Chromosomes unwinding

Topoisomerases - Bidirectional replication of the circular E. coli chromosome unwinds about 100,000 base pairs per minute. Relief of this torsional stress is essential for DNA replication to occur. Topoisomerases are enzymes with a "swivel" mechanism that can relieve this stress. There are two general classes of topoisomerases, type I (Figure 24.30) and type II (Figure 24.31). Type I enzymes change the DNA linking number (see here for reference) in units of 1, whereas type II enzymes change the linking number in units of 2. 

As described in section 4.1, the DNA double helix must unwind to allow access ofthe polymerase enzymes to produce two new strands ofDNA. This is facilitated by DNA gyrase, the target of the quinolones. Some agents interfere with the unwinding of the chromosome by physical obstruction. These include the acridine dyes, of which the topical antiseptic proflavine is the most familiar, and the antimalarial acridine, mepacrine. They prevent strand separation by insertion (intercalation) between base pairs from each strand, but exhibit very poor selective toxicity. 

DNA replication begins with protein binding to the origin of replication, a unique sequence in the bacterial chromosome, causing a short region of double-stranded DNA (dsDNA) to unwind (Figure 11-2). 

Although the action of the DNA polymerase I, according to Eq. 27-3, provided a straightforward way to form a complementary strand of DNA, it did not explain how double-stranded DNA could be copied. One problem is that the two strands must be separated and unwound. If unwinding and replication occured at a single replication fork in the DNA, as indicated by Caims experiment, the entire molecule would have to spin at a speed of 300 revolutions per second to permit replication of the E. coli chromosome in 20 min. It also required that some kind of a swivel, or at least a... 

The elongation step in E. coli chromosomal DNA synthesis is depicted in figure 26.12. As the DNA gradually unwinds new synthesis takes place on the two strands. At the heart of... 

Many proteins are required for DNA synthesis and chromosomal replication. These include polymerases helicases, which unwind the parental duplex enzymes that fill in the gaps and join the ends in the case of lagging-strand synthesis enzymes that synthesize RNA primers at various points along the DNA tem-... 

RNA formation, from a DNA template, is initiated at sites on DNA known as promoters (Chap. 17). For primer formation at the moving replication fork the cell provides a mobile promoter that moves along the 5 —>3 template and functions at regular intervals to promote the initiation of RNA primer synthesis by the primase. The role of the mobile promoter (sometimes referred to as the primosome) on the E. coli chromosome is performed by the protein/enzyme DnaB. DnaB facilitates the action of primase (DnaG), with which it is presumably loosely associated, at regular intervals. (DnaB also has an important role in unwinding the DNA, see below.)... 

DNA damage (unwinding rate) in hepatocytes Chromosomal aberrations in bone marrow cells Chromosomal aberrations in male germinal cells Chromosomal aberrations in male germinal cells Dominant lethality... 

Rothmund-Thomson (Recql 4) proteins. The genes that encode these proteins are altered in human syndromes of the same name. Patients with these diseases are predisposed to different forms of cancer. Cell lines derived from patients with these diseases exhibit chromosome instability and accumulate abnormal replication intermediates. When these helicase proteins are studied in vitro, they can unwind Holliday junction-like structures. A characteristic of many proteins involved in homologous recombination is that they can be visualized as forming subnuclear structures in cells in response to DNA damage. This can be directly visualized using immunofluorescence and the structures that are formed are called foci (see Section 23.8.2). 

E. coli can divide every 40 minutes. Thus, its DNA (MW = 2.2x 10 ) can be duplicated in 40 minutes (or less). Calculate (a) the number of internucleotide bonds made per minute, (b) the rate of chromosome duplication in terms of mm/min and /xm/min (assuming only one growing point), and (c) the rate at which the double helix unwinds (turns/min) during duplication. 

The answer is c. (Murray, pp 412-434. Scriver, pp 3-45. Sack, pp 3—29. Wilson, pp 99-121.) Despite the great length of the chromosomes of eukaryotic DNA, the actual replication time is only minutes. This is because eukaryotic DNA is replicated bidirectionally from many points of origin. The hundreds of initiation sites for DNA replication on chromosomes share a consensus sequence called an autonomous replication sequence (ARS). Thus, while the process of DNA replication in mammals is similar to that in bacteria, with DNA polymerases of similar optimal temperatures and speed, the many replication forks allow for a rapid synthesis of chromosomal DNA. Proteins such as histones, which are bound to mammalian chromosomes, inhibit DNA replication or transcription. Dissociation of the protein-DNA complex (chromatin) and unwinding of DNA supercoils (followed by chromatin reassembly) is part of the replication process. 

FIGURE 124-9. Structure and function of DNA. Within the cellular nucleus, tightly coiled strands of DNA are packaged in units called chromosomes. Working subunits of chromosomes are called genes. During DNA replication, the double-stranded DNA helix unwinds, exposing individual nucleotides. Complementary nucleotides are retrieved and assembled by DNA polymerases to form new strands of DNA. 

Classical Method. This method (Rl) involves isopycnic centrifugation of cleared lysate in a solution of CsCl containing ethidium bromide (EtBr). EtBr binds by intercalating between DNA base pairs, which causes the DNA to unwind. A covalently closed circular (ccc) DNA molecule such as a plasmid has no free ends and can only unwind to a limited extent, thus limiting the amount of bound EtBr. Linear DNA, such as fragmented chromosomal DNA, has no such topological constraints and can therefore bind more of the EtBr molecules. Because the density of the DNA/EtBr complex decreases as more EtBr is bound, and because more EtBr can be bound to a linear molecule than a covalent circle, the... 

The replication of the circular E. coli chromosome (Figure 18.5) begins at a precise initiation site referred to as oriC and proceeds in two directions. Helicases unwind the DNA duplex, two replisomes assemble, and replication proceeds outward in both directions. As the two sites of active DNA synthesis (referred to as replication forks) move farther away from each other, a replication eye forms. Because an E. coli chromosome contains one initiation site, it is considered a sin-... 

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