair; BER, base excision repair; MMR, mismatch repair; DNA pol b and mutagenesis studies have NER, nucleotide excision repair. revealed that three aspartic acid d 2 Schizosaccharomyces pombe has a fourth, possibly even a fifth subunit residues in the active site are involved in eA new category of a nucleotide-polymerizing enzyme38 dNMP transfer – like corresponding as- partic acid residues in the active site of The DNA pol a–prim complex consists of DNA pol a–prim also plays an impor- Escherichia coli DNA pol I Klenow frag- four subunits, with molecular masses of tant role in coordinating DNA repli- ment.