Thymidine mimic
5-Bromodeoxyuridine (BUdR) is similar to thymidine and is efficiently phosphorylated and incorporated in its place into DNA. However, BUdR incorporated into DNA often mispairs and increases replication errors. DNA containing BUdR is also more sensitive to some wavelengths of ultraviolet (UV) light (which makes BUdR useful in enrichment schemes for isolating mutants [see chapter 3]). Moreover, DNA containing BUdR has a different density from DNA exclusively containing thymidine (another feature of BUdR that is useful in experiments).
Antibiotics That Affect Gyrase
Many antibiotics and antitumor drugs affect topoisomerases. The type II topoisomerase, gyrase, in bacteria is a target for many different antibiotics. Because this enzyme is similar among all bacteria, these antibiotics have a broad spectrum of activity and kill many types of bacteria.
GyrA inhibition
Nalidixic acid specifically binds to the GyrA subunit, which is involved in cutting the DNA and in strand passage. This activity makes nalidixic acid and its many derivatives, including oxolinic acid and chloromycetin, very useful antibiotics. Another antibiotic that binds to the GyrA subunit, ciprofloxacin, is used for treating gonorrhea, anthrax, and bacterial dysentery. However, because these antibiotics can induce prophages, they may actually make some diseases worse (see chapter 7).
The mechanism of killing by these antibiotics is not completely understood. They are known to cause degradation of DNA and can cause the DNA to become covalently linked to gyrase, presumably by trapping it in an intermediate state in the process of strand passage. Bacteria resistant to nalidixic acid have an altered gyrA gene.
GyrB inhibition
Novobiocin and its more potent relative coumermycin bind to the GyrB subunit, which is involved in ATP binding. These antibiotics do not resemble ATP, but by binding to the gyrase they somehow prevent ATP cleavage, perhaps by changing the conformation of the enzyme. Mutants resistant to novobiocin have an altered gyrB gene.
The Bacterial Genome
The discussion of the replication and structure of the bacterial genome ignores the complexity of the sequences and the functions they encode. Advances in DNA-sequencing technologies are drastically reducing both the time it takes to sequence DNA and the cost of DNA sequencing. We are now at a time when determining the genomic sequence of a newly discovered bacterium is often one of the earliest steps in trying to fully characterize the functions of a new species. In addition, there are also a large number of studies where DNA from a particular environment or a consortium of organisms is chosen for DNA sequencing instead of that of a single organism derived from pure culture. Bacterial and archaeal genomes are in some ways more amenable to genome analysis than eukaryotic genomes because they are relatively small, ranging from ca. 0.5 megabases (Mb) for some obligate parasites to around 10 Mb for some free-living bacteria. They contain few introns and much less repetitive DNA than eukaryotic genomes. Chapter 13 deals with ideas for how genomes are structured and the tools we use to study them.
Summary
1 DNA consists of two strands wrapped around each other in a double helix. Each strand consists of a chain of nucleotides held together by phosphates joining their deoxyribose sugars. Because the phosphate joins the third carbon of one sugar to the fifth carbon of the next sugar, the DNA strands have directionality, or polarity, and have distinct 5′ phosphate and 3′ hydroxyl ends. The two strands of DNA are antiparallel, so that the 5′ end of one is on the same end as the 3′ end of the other.
2 DNA is synthesized from the precursor deoxynucleoside triphosphates by DNA polymerase. The first phosphate of each nucleotide is attached to the 3′ hydroxyl of the next deoxynucleotide, giving off the terminal two phosphates to provide energy for the reaction.
3 DNA polymerases require both a primer and a template strand. The pairing of the bases between the incoming deoxynucleotide and the base on the template strand dictates which deoxynucleotide will be added at each step, with A always pairing with T and G always pairing with C. The DNA polymerase synthesizes DNA in the 5′-to-3′ direction, moving in the 3′-to-5′ direction on the template.
4 DNA polymerases cannot put down the first deoxynucleotide, so RNA is usually used to prime the synthesis of a new strand. Afterward, the RNA primer is removed and replaced by DNA, using upstream DNA as a primer. The use of RNA primers helps reduce errors by allowing editing.
5 DNA polymerase does not synthesize DNA by itself but needs other proteins to help it replicate DNA. These other proteins are helicases that separate the strands of the DNA, ligases to join two DNA pieces together, primases to synthesize RNA primers, and other accessory proteins to keep the DNA polymerase on the DNA and reduce errors.
6 Both strands of double-stranded DNA are replicated from the same end, so that the overall direction of DNA replication is from 5′ to 3′ on one strand and from 3′ to 5′ on the other strand. Because DNA polymerase can polymerize only in the 5′-to-3′ direction, it must replicate one strand in short pieces and ligate them afterward to form a continuous strand. The short pieces are called Okazaki fragments. The two DNA polymerases replicating the leading and lagging template strands remain bound to each other in a process called the trombone model of replication.
7 Lesions on DNA, proteins bound to DNA, and transcription can stall DNA replication. Primase can reinitiate replication to prevent the entire holoenzyme from stalling but leaving a gap that is repaired by other mechanisms. DNA helicases and other enzymes can be used to help DNA polymerase past bound proteins and to allow the polymerase to continue after it stalls from collisions with RNA polymerase.
8 The DNA in a bacterium that carries most of the genes is called the bacterial chromosome. The chromosome of most bacteria is a long, circular molecule that replicates in both directions from a unique origin of replication, oriC. Replication of the chromosomes initiates each time the cells reach a certain size. For fast-growing cells, new rounds of replication initiate before old ones are completed. This accounts for the fact that fast-growing cells have a higher DNA content than slowergrowing cells.
9 Chromosome replication terminates, and the two daughter DNAs separate, when the replication forks meet. In some bacteria, multiple ter sites that act as “one-way gates” delay movement of the replication forks on the chromosome. Proteins that bind to the ter sites that are inhibitors of the DnaB helicase stop replication at these sites.
10 To separate the daughter DNAs after replication, dimerized chromosomes, created by recombination between the daughter DNAs, are resolved by XerC and XerD, a site-specific recombination system that promotes recombination between the dif sites on the daughter chromosomes. The FtsK protein is a DNA translocase that promotes XerC-XerD recombination at dif sites to prevent dimerized chromosomal DNA from being guillotined by the forming septum. Topo IV decatenates the intertwined daughter DNAs by passing the double-stranded DNAs through each other, and its activity is also regulated temporally and spatially via an interaction with FtsK.
11 The