Replication. Replication fork

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Lynne Baker
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1 Replication In their seminal paper on the DNA double helix published in Nature on April 25 th, 1953, Watson and Crick wrote: It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material. Indeed, during DNA replication within the cell, the two strands of the parent DNA molecule are separated and on each of them a special enzyme, DNA polymerase, synthesizes the complementary strands using deoxynucleoside triphosphates (dntps) as precursors (see Chemical Structure of Proteins and Nucleic Acids). The processes of separation of the strands and the synthesis of new strands proceed in parallel so that the replication fork is formed and it moves along the parent dsdna molecule. There are two fundamental facts about all DNA polymerases, which are crucial for the process of DNA replication: 1. Since in dntps used by the DNA polymerase during synthesis of the new DNA strand the ppp group is attached to the 5 end of the nucleoside, the new nucleotide can be added only to the 3 end of the growing DNA chain. This fact determines the strict 5 3 direction of the extension of the DNA strand. 2. Similarly to RNA polymerase, DNA polymerase requires template for the complementary chain synthesis but, in contrast to RNA polymerase, DNA polymerase cannot initiate synthesis and requires a primer. Both DNA and RNA chains can serve as primers, the only requirement is the presence of the 3 OH group so that DNA polymerase could attach the next nucleotide to it. The reaction of extension of the primer by DNA polymerase is called the primer extension reaction. This reaction is crucial for DNA replication and for two major techniques responsible for the current revolution in biomedicine: PCR and DNA sequencing. But how can DNA be gradually replicated if the strands are antiparallel while primer extension reaction can proceed only in the 5 3 direction? Replication fork Well, replication indeed is a process that is far from trivial. The replication fork is shown in the sketch and the cartoon below. One of the strands, called the leading strand, is synthesized to its (almost) full length since the direction of synthesis coincides with the 5 3 direction. The other strand, called the lagging strand, cannot be synthesized this way. So it is synthesized in the form of very short fragments (consisting from a hundred nt in case of eukaryotes to a thousand nt in case of prokaryotes), which are

The replication fork. Above: schematics in which DNA strands are shown in solid lines, RNA primers are shown in waving lines. Below: cartoon showing most of players participating in replication.

2 The replication fork. Above: schematics in which DNA strands are shown in solid lines, RNA primers are shown in waving lines. Below: cartoon showing most of players participating in replication. known as Okazaki fragments, called after the Japanese molecular biologist who first discovered them back in 1960s. Unlike in case of PCR and sequencing methods, the cell cannot utilize DNA primers since

3 DNA polymerase cannot synthesize primers. The cell uses for this purpose the RNA polymerase enzyme, which does not require a primer (see section Transcription and Open Reading Frame). This special RNA polymerase, which is not sequence-selective, is called primase (see cartoon above). For DNA polymerase there is no difference whether the primer is DNA or RNA, it requires only the 3 OH group to perform the primer extension reaction. There are more players in the process of replication, which are shown on the cartoon above. Most significantly, since DNA strands must be separated at ambient conditions at which the double helix is very stable, a special molecular motor, called helicase moves along DNA and separates the complementary strands consuming the ATP energy, of course. Special small proteins, called SSB (for single-strand-binding), grab strands separated by helicase preventing them from reannealing prior to synthesis on both of them new strands by DNA polymerase. Thus, when the replication fork reaches the other end of the molecule, the original strands fully separate and the two daughter molecules shown at the top of the following cartoon are formed: Then two additional enzymes enter the scene. One is called DNA polymerase I (DNApol I), which extends each available 3 end of Okazaki fragments. In doing so it digest RNA primers ahead of itself since, as every DNA polymerase, DNApol I has a special domain exhibiting the 5 exonuclease activity. The rest of the job, consisting in sealing the gaps between adjacent Okazaki fragments extended by DNApol I, is fulfilled by yet another enzyme called DNA ligase. As a result both daughter molecules end up being

4 very similar, as shown at the bottom of the above cartoon. It is essentially the end of the replication process. The end problem and its solution One can ask: but who removes the two 5 -terminal primers, one per each daughter molecule? Of course, there is no problem with removing the primer. It can be done by a special ribonuclease known as RNase H, which specifically recognizes DNA/RNA heteroduplex but digests only RNA (that is why it is called RNase; there are all sorts of nucleases, enzymes digesting nucleic acids: some of them digest only DNA, some only RNA, others digest both (like the exonuclease domain of DNApol I), some of them digest inside chain (they are called endonucleases), others only from the end (they are called exonucleases; some of them like 3 -end others 5 -end)). However, after the removal of the primer there is no enzyme, which can replace the primer with DNA since no DNA polymerase can extend the 5 end. This end problem is a fundamental problem of DNA replication: at each round of replication the double helix becomes shorted on the size of the primer, which is several dozens bp. How is the end problem resolved by the cell? Prokaryotes and eukaryotes approach the problem very differently. Here we encounter one of many points where there these two major kingdoms of living organisms demonstrate totally different approach. Prokaryotes

