Conservative and semiconservative mechanisms of DNA replication differ in whether the newly synthesized strands pair with each

other (conservative) or with an old strand (semiconservative). All available evidence supports semiconservative replication in both prokaryotic and eukaryotic cells.

Three mechanisms of DNA strand growth that are consistent with semiconservative replication. The third mechanism bidirectional growth of both strands from a single origin appears to be the most common in both eukaryotes and prokaryotes.

2000 by W. H. Freeman and Company

DNA replication and cell division in a prokaryote. (a) In a bacterial cell, the partially replicated circular chromosome (blue) is attached to the plasma membrane at the origins of the two daughter DNAs (step 1 ). The origins of the replicated chromosomes have independent points of attachment to the membrane and thus move farther apart as new membrane and cell wall forms midway along the length of the cell (step 2). Continued formation of more sections of membrane and cell wall gives rise to a septum dividing the cell (step 3), leading to division of the cytoplasm into two daughter cells, each with a chromosome attached to the plasma membrane (step 4).

Model of initiation of replication at E. coli oriC. The 9-mers and 13-mers are repetitive sequences. Multiple copies of DnaA protein bind to the 9-mers at the origin and then "melt" (separate the strands of) the 13-mer segments. The sole function of DnaC is to deliver DnaB, which is composed of six identical subunits, to the template. One DnaB hexamer clamps around each single strand of DNA at oriC, forming the prepriming complex. DnaB is a helicase, and the two molecules then proceed to unwind the DNA in opposite directions away from the origin. [Adapted from C. Bramhill and A. Kornberg, 1988, Cell 52:743, and S. West, 1996, Cell 86:177.]

At a growing fork, one strand is synthesized from multiple primers. (a) The overall structure of a growing fork. Synthesis of the leading strand, catalyzed by DNA polymerase III, occurs by sequential addition of deoxyribonucleotides in the same direction as movement of the growing fork. (b) Steps in the discontinuous synthesis of the lagging strand. This process requires multiple primers, two DNA polymerases, and ligase, which joins the 3 -hydroxyl end of one (Okazaki) fragment to the 5 -phosphate end of the adjacent fragment. (c) DNA ligation. During this reaction, ligase transiently attaches covalently to the 5 phosphate of one DNA strand, thus activating the phosphate group. E. coli DNA ligase uses NAD+as cofactor, generating NMN and AMP, whereas bacteriophage T4 ligase, commonly used in DNA cloning, uses ATP, generating PPi and AMP.

Schematic model of the relationship between E. coli replication proteins at a growing fork. (1) A single DnaB helicase moves along the lagging-strand template toward its 3 end, thereby melting the duplex DNA at the fork. (2) One core polymerase (core 1) quickly adds nucleotides to the 3 end of the leading strand as its singlestranded template is uncovered by the helicase action of DnaB. This leading-strand polymerase, together with its b-subunit clamp, remains bound to the DNA, synthesizing the leading strand continuously. (3) A second core polymerase (core 2) synthesizes the lagging strand discontinuously as an Okazaki fragment (see Figure 12-9b). The two core polymerase molecules are linked via a dimeric t protein. (4) As each segment of the single-stranded template for the lagging strand is uncovered, it becomes coated with SSB protein and forms a loop. Once synthesis of an Okazaki fragment is completed, the lagging-strand polymerase dissociates from the DNA but the core remains bound to the t-subunit dimer. The released core polymerase subsequently rebinds with the assistance of another b clamp in the region of the primer for the next Okazaki fragment. See the text for additional details. [Adapted from A. Kornberg, 1988, J. Biol. Chem. 263:1; S. Kim et al., 1996, Cell 84:643.]

Action of E. coli type I topoisomerase (Topo I). The DNA-enzyme intermediate contains a covalent bond between the 5 -phosphoryl end of the nicked DNA and a tyrosine residue in the protein (inset). After the free 3 -hydroxyl end of the red cut strand passes under the uncut strand, it attacks the DNA-enzyme phosphoester bond, rejoining the DNA strand. During each round of nicking and resealing catalyzed by E. coli Topo I, one negative supercoil is removed. (The assignment of sign to supercoils is by convention with the helix stood on its end; in a negative supercoil the "front" strand falls from right to left as it passes over the back strand (as here); in a positive supercoil, the front strand falls from left to right.)

Action of E. coli DNA gyrase, a type II topoisomerase. (a) Introduction of negative supercoils. The initial folding introduces no stable change, but the subsequent activity of gyrase produces a stable structure with two negative supercoils. Eukaryotic Topo II enzymes cannot introduce supercoils but can remove negative supercoils from DNA. (b) Catenation and decatenation of two different DNA duplexes. Both prokaryotic and eukaryotic Topo II enzymes can catalyze this reaction. [See N. R. Cozzarelli, 1980, Science 207:953.]

Movement of the growing fork during DNA replication induces formation of positive supercoils in the duplex DNA ahead of the fork. In order for extensive DNA synthesis to proceed, the positive supercoils must be removed (relaxed). This can be accomplished by E. coli DNA gyrase and by eukaryotic type I and type II topoisomerases. [Adapted from A. Kornberg and T. Baker, 1992, DNA Replication, 2d ed., W. H. Freeman and Company, p. 380.]

Completion of replication of circular DNA molecules. Denaturation of the unreplicated terminus followed by supercoiling overcomes the steric and topological constraints of copying the terminus. At least with SV40 DNA, the final two steps (synthesis and decatenation) can occur in either order depending on experimental conditions. Parental strands are in dark colors; daughter strands in light colors. (Inset) Electron micrograph of two fully replicated SV40 DNA molecules interlocked twice. This structure would result if synthesis was completed before decatenation. Topo II can catalyze decatenation of such interlocked circles in vitro. [Drawing adapted from S. Wasserman and N. Cozzarelli, 1986, Science 232:951. Micrograph from O. Sundin and A. Varshavsky, 1981, Cell 25:659; courtesy of A. Varshavsky.]

the horizontal black arrows show the direction that the replication forks are moving. Wherever the replication fork of a strand is moving towards the 3' end, the newly-synthesized DNA (red) begins as Okazaki fragments (red dashes). This continues until close to the end of the chromosome. Then, as the replication fork nears the end of the DNA, there is no longer enough template to continue forming Okazaki fragments. So the 5' end of each newly-synthesized strand cannot be completed. Thus each of the daughter chromosomes will have a shortened telomere. It is estimated that human telomeres lose about 100 base pairs from their telomeric DNA at each mitosis. This represents about 16 TTAGGG repeats. At this rate, after 125 mitotic divisions, the telomeres would be completely gone. Is this why normal somatic cells are limited in the number of mitotic divisions before they die out?

Telomere replication. Shown here are the reactions involved in synthesizing the repeating G-rich sequences that form the ends of the chromosomes (telomeres) of diverse eucaryotic organisms. The 3 end of the parental DNA strand is extended by RNA-templated DNA synthesis; this allows the incomplete daughter DNA strand that is paired with it to be extended in its 5 direction. This incomplete, lagging strand is presumed to be completed by DNA polymerase , which carries a DNA primase as one of its subunits. The telomere sequence illustrated is that of the ciliate Tetrahymena, in which these reactions were first discovered. The telomere repeats are GGGTTG in the ciliate Tetrahymena, GGGTTA in humans, and G13A in the yeast S. cerevisiae.