The integrity of the structure and nucleotide sequence of DNA is of utmost importance to the cell. This is reflected in the complexity and redundancy of the enzyme systems that participate in DNA replication, repair, and recombination.
Replication of DNA occurs with very high fidelity and within a designated time period in the cell cycle. Replication is semiconservative, with each strand acting as a template for a new daughter strand. The reaction starts at a sequence in the DNA called the origin, and usually proceeds bidirectionally from that point. DNA is synthesized in the 5'?3' direction by DNA polymerases. At the replication fork, the leading strand is synthesized continuously and in the same direction as replication fork movement. The lagging strand is synthesized discontinuously. The fidelity of DNA replication is maintained by (1) base selection by the polymerase, (2) a 3'?5' proofreading exonuclease activity that is part of most DNA polymerases, and (3) a specific repair system that repairs any mismatches left behind after replication.
Most cells have several DNA polymerases. In E. coli, DNA polymerase III is the primary replication enzyme. DNA polymerase I is responsible for special functions during replication, recombination, and repair. DNA polymerase II has a specialized replication activity that allows it to replicate past DNA lesions in error-prone DNA repair. Replication of the E. coli chromosome involves many enzymes and protein factors organized into complexes. Initiation of replication requires binding of DnaA protein to the origin, strand separation, and the entry of the DnaB and DnaC proteins to set up two replication forks. The action of DnaA is associated with the E. coli membrane and is regulated by the action of acidic phospholipids. Initiation is the only phase of replication that is regulated. The process of elongation has different requirements for each strand. DNA strands are separated by helicases, and the resulting topological strain is relieved by topoisomerases. Single-strand DNA binding proteins stabilize the separated strands. In synthesis of the lagging strand, the primosome protein complex moves with the fork and regulates the synthesis of RNA primers by primase. Synthesis of the leading and lagging strands by DNA polymerase III may be coupled. RNA primers are removed and replaced with DNA by DNA polymerase I, and nicks are sealed by DNA ligase.
A similar pattern of replication occurs in eukaryotic cells, but eukaryotic chromosomes have multiple replication origins. Several eukaryotic DNA polymerases have been identified.
Every cell also has multiple and sometimes redundant systems for DNA repair. Mismatch repair in E. coli is directed by transient undermethylation of (5')GATC sequences on the newly synthesized strand after replication. Other systems recognize and repair damage caused by environmental agents such as radiation and alkylating agents, and damage caused by spontaneous reactions of nucleotides. Some repair systems recognize and excise only damaged or incorrect bases te.g., uracil), leaving an AP (apurinic or apyrimidinic) site in the DNA. This is repaired by excising and replacing the segment of DNA containing the AP site. Other excision repair systems recognize and remove pyrimidine dimers and other modified nucleotides. Some types of DNA damage can also be repaired by direct reversal of the reaction causing the damage: pyrimidine dimers are directly converted to monomeric pyrimidines by photolyase, and the methyl group in O6-methylguanine is removed by a specific methyltransferase. Errorprone repair is a specialized and mutagenic replication process observed when DNA damage is so heavy that the need for some replication outweighs the need to avoid errors.
DNA sequences are rearranged in recombination reactions. Homologous genetic recombination occurs between any two DNAs that share sequence homology. This reaction takes place in meiosis (in eukaryotes) and is one of the processes that creates genetic diversity. Homologous recombination also is needed for repair of some types of DNA damage. A Holliday intermediate in which a crossover has occurred between the strands of two homologous DNAs is formed during the process. In E. coli, the RecA protein promotes formation of Holliday intermediates and branch migration to extend heteroduplex DNA.
Site-specific recombination occurs only at specific target sequences and can also involve a Holliday intermediate. The recombinases cleave the DNA at specific points and ligate the strands to new partners. This type of recombination is found in virtually all cells, and its many functions include DNA integration and regulation of gene expression. In vertebrates, a programmed recombination reaction related to site-specific recombination is used to join immunoglobulin gene segments to form immunoglobulin genes during B-lymphocyte differentiation. Some small segments of DNA, called transposons, are capable of moving from one point in a chromosome to another point in the same or another chromosome. These elements are found in virtually all cells.
General
Kornberg, A. & Baker, T.A. (1991) DNA Replication, 2nd edn, W.H. Freeman and Company, New York.
An excellent primary source.
Kucherlapati, R. & Smith, G.R. (eds) (1988) Genetic Recombination, American Society of Microbiology, Washington, DC.
Excellent reviews on a wide ~ssortment of recombination topics.
Richardson, C.C. & Lehman, I.R. (eds) (1990) Molecular Mechanisms in DNA Replication and Recombination, Alan R. Liss, Inc., New York.
A collection of a papers from a major symposium on the topic.
Replication
Bramhill, D. & Kornberg, A. (1988) A model for initiation at origins of DNA replication. Cell 54, 915-918.
Burgers, P.M.J. (1989) Eukaryotic DNA polymerases a and d conserved properties and interactions, from yeast to mammalian cells. Prog. Nucleic Acid Res. Mol. Biol. 37, 235-280.
