Prokaryote, DNA

We doubt that there will prove to be a generally close linkage between DNA modules (exons) and protein modules (domains or sub-domains). In addition to the above arguments, the proposed linkage must also deal with the awkward problem that is posed by prokaryotic DNA, for which there is no evidence of introns. Nonetheless, we will continue to watch with great interest for additional relevant evidence. 

The primary structure of DNA is a one-dimensional system similar to four-letter text and can be subjected to the simplest combinatory rules. The particular motifs can be combined with one or several other motifs in away similar to using building blocks. For instance, G-rich motif can be added to one or both ODN flanks. A certain sequence, e.g., a sequence containing unmethylated deoxyribodi-nucleotide CpG motifs that mimic prokaryotic DNA (I), can be placed between similar or different motifs, like GC-rich palindrome and/or G-rich motifs (Fig. I). Various motif combinations will yield a number of putative DNA sequence variants that can be used for further tests and selection of perspective ODN compounds (see Notes 1-4). 

B. Prokaryotic DNA replication is accomplished by DNA polymerases, large multienzyme complexes that move out bidirectionally from the origin of replication. 

Figure 11-2. The prokaryotic DNA replication fork. A schematic representation of semi-conservative replication of DNA by different mechanisms on the leading and lagging strands by DNA polymerase III (DNA pol III) is shown. Other enzymes and accessory proteins that participate in initiation, elongation, and ligation phases of the process are indicated, with DNA pol I depicted as having just dissociated from a completed Okasaki fragment. SSBs, single-stranded DNA binding proteins.

The process of eukaryotic DNA replication closely follows that of prokaryotic DNA synthesis. Some differences, such as the multiple origins of replication in eukaryotic cells versus single origins of replication in prokaryotes, have already been discussed. Eukaryotic single-stranded DNA-binding proteins and ATP-dependent DNA helicases have been identified, whose functions are analogous to those of the prokaryotic enzymes previously discussed. In contrast, RNA primers are removed by RNase H. 

Prokaryotic DNA polymerases are so accurate that special kinetic assays have had to be introduced to detect errors in vitro. These depend on replicating under controlled conditions the circular DNA of a small bacteriophage that contains a... 

DNA synthesis proceeds in the 5 — 3 direction, with the nucleotides being added to the 3 -hydroxyl of the polynucleotide. At the same time, all prokaryotic DNA polymerases have a 3 — 5 exonuclease activity that works in the... 

There are many different types of DNA polymerases, and they vary greatly in their activities and in the nature of the reactions they catalyze. Some polymerases are involved mainly in the replication of DNA. Others are used for the repair of damaged DNA. There is also an important difference between the enzymes isolated from eukaryotes and those isolated from prokaryotes. Most of the eukaryotic DNA polymerases that have been isolated so far have just the simple 5 —> 3 polymerization activity shown in equation 14.1. Prokaryotic DNA polymerases, however, are multifunctional. In addition to their 5 — 3 polymerase activity, they possess a 3 5 exonuclease activity that can excise incorporated... 

Stanford University received a product patent for prokaryote DNA. 

Eukaryotic DNA is replicated at a slower rate than prokaryotic DNA. One reason may be the requirement for the deposition of histone proteins on DNA (histone synthesis and DNA replication are coupled). Describe a model for the replication of eukaryotic DNA and nucleosome formation. 

In prokaryotes DNA, RNA, and protein synthesis all take place in the same cellular compartment. In eukaryotes the DNA is compartmentalized in the cell nucleus, and it became clear long before the biochemistry of these three processes was understood that DNA synthesis takes place in the nucleus, whereas the bulk of protein synthesis takes place in the cytoplasm. From these observations on eukaryotes it was self-evident that DNA cannot be directly involved in the synthesis of protein but must somehow transmit its genetic information for protein synthesis to the cytoplasm. Careful experiments with radioactive labels were used to demonstrate that RNA synthesis takes place in the nucleus much of this RNA is degraded rather quickly, but the portion that survives is mostly transferred to the cytoplasm (fig. 28.1). From observations of this kind it became clear that RNA was the prime candidate for the carrier of genetic information for the synthesis of proteins. 

Mannheim market a combination of two antibiotics for sequential use (BM cycle) and quinolones such as ciprofloxacin (Schmitt et al., 1988) and MRA (available from Flow Laboratories for use at 0.5-10 fig/ml) are very effective at eliminating mycoplasma. The quinolones inhibit the prokaryotic DNA gyrase. 

The above description of prokaryotic DNA replication has been pieced together by the enormous efforts of a large number of laboratories. The replication of eukaryotic DNA appears to be much more complex, and therefore much less is known about it. 

In E. coli and other prokaryotes, DNA is localized in a nucleoid region with no surrounding membrane. All genes are contained on a single, double-stranded, supercoiled, circular DNA molecule. The extended length of the "circle" is about 1300 pm, with a diameter of 2 x 10 3 pm. Because E. coli has a diameter and a length of about 1 and 2-3 pm, respectively, it is obvious that its DNA must be highly coiled and folded to reside in the cell. 

In prokaryotes DNA polymerase I has a 5 — 3 DNA polymerase activity as well as a proof reading capacity to chop out nucleotides in either direction through a 5 — 3 and a 3 — 5 direction exonuclease activity DNA polymerases II and III have 5 — 3 ... 

The initiation codon, usually an AUG, signals the start of translation, and a termination codon marks the end of the translated region. In the analysis of prokaryotic DNA sequences, the signals include the transcriptional and translational initiation sites, the ribosome-binding site, and the transcriptional and translational termination sites. Due to the interrupted nature of the eukaryotic genes, the signals include the translation initiation sites, the intron/exon boundaries (splice sites), translational termination sites, and the polyadenylation sites. 

In addition, three enzymes involved in DNA replication, including DNA primases, prokaryotic DNA topoisomerase I and some hexameric DNA helicases, are also classic zinc-ribbon proteins. In bacteriophage DNA primases, mutations of the zinc-binding residues abrogate the synthesis of RNA primers for lagging strand DNA synthesis. Strikingly, each subunit of the mini-chromosomal maintenance (MCM) protein, a heterohexameric helicase that initiates DNA replication in S. cerevisiae, contains an independently folded zinc-ribbon domain that appears to stabilize the dodecameric structure (a dimer of hexamers) of this replication complex. ... 

Making the link from structural organization is sometimes considered trivial. It is not, however. The intracellular structures themselves are not constants but depend on the effect of a multimde of cooperating non-equilibrium processes. This is clear for the supercoiling state of prokaryotic DNA (Snoep et al. 2002), but also for the complex structure of chromatin in higher eukaryotes. 

To date most of the proteins expressed with 5-OHTrp or 7-ATrp have been prokaryotic. Those that we are aware of are listed in Table III. The prokaryotic DNA-binding proteins listed have been prepared to provide absorption spectra that can be easily resolved from that of DNA. Of these, only BirA has an enzymatic activity, and it has a Trp in its active site. It is therefore of interest that the 5-OHTrp-containing protein has no enzymatic activity. Its DNA-binding properties have not yet been tested. The other DNA-binding proteins all appear to have wild-type function. To date, there is not enough data available to make similar generalizations about eukaryotic proteins. 

Prokaryotic DNA-Binding Proteins Bind Specifically to Regulatory Sites in Operons... 

The Helix-Turn-Helix Motif Is Common to Many Prokaryotic DNA-Binding Proteins... 

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