This essentially means that it cannot add nucleotides if a free 3'-OH group is not available. Then how does it add the first nucleotide? The problem is solved with the help of a primer that provides the free 3'-OH end. Because this sequence primes the DNA synthesis, it is appropriately called the primer. You isolate a cell strain in which the joining together of Okazaki fragments is impaired and suspect that a mutation has occurred in an enzyme found at the replication fork.
Which enzyme is most likely to be mutated? The replication fork moves at the rate of nucleotides per second. DNA polymerase can only extend in the 5' to 3' direction, which poses a slight problem at the replication fork. As we know, the DNA double helix is anti-parallel; that is, one strand is in the 5' to 3' direction and the other is oriented in the 3' to 5' direction.
One strand, which is complementary to the 3' to 5' parental DNA strand, is synthesized continuously towards the replication fork because the polymerase can add nucleotides in this direction. This continuously synthesized strand is known as the leading strand.
The other strand, complementary to the 5' to 3' parental DNA, is extended away from the replication fork, in small fragments known as Okazaki fragments , each requiring a primer to start the synthesis. Okazaki fragments are named after the Japanese scientist who first discovered them. The strand with the Okazaki fragments is known as the lagging strand. The leading strand can be extended by one primer alone, whereas the lagging strand needs a new primer for each of the short Okazaki fragments.
The overall direction of the lagging strand will be 3' to 5', and that of the leading strand 5' to 3'.
A protein called the sliding clamp holds the DNA polymerase in place as it continues to add nucleotides. The sliding clamp is a ring-shaped protein that binds to the DNA and holds the polymerase in place.
Topoisomerase prevents the over-winding of the DNA double helix ahead of the replication fork as the DNA is opening up; it does so by causing temporary nicks in the DNA helix and then resealing it. The primers are removed by the exonuclease activity of DNA pol I, and the gaps are filled in by deoxyribonucleotides.
The nicks that remain between the newly synthesized DNA that replaced the RNA primer and the previously synthesized DNA are sealed by the enzyme DNA ligase that catalyzes the formation of phosphodiester linkage between the 3'-OH end of one nucleotide and the 5' phosphate end of the other fragment. Once the chromosome has been completely replicated, the two DNA copies move into two different cells during cell division. The process of DNA replication can be summarized as follows.
The leading strand is the template for lagging strand synthesis, and a CTG on the leading strand serves as a primase binding site and a primer initiation site.
Hence the oligonucleotide bias on leading versus lagging strand fits with the needs for multiple priming events during discontinuous replication. The recombination hot-spot Chi is more frequent along the leading strands. Finally, most genes are transcribed in the same direction as the replication fork moves in these replicores.
The full significance of some of these observations is still not clear, but they point to an overall organization of the genome with respect to replication. It will be of considerable interest to see whether these patterns are found in replicores in other organisms.
Replicores on the E. This is the same strand as is listed in the standard E. The two replication forks meet on the side of the chromosome opposite oriC. Termination occurs in a zone where the forks meet Fig. It is restricted to this zone by the action of the Tus protein at ter sequences. The ter sequences block further progression of the replication fork, with a clear polarity. In contrast, terC and terB block the progress of the clockwise fork Fork 2 in Fig.
The ter sequences are 23 bp and are binding sites for the Tus protein, the product of the tus gene "ter utilization substance" , which is required for termination.
It prevents further helicase action from the replication fork. Resolution of the replicated chromosomes occurs when the two replication forks meet. Since these are moving in opposite directions, the distribution of ter sites roughly opposite to the ori insures that the two replication forks will meet in the zone between the oppositely oriented ter sites. One scenario is illustrated in Fig. Let Fork 1, moving in a counter-clockwise direction, proceed as far as it can, i.
Fork 2, moving in a clockwise direction, can proceed past these ter sites, and will it will meet Fork 1. The two sets of products from each replication fork are then joined. The leading strand synthesized from Fork 2 joins the lagging strand synthesized from Fork 1.
Likewise, the lagging strand from Fork 2 joins the leading strand from Fork 1. Resolution of replication forks in the termination zone.
Use the model for lagging strand synthesis to explain how the leading strand is joined to the lagging strand when the replication forks meet and resolve. Control of initiation at oriC by methylation.
A new round of replication will initiate on the E. The dam methylase and features of its sites of action are used to prevent premature re-initiation. The dam methylase of E. Thus a GATC in duplex DNA can be unmethylated on either strand, methylated on only one strand referred to as hemimethylated or methylated on both strands referred to as fully methylated , as shown in Fig.
