Replicating And Repairing The Genome: From Basic Mechanisms To Modern Genetic Technologies
if the DNA is cleaved with restriction enzyme SmaI prior to the column (filled triangles), but still bound to DNA without restriction enzyme (filled circles).4 In panel B, clamp was retained on circular DNA even if ATP and the clamp loader (γ complex) were removed; however, clamp again fell off the DNA if the DNA was cleaved with a (different) restriction enzyme. In panel C, clamp remained bound to circular DNA when the DNA nick was sealed with DNA ligase, showing that the clamp does not need a nick to remain bound. Panel D showed that multiple clamps (roughly 20) can be loaded onto DNA; DNA cleavage with SmaI released the loaded clamps. The data in panel E are remarkable. DNA-binding protein EBNA1 was bound in two locations to the loaded clamp–DNA complex, and then the DNA was cleaved in between. Now, the clamp no longer dissociated when the DNA was linearized, showing that another bound protein can block the clamp from sliding off the ends of linearized DNA! (The insert gel shows that the restriction enzyme did indeed cleave the DNA.) These experiments provided strong evidence that clamp encircles DNA, and later crystal structures showed that the clamp is shaped like a donut with a hole that can accommodate duplex DNA. The figure was reproduced from Stukenberg et al. (1991), with permission from the American Society of Biochemistry and Molecular Biology; permission conveyed by Copyright Clearance Center, Inc.
1Some evidence suggests that the functioning Escherichia coli holoenzyme contains three copies of DNA polymerase, including a spare like the one discussed in the T7 replisome in the previous chapter, but this point is still controversial.
2We will discuss an exception in the section below on replication restart.
3The resulting enzyme free of 5′ to 3′ exonuclease activity, called “Klenow enzyme,” has been used extensively in biotechnology and DNA manipulation in vitro.
4Only a subset of clamp is loaded onto DNA, explaining the peak in the unboundprotein fractions. The dimeric Escherichia coli clamp is named β, explaining the axis label.
Chapter 4
Eukaryotic DNA replication
Normal diploid human cells carry 46 linear chromosomes: two each of the 22 autosomes and two sex chromosomes. Chromosome numbers in this range are quite typical throughout the animal kingdom, although cells of some animal species have more than 100 chromosomes, and certain plant cells even more (e.g., well over 1000 in a particular fern species). While the number of chromosomes per cell can be large depending on the species, in every case, each and every chromosome must be replicated once and only once per cell cycle. The two replicated copies of each chromosome must also be segregated correctly, one into each of the two daughter cells. Both of these processes have a very low error rate. However, failures do occur, often resulting in non-viable daughter cells or contributing to cancer formation in metazoans.
The machinery that replicates chromosomes in eukaryotic cells is significantly more complex than that of prokaryotes. As we will see, this complexity allows a high degree of flexibility in the replication program and a remarkable degree of regulation to ensure a high probability of correctly replicating each chromosome once and only once. Some of the basic machinery involved in replication is conserved throughout evolution, and proteins involved in replication from bacteria to humans show many commonalities in protein structure and molecular mechanisms. In this Chapter, we will focus on the eukaryotic replisome machine and how it functions to duplicate the chromosomal DNA. In the following Chapter, we will shift our attention to how replication is initiated at replication origins, regulated both at the initiation and later stages, and how replication is completed prior to cell division.
The exact number of proteins involved in eukaryotic DNA replication is still not known and almost certainly varies between disparate species. Nonetheless, the number of different proteins is clearly much higher than in prokaryotes, for at least three major reasons (described in more detail in ensuing sections). First, multimeric proteins that are composed of identical subunits in prokaryotes are instead composed of different but related subunits in eukaryotes (for example, the hexameric replicative helicase). Second, eukaryotes were found to have multiple forms of certain key replication protein complexes, each with specialized functions, in contrast to a singular form in prokaryotes (for example, both clamps and clamp loaders). Third, eukaryotic replication systems must deal with additional complexities such as replicating DNA that contains nucleosomes and preparing their multiple chromosomes for accurate segregation. Many additional proteins, including some that travel with the replication fork, are involved in these processes. The growing list of eukaryotic replication proteins is quite staggering, as indicated by Tables 1 and 2 in the Appendix. In these Chapters, we will again try to minimize the nomenclature to keep the focus on important concepts and functions. A few important and commonly used names will be introduced, particularly when these proteins are also central in other cellular functions such as DNA damage responses and DNA repair.
4.1Special challenges of replicating multiple linear chromosomes
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