Читать книгу Replicating And Repairing The Genome: From Basic Mechanisms To Modern Genetic Technologies - Kenneth N Kreuzer - Страница 41

One of the major challenges of DNA replication in eukaryotes is the large amount of DNA to be replicated. As mentioned above, eukaryotic cells have multiple chromosomes. Each chromosome is believed to contain one double helix of DNA,¹ which can be quite long. For example, the longest human chromosome, number 1, is just under 250 million base pairs in length, more than 50 times longer than the E. coli chromosome. At the same time, the eukaryotic replication machinery travels about 20 times slower than that of bacteria. If human chromosome 1 had only one origin near its center, which initiated bidirectional replication (like in E. coli), it would take about a month to replicate the entire chromosome (40 minutes × 50 × 20; recall that chromosomal replication in E. coli takes 40 minutes). However, we know that human cells can replicate their DNA and divide in as little as 24 hours. The solution to this riddle is that all known eukaryotic chromosomes contain multiple replication origins that must fire in order for DNA replication to keep up with cell division. Thus, during cellular DNA replication, any given chromosome can have multiple replication complexes, some moving towards each other relative to the parental DNA and some away from each other. Particularly in the next Chapter, we will see how regulation of this origin firing is used to achieve different rates of cell division and to overcome problems, such as replication-fork blockage, that occur during replication.

The slower rate of fork movement in eukaryotes probably relates largely to the complex structure of eukaryotic chromosomes. The DNA is wrapped around nucleosomes in 200-base pair segments, and these nucleosomes are packed into complex higher-order structures. As the DNA is replicated, the replication fork needs to access the duplex DNA wrapped into the nucleosome in order to duplicate the DNA, and then a new nucleosome must be assembled so that each daughter chromosome has the full nucleosome complement. Furthermore, the histones within nucleosomes carry multiple and complex modifications that are particularly important in controlling whether the nearby genes are expressed or not, and these modifications must also be regenerated in the nucleosomes of both daughter DNA molecules. Details about how nucleosomes become duplicated and maintain their proper modifications are the subject of extensive current investigations, but are beyond the scope of this book. It is worth mentioning that a complex set of proteins are needed to manipulate the histones during and immediately after the replication event, and some of these proteins indeed are directly associated with the replication machinery.

As we will discuss in the next Chapter, the multiple origins of eukaryotic chromosomes do not all fire in every cell division. Furthermore, the subset of origins that do fire varies by developmental stage, tissue type, and time during the cell cycle, and DNA damage responses can activate additional origins that would otherwise be inactive. This extreme flexibility in origin usage means that replication forks terminate in very diverse, perhaps random, locations throughout the chromosome. In a large majority of the genome, two replication forks traveling towards each other simply terminate wherever they meet. This process must involve disassembly of the replicative helicase, to prevent replication forks from essentially “passing” each other and replicating DNA that has already been replicated. Two proteins have been implicated in replicative polymerase unloading, and the process is under active investigation.

All known DNA polymerases synthesize DNA in the 5′ to 3′ direction and nearly all require a pre-existing primer to initiate synthesis. These features lead to the “end-replication problem”, that is, the need for a special mechanism to allow replication of the 3′ end of the parental strand of a linear DNA. To illustrate the problem, imagine that the RNA primer is synthesized by a primase exactly at the 3′ end of the parental DNA to allow DNA polymerase to begin synthesis (Figure 4.1A). Since RNA primers are erased during Okazaki fragment processing, the new 5′ end will be left with a gap of several unreplicated bases after RNA removal. During the next round of replication, the 5′ end of this daughter chromosome can be replicated up to its 5′ end, but the duplex DNA will be several nucleotides shorter because of the gap that was left behind. Progressive rounds of replication will make the daughter chromosomes shorter and shorter. The problem is worse than this, because all known primase enzymes have some modest sequence specificity. Even if the very end of the chromosome initially has a primase site, this site will be lost with the first shortening of the chromosome, requiring the use of a primase site further down the chromosome. Thus, the shortening will occur much faster than just a few base residues per replication cycle.

Figure 4.1.The end-replication problem. Replicative DNA polymerases require a pre-existing primer, usually RNA. Even if an RNA primer is synthesized at the precise end of a duplex DNA, removal of the primer after replication and a second round of replication results in shortening of the chromosome end (panel A). Two solutions to the end-replication problem are circular chromosomes (and plasmids) and terminal protein priming (panel B).

Nature has evolved several solutions to the end-replication problem (Figure 4.1B). The first is to avoid ends by using circular DNA, as found in bacteria, some viruses, and mitochondria. Another solution, used by certain viruses including adenovirus, involves the attachment of a special terminal-priming protein to the end of the genome. This terminal protein does not synthesize primers, but rather itself serves as the primer for DNA polymerase. The viral DNA polymerase uses a specific serine-hydroxyl group on the terminal protein to form a phosphodiester bond with the α-phosphate of the first deoxynucleotide to be added. Once the first deoxynucleotide residue is incorporated, the remainder of the strand can be synthesized by the usual 5′ to 3′ reaction of DNA polymerase. The process has additional complexities, and the reader is encouraged to explore this interesting topic with further reading.

The third solution to the end-replication problem, used by virtually all eukaryotic chromosomes, involves the use of special structures called telomeres at the ends of the chromosome. The mechanisms involved in telomere replication will be discussed in the next Chapter, along with the implications of telomere biology with regard to stem cells, aging and cancer.