5 The solution presented by prokaryotes is very elegant, I would say, it demonstrates mathematical elegance: if there is problems with end, let us get rid of them. Let us make DNA circular: in replicating the circle no end problem arises since the gap after removal of primer can be filled by DNApol I (actually, RNase H is also not required since the removal of last primer is performed by DNApol I via its 5 - exonuclease activity). ALL prokaryotic (bacterial) DNAs are always circular. It is the case not only for bacterial genomic DNA but also for plasmids, of course. Eukaryotes By whatever reason in eukaryotes genomic DNA molecules are always linear. May be it is because they are normally much longer than bacterial DNAs. Bacterial genomes consist of several million bp while in humans we have the whole genome (consisting of 3 billion bp) in the form of 23 chromosomes, each chromosome carrying exactly one DNA molecule. Therefore one human DNA molecule carries about 100 million bp. DNA molecule of such enormous length, if being circular, could probably encounter unsurpassable topological problems, such as knotting. Of course, we do not know for sure the reason but the fact is that ALL eukaryotic genomic DNA molecules are linear. BOX: The laws of biology One of many reasons while studying biology is so frustrating for engineers and physicists is the shortage of solid laws, which could not be violated, similar to, say, conservation laws (conservation of momentum, angular momentum and energy, for instance). In biology we find that as soon as a specific statement is made, numerous exceptions immediately pop up. I remember when back in Russia I learned biology going every winter to 2-week-long school on Molecular Biology near Moscow, the motto of the school was: the coyote hunts ONLY at moon nights; but if there no moon, it also hunts if it is hungry. Over the years we have learned that there are laws in molecular biology, at least with respect of life as we know it. Of what we have learned already in the course, we can formulate the following laws, which do not know exceptions: 1. In all living organisms genetic information is stored in the form of the DNA double helix 2. In all prokaryotes, DNA is always circular 3. In all eukaryotes, genomic (nuclear) DNA is always linear 4. All proteins synthesized via the ribosome pathway carry only L amino acids when they are exiting the ribosome Comments: To law #3: In addition to DNA in nucleus, the eukaryotic cell also carries DNA in the cytoplasm, within mitochondria, which is a very short circular dsdna molecule. This is because, beyond doubts, mitochondria used to be bacteria, which co-existed with pre-eukaryotic cells living in them (biologists call such a mutually profitable relationship between different species symbiosis). Gradually, mitochondria passed almost all their genes to the nucleus. The process stopped only because at some point the mitochondrial genetic code changed (see pp of Unraveling DNA) To law #4: Via posttranslational changes and via non-ribosome synthesis D amino acids pop up in the cell (see Background: Chirality)

6 How is it possible to avoid shortening of linear DNA during the every round of replication? Well, the truth is that it is exactly what happens in eukaryotes, I mean the shortening. So to protect genes from being truncated, the chromosomal DNAs carry special buffer regions at their termini, called telomeres. Telomeres are repeats, many thousand times, of a very simple motif. For all chromosomes in all humans (actually, in all vertebrates) the repeating sequence is: 5 TTAGGG3. Mostly it is dsdna but at the very end there is a single-stranded 3 overhang (TTAGGG)n consisting of several dozen repeats. During individual development of the organism telomeres indeed become shorter and shorter. This results in the phenomenon of somatic cell mortality: normal somatic cell can sustain only about 50 divisions after which it demonstrates clear signs of senescence and then dies (the phenomenon is dubbed the Hayflick limit). But what prevents our genomes from complete annihilation over many generations? The mechanism of telomere extension has been unraveled mostly by the efforts of Elizabeth Blackburn who was awarded, together with two other scientists, the Nobel Prize in Physiology or Medicine for She discovered a special enzyme, which she called telomerase and which extends telomeres. Since it is impossible to extend 5 -end, telomerase extends the 3 end instead. It is a good idea: if the 3 -end is extended significantly, the opposite strand can be synthesized via Okazaki fragments, similarly to the synthesis on the lagging strand in the replication fork (see above). That is OK but where is the template?! A sensational discovery Blackburn made consisted in the fact that the telomerase brings the template with itself: the enzyme is a complex of a protein, reverse transcriptase, Telomere extension by telomerase (the enzyme itself is not shown). The scheme is done for telomere sequence of single-cell eukaryote Tatrahymena, which has telemetric repeat TTGGGG. The telomerase was originally discovered by Blackburn in Tetrahymena.

7 which synthesizes DNA on the RNA template, and a pretty long ssrna molecule (about 500 nt). Somewhere inside this long RNA there are several telomeric repeats, which serve as the template for the telomere extension (see the scheme above). The telomeric 3 -overhang serves as primer for reverse transcriptase. The discovery of telomerase resolved the mystery of how the replication end problem is resolved for linear, noncircular DNA molecules of eukaryotes. In normal somatic cells the telomerase gene is completely shut down: hence the telomere s shortening with aging and the Hayflick limit. By contrast, during development of germ cells, in both males and females, the telomerase gene is switched on and telomerase extends telomeres preparing them for performing their buffer duties in the next generation. The fact of enormous importance is that in cancer cells the telomerase gene is activated again. This is why, in contrast to normal somatic cells, cancer cells are immortal: they can divide indefinitely. Most dramatic example of this immortality is so called HeLa cell line: a cell line based on a sample of cells taken from a tumor of a female patient in 1951 (her name was Henrietta Lacks, hence HeLa). The cell line is still in extensive use in many laboratories over the world.

Molecular Biology, Lecture 3 DNA Replication

Molecular Biology, Lecture 3 DNA Replication We will continue talking about DNA replication. We have previously t discussed the structure of DNA. DNA replication is the copying of the whole DNA content