DNA‐binding Enzymes: Structural Themes

Abstract

DNA‐binding enzymes play pivotal roles in nucleic acid biosynthesis. Their structural and functional resemblance bears the imprint of DNA evolution.

Keywords: helicase; polymerase; recombinase; restriction enzyme; topoisomerase

Figure 1.

Model of a DNA replication fork with representative DNA‐binding enzymes in action. An enlarged electron micrograph of a negatively stained hexamer complex of Escherichia coli Rho RNA helicase is shown. ssDBP, single‐stranded DNA‐binding protein. From Knopf KW (1974) PhD thesis, Universität Heidelberg.

Figure 2.

Structure and operation of DNA restriction enzymes. Schemes show the DNA interaction of major ENases as follows. The 440‐kDa multifunctional complex of EcoKI, representative of type I enzymes, acts as a modification methyltransferase on the recognition sequences in which one of the underlined adenine residues is already methylated, or as an ENase on DNA containing unmethylated recognition sequences. DNA binding and recognition of the methylation status is operated by the M/S trimer core complex, to which two R subunits are C‐terminally bound, conferring the ENase function that is encoded by an N‐terminal domain. During DNA restriction both R subunits perform ATP‐dependent DNA translocation followed by single‐strand cleavage, producing random double‐strand breaks in a varying distance and on both sides of the recognition sequence. The 24.6‐kDa ENase of BamHI, a type II enzyme, binds the stated palindromic recognition site in the form of an U‐shaped homodimer complex, and simultaneously cleaves both DNA strands in a sequence specific manner, generating double‐stranded DNA fragments with identically staggered ends. The functional 66.2‐kDa monomer of FokI, a type IIs enzyme, possesses two domains: the N‐terminal DNA recognition domain binds to the recognition site (shaded). The C‐terminal restriction domain, flexibly connected with the N‐terminal domain, is positioned alongside the DNA cleavage signal. The ENase also cleaves hemi‐methylated DNA, is specific but generates double‐stranded DNA fragments with staggered ends of unique sequence composition. EcoP15I, a type III enzyme, is composed of separate restriction (R) and modification (MS) subunits. The latter subunit is required for binding to the stated inversely oriented recognition site (shaded) as well as for its methylation. Cleavage is performed by the R subunit only if the recognition site is unmethylated, and occurs a distance of 25 nucleotides downstream from the recognition signal, resulting in double‐stranded DNA fragments with staggered ends of random sequence composition.

Figure 3.

Model of site specific recombination mechanism by Int family recombinases. Recombinase subunits on different DNA strands are marked in yellow and blue circles, and the conserved catalytic tyrosine (Y) is indicated. Two recombinase subunits bind to each recombination site at inverted repeat sequences that are separated by a short ‘crossover’ region, and synapse the duplex DNA substrates by protein–protein interactions (step I). After the formation of a tetrameric bihelical complex, one strand of each DNA target is cleaved at the 5′ ends of the crossover region forming a covalent 3′ phosphotyrosine intermediate (step II). The freed 5′ ends are exchanged and ligated (step III). During an isomerization event the recombinase dimers dissociate and form a four‐way Holliday junction intermediate (step IV). The Holliday junction is resolved when the second pair of recombinase subunits at the other end of the crossover region carries out the cleavage and strand exchange reaction on the unexchanged strands (steps V and VI).

Figure 4.

Operational modes of topoisomerases of type I and II. A type IB enzyme is shown that binds DNA as a monomer, cleaves and reseals only one strand by forming a covalent 3′‐phosphotyrosine linkage during transient linkage. Type II enzymes bind DNA as a dimer or heterotetramer and perform cleavage and resealing of both strands. Transient cleavage involves two covalent 5′‐phosphotyrosine linkages.

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References

Aggarwal AK (1995) Structure and function of restriction endonucleases. Current Opinion in Structural Biology 5: 11–19.

Aggarwal AK and Wah DA (1998) Novel site‐specific DNA endonucleases. Current Opinion in Structural Biology 8: 19–25.

Belfort M and Roberts RJ (1997) Homing endonucleases: keeping the house in order. Nucleic Acids Research 25: 3379–3388.

Berger JM (1998) Structure of DNA topoisomerases. Biochimica et Biophysica Acta 1400: 3–18.

Bird LE, Subramanya HS and Wigley DB (1998) Helicases: a unifying structural theme ? Current Opinion in Structural Biology 8: 14–18.

Brautigam CA and Steitz TA (1998) Structural and functional insights provided by crystal structures of DNA polymerases and their substrate complexes. Current Opinion in Structural Biology 8: 54–63.

Budd ME, and Campbell JL (1997) The roles of the eukaryotic DNA polymerases in DNA repair synthesis. Mutational Research 384: 157–167.

Capriciano G and Binaschi M (1998) DNA sequence selectivity of topoisomerases and topoisomerase poisons. Biochimica et Biophysica Acta 1400: 185–194.