However, when the GATCs are hemimethylated, it is not active as an origin. The reason for thnis is not fully known. One hint comes from the behavior of unmethylated oriC from dam - strains. This unmethylated oriC is active, showing that methylation of the GATC is not a requirment for initiation, and further suggesting that some inhibitor of initiation recognizes the hemimethylated form.
How do these results lead to this conclusion? Re-methylation of oriC by the dam methylase is quite slow. This provides a means to delay the use of oriC to start another round of replication. In the next chapter, we will also see the use of methylation of GATCs in post-replicative repair. Eukaryotic organisms usually have to synthesize much more genomic DNA than is found in bacteria, and the template for replication is chromatin, not just DNA.
Also, and perhaps related to the effects of this protein-DNA template, replication fork movement is considerably slower in eukaryotes, being only about 1, to 3, bp per min, compared to the very rapid rate of 50, bp per min in bacteria. Consequently, eukaryotic organisms take more time to replicate their genomes, and they use many origins per chromosome.
Much is now known about the genetics and some biochemistry of replication in the budding yeast Saccharomyces cerevisiae , whereas in plants and animals, more detailed biochemical information is derived largely from viral systems. In this section, we will examine some aspects of the replication origins in yeast and proteins that act at those origins. ARS core consensus. Matches to the core consensus sequences of ARS s are underlined doubly for an exact match and singly if the segment has a single mismatch from the consensus.
Two matches to the consensus overlaps for 5 bp from positions through Replication in S. Many, if not all, of these origins are also autonomously replicating sequences , or ARSs. ARSs were isolated in a similar approach to that used for isolating the bacterial oriC. Yeast plasmids carrying a selectable marker were mutationally inactivated in their plasmid origins, genomic yeast DNA fragments were ligated into the mutated plasmids, and transformed yeast were screened for the selectable marker, which should only be present in strains carrying a replicating plasmid, i.
These ARSs have the genetic properties of replicators. Many have been isolated and mapped, and of course their positions along the chromosome are known because of the complete genomic DNA sequence. Some of these ARSs have now been shown to function biochemically as origins of replication.
One exact and one partial match to this consensus are shown in Fig. The core sequences a and b comprises an ARS consensus sequence that is essential for origin function, but it is not sufficient. Additional sequences surrounding the consensus are also needed. The core consensus sequences are binding sites for proteins involved in replication. The major protein is the origin recognition complex , or ORC.
The complex binds to origins of replication in an ATP-dependent manner and directs DNA replication to start at the origin. ORC was initially isolated on the basis of its ability to bind to ARSs, and subsequent studies have shown that it is required for replication and cell viability. The critical role of the ORC is not restricted to budding yeast. Homologs to the largest subunit, ORC1, have been identified in other fungi, in Drosophila , in amphibians and in humans.
It binds to the specific DNA sequences in the origin, it induces local unwinding of the DNA at the origin, and it recruits other replication enzymes. At least in yeast, the ORC binds stably to the origin even after it has fired , and it is thought that the recruitment of additional proteins, such as Cdc6p and the Mcm proteins see below , is a critical point of control on replication.
Once DNA synthesis has initiated at the origin, new replication forks move bidirectionally in most cases away from the origin, and terminate when they meet opposing replication forks from adjacent replicons.
In this manner, almost all of a linear chromosome is replicated by the many replciation forks that start at multiple origins. However, a problem arises at the ends of the chromsomes, as will be explored in the next section. The requirement of DNA polymerases to have a primer causes a problem at the ends of linear templates. As illustrated in Fig.
If nothing else were done, the chromosome would become progressively shorter after each round of replication. Lagging strand synthesis cannot copy the end of a linear chromosome. At least three different types of solution to this problem have been discovered in various organisms. One, utilized by bacteriophage such as l and T4, is to convert the linear template to a circle. For instance, the linear chromosome of bacteriophage l has cohesive ends complementary single strands at each end, generated by a phage endonuclease that can anneal upon infection, thereby forming a cirucular template for replication.
Other viruses, such as adenovirus, attach a protein to the end of unreplicated DNA to serve as a primer. Such an attached protein obviates the requirement for using the unreplicated DNA as a template, and the entire viral chromosome can be replicated.
A third solution is to make the ends a series of simple repeats that are synthesized in a process distinct from DNA replication. Indeed, the ends of the linear chromosomes of most perhaps all eukaryotes, called telomeres , are composed of many copies of a simple repetitive sequence. This sequence is distinctive for different organisms, but in all cases one strand is rich in G and the other is rich in C.