Campbell, J. (1988) Eukaryotic DNA replication: yeast bares its ARSs. Trends Biochem. Sci. 13, 212-217.
Echols, H. & Goodman, M.F. (1991) Fidelity mechanisms in DNA replication. Annu. Rev. Biochem. 60, 477-511.
Marians, K.J. (1992) Prokaryotic DNA replication. Annu. Reu. Biochem. 61, 673-719.
MeHenry, C.S. (1991) DNA polymerase III holoenzyme. J. Biol. Chem. 266, 19127-19130.
Radman, M. & Wagner, R. (1988) The high fidelity of DNA duplication. Sci. Am. 259 (August), 40-46.
Wang, T.S.-F. (1991) Eukaryotic DNA polymerases. Annu. Reu. Biochem. 60, 513-552.
Repair
Friedberg, E.C. (1985) DNA Repair, W.H. Freeman and Company, New York.
Modrich, P. (1991) Mechanisms and biological ef fects of mismatch repair. Annu. Rev. Genet. 25, 229-253.
Sancar, A. & Sancar, G.B. (1988) DNA repair enzymes. Annu. Rev. Biochem. 57, 29-67.
Recombination
Berg, D.E. & Howe, M.M. (eds) (1989) Mobile DNA, American Society for Microbiology, Washington, DC.
Reviews covering mezny topics related to transposition.
Cox, M.M. & Lehman, I.R. (1987) Enzymes of genetic recombination. Annu. Rev. Biochem. 56, 229262.
Craig, N.L. (1988) The mechanism of conservative site-specific recombination. Annu. Reu. Genet. 22, 77-105.
Landy, A. (1989) Dynamic, structural, and regulatory aspects of l site-specific recombination. Annu. Rev. Biochem. 58, 913-949.
A thorough description of the protein-DNA complexes involved in this reaction.
Mizuuchi, K. (1992) Transpositional recombination: insights from Mu and other elements. Annu. Rev. Biochem. 61, 1011-1051.
Radding, C.M. (1991) Helical interactions in homologous pairing and strand exchange driven by RecA protein. J. Biol. Chem. 266, 5355-5358.
A good, short summary.
Roca, A.I. & Cox, M.M. (1990) The R.ecA protein: structure and funetion. Crit. Rev. Biochem. Mol. Biol. 25, 415-456.
Taylor, A.F. (1992) Movement and resolution of Holliday junctions by enzymes from E. coli. Cell 69, 1063-1065.
1. Conclusions from the Meselson-Stahl Experiment The Meselson-Stahl experiment proved that DNA undergoes semiconservative replication in E. coli. In the "dispersive" model of DNA replication, the parent DNA strands are cleaved into pieces of random size and are then joined with pieces of the newly replicated DNA to yield daughter duplexes in which, in the Meselson-Stahl experiment, both strands would contain random segments of both heavy and light DNA. Explain how the results of the Meselson-Stahl experiment ruled out such a model.
2. Number of Turns in the E. coli Chromosome How many turns must be unwound during replication of the E. coli chromosome? The chromosome contains about 4.7 × 106 base pairs.
3. Replication Time in E. coli From the data in this chapter, how long would it take to replicate the E. coli chromosome at 37 °C, if two replication forks start from the origin? Under some conditions E. coli cells can divide every 20 min. Can you suggest how this is possible?
4. Base Composition of DNAs Made from SingleStranded Templates Determine the base composition you might expect in the total DNA synthesized by DNA polymerase on templates provided by an equimolar mixture of the two complementary strands of circular bacteriophage fX174 DNA. The base composition of one strand is A, 24.7%; G, 24.1%; C, 18.5%; and T, 32.7%. What assumption is necessary to answer this problem?
5. Okazaki Fragments In the replication of the E. coli chromosome, about how many Okazaki fragments would be formed? What factors guarantee that the numerous Okazaki fragments are assembled in the correct order in the new DNA?
6. Leading and Lagging Strands List and compare the precursors and enzymes needed to make the leading versus lagging strands during DNA replication in E. coli.
7. Fidelity of Replication of DNA What factors participate in ensuring the fidelity of replication during the synthesis of the leading strand of a new DNA? Would you expect the lagging strand to be made with the same fidelity as the leading strand? Give reasons for your answers.
8. DNA Repair Mechanisms Vertebrate and plant cells often methylate cytosine in DNA to form 5-methylcytosine (see Fig. 12-5a). In these same cells, there is a specialized repair system that recognizes G-T mismatches and repairs them to G=C base pairs. Rationalize this repair system m terms of the presence of 5-methylcytosine in the DNA.
9. Holliday Intermediates How are the Holliday intermediates formed in homologous genetic recombination and in site-specific recombination dif ferent?
10. DNA Recombination A circular DNA molecule is converted to two smaller circles by an enzyme or enzymes in a crude cellular extract. What types of recombination could account for this reaction, and what else must you know to determine which type it is?