Davies GP, Martin I, Sturrock SS, Cronshaw A, Murray NE and Dryden TF (1999) On the structure and operation of type I DNA restriction enzymes. Journal of Molecular Biology 290: 565–579.

Gobert C, Skladanowski A and Larsen AK (1999) The interaction between p53 and DNA topoisomerase I is regulated differently in cells with wild‐type and mutant p53. Proceedings of the National Academy of Sciences of the USA 96: 10355–10360.

Gopaul DN and Van Duyne GD (1999) Structure and mechanism in site‐specific recombination. Current Opinion in Structural Biology 9: 14–20.

Hallet B and Sherratt DJ (1997) Transposition and site‐specific recombination: adapting DNA cut‐and‐paste mechanisms to a variety of genetic rearrangements. FEMS Microbiological Reviews 21: 157–178.

Ito J and Braithwaite DK (1991) Compilation and alignment of DNA polymerase sequences. Nucleic Acids Research 19: 4045–4057.

Johnson RE, Washington MT, Prakash S and Prakash L (1999) Bridging the gap: A family of novel DNA polymerases that replicate faulty DNA. Proceedings of the National Academy of Sciences of the USA 96: 12224–12226.

Joyce CM and Steitz TA (1994) Function and structure relationships in DNA polymerases. Annual Review of Biochemistry 63: 777–822.

Kimura K, Rybenkov VV, Crisona NJ, Hirano T and Cozzarelli NR (1999) A protein named ‘condensin’ converts interphase chromatin to mitotic chromosomes together with Top I and introduces positive supercoils in presence of ATP. Cell 98: 239–248.

Krüger DH, Kupper D, Meisel A, Reuter M and Schroeder C (1995) The significance of distance and orientation of restriction endonuclease recognition sites in viral DNA genomes. FEMS Microbiological Reviews 17: 177–184.

Nitiss JL (1998) Investigating the biological functions of DNA topoisomerases in eukaryotic cells. Biochimica et Biophysica Acta 1400: 63–81.

Nunes‐Düby SE, Kwon HJ, Tirumalai RS, Ellenberger T and Landy A (1998) Similarities and differences among 105 members of the Int family of site‐specific recombinases. Nucleic Acids Research 26: 391–406.

Roberts RJ and Macelis D. The Restriction Enzyme Database. Massachusetts, USA: New England Biolabs. [http://rebase.neb.com]

Roberts RJ and Macelis D (1998) REBASE – restriction enzymes and methylases. Nucleic Acids Research 26: 338–350.

Roberts RJ and Halford SE (1993) Type II restriction endonucleases. In: Linn SM, Lloyd RS and Roberts RJ (eds) Nucleases, 2nd edn, pp. 35–88. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.

Smith BT and Walker GC (1998) Mutagenesis and more: umuDC and the Escherichia coli SOS response. Genetics 148: 1599–1610.

Szybalski W, Kim SC, Hasan N and Podhajska AJ (1991) Class‐IIS restriction enzymes – a review. Gene 100: 13–26.

Wang TS‐F (1991) Eukaryotic DNA polymerases. Annual Review of Biochemistry 60: 513–552.

Woodgate R (1999) A plethora of lesion‐replicating DNA polymerases. Genes and Development 13: 2191–2195.

Further Reading

Viewing enzyme structural models as well as substrate interactions on the molecular level is best achieved by implementing a molecular viewer like RASMOL in the Internet browser. Necessary PDB.files with the molecular data of the enzymes are obtained through links from conventional sequence databanks (Swiss‐Protein Data Library, Gene Bank, Brookhaven Data Bank, Embl Sequence Library). A recommended site containing various model enzymes is: [http://pdb.weizman.ac.il/scop/data/scop.1.005.html]

Alberts B (1995) Molecular Biology of the Cell, 3rd edn. New York: Garland Publishers.

Aravind L and Koonin EV (1998) Phosphoesterase domains associated with DNA polymerases of diverse origins. Nucleic Acids Research 26: 3746–3752.

Cotterill S (1999) Eukaryotic DNA Replication: A Practical Approach. Practical approach series vol. 199. Oxford and New York: Oxford University Press.

DePamphilis ML (1996) DNA Replication in Eukaryotic Cells. CSH Monograph Series vol. 31. Plainview, New York: Cold Spring Harbor Laboratory Press.

Knippers R (1997) Molekulare Genetik. 7. Auflage. Stuttgart: Thieme‐Verlag.

Kornberg A and Baker TA (1992) DNA Replication, 2nd edn. San Francisco: WH Freeman.

Kucherlapati R and Smith GR (1988) Genetic Recombination. Washington, DC: American Society for Microbiology.

Lewin B (2000) Genes VII, 4th edn. Oxford and New York: Oxford University Press.

Stryer L (1995) Biochemistry, 4th edn. San Francisco: WH Freeman.

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Knopf, Charles W, and Waldeck, Waldemar(Apr 2001) DNA‐binding Enzymes: Structural Themes. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0002717]