New copies of the telomeric repeats can be synthesized each time the chromosome replicates Fig. This re-synthesis of the telomeric repeats counteracts the progressive shortening of the linear chromosomes that would occur if only the replication forks were used to synthesize new chromosomes.
Addition of new telomeric repeats to the ends of replicated chromosomes. In this figure, the string of "a" at the ends of the chromosome is the tandem repeat of simple sequence, in duplex form.
For instance, for a human chromosome, "a" would be. In each case, the "a" or monomer is repeated thousands of times in tandem. Addition of new telomeric repeats is catalyzed by the enzyme telomerase. The enzyme is a ribonucleoprotein, i. Then the enzyme shifts over and synthesizes another hexanucleotide. This exchnage led to the addition of telomeres with sequences characteristic of that of the second species, showing that the telomerase RNA is the determinant of the sequence of the telomere.
The protein component provides the reverse transcriptase activity. Examination of the ends of replicating chromosomes in the electron microscope show a circular structure. Some processing, e. Synthesis of new telomeric repeats catalyzed by telomerase.
This enzyme is a ribonucleoprotein complex. The RNA component is the template for synthesis of telomeric repeats. How processive is telomerase? Not all replicating cells have telomerase activity. This activity is higher in some transformed cells than in nontransformed cells.
Also, older cells tend to have shorter telomeres. Thus telomeres are being actively investigated as possibly playing roles in both aging and in tumorigenic transformation. Telomeres are important for stabilizing chromsomes. Some chromosomal deletions remove the ends of the chromosome, including the telomere, and these shortened chromosomes are less stable than their wild-type counterparts. Directed mutations have been made in mice to eliminate telomerase activity. These mice are viable for several generations, but they show many broken and abnormal chromosomes, demonstrating the importance of this activity.
We have seen that the initiator protein DnaA and the replicator element oriC are needed for the initiation of replication, and that the slow rate of methylation at GATC motifs prevents re-initiation for some time. The bacterial cell can sense when the nutritional conditions, levels of nucleotide pools, and protein concentrations are adequate to support a round of replication.
The details of this monitoring are beyond the scope of this presentation, and can be explored in references such as Niedhart et al. In general, initiation is triggered by the increase in cell mass. Initiation occurs at a constant ratio of cell mass to the number of origins. This suggests that a mechanism exists to titrate out some regulatory molecule as the cell mass increases, but the molecule and mechanism have not been elucidated.
The result of this monitoring and signalling is the formation of an active DnaA complex at oriC , followed by unwinding the DNA and the other events discussed above. Depending on the growth conditions, bacteria can divide rapidly or slowly. In rich media, the cell number can double every 18 min, whereas when nutrients are scare, the doubling time can be long as min.
The bacterial cells accomplish this by varying the rate of re-initiation of replication. Re-initiation has to occur at the same frequency as the cell doubling time. Although the frequency of re-initiation can be varied fold, the time required for the replication cycle is constant.
This cycle consists of two periods called C and D. The elongation time , or C period, is the time required to replicate the bacterial chromosome. From initiation to termination, this is about 40 min. The division time , or D period, is the time that elapses between completion of a round of DNA replication and completion of cell division. This is about 20 min. Hence the time for the replication cycle C period plus D period is essentially constant in bacterial cultures with doubling times shorter than 60 min.
The constant replication cycle time means that a round of replication must be initiated 60 min i. However, re-initiation can occur before 60 min has past. This is illustrated in Fig. When the cell doubling time is less than 60 min, a cycle of replication must initiate before the end of the preceding cycle. This results in chromosomes with more than one replication fork. Multiple replication forks per chromosome allow bacteria to divide more rapidly than the replication cycle time.
This diagram illustrates a bacterial cell dividing every 30 min, and hence initiating a new cycle of replication every 30 min. If the time required for two replication forks traveling in opposite directions to traverse the entire E. Actively growing or dividing eukaryotic cells pass through a cell cycle that is divided into four phases Fig.
Classic studies showed that cells in two of these phases are discernable in the light microscope. Cells undergoing mitosis have condensed chromosomes, and in most organisms but not yeast , the nuclear membrane breaks down. Cells with this appearance are in M phase for m itosis. The other observable phase is S phase for DNA s ynthesis. Cells in S phase can be marked by the incorporation of labeled thymidine into the nuclear DNA.
These two phases are separated by two "gaps", G1 and G2. G2 follows completion of DNA replication and precedes the initiation of mitosis. Nonreplicating, or quiescent cells, can be considered to be "out of the cycle" or in a state referred to as G0.
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