Replication

Central Dogma of Molecular Biology The flow of information in the cell starts at DNA, which replicates to form more DNA. Information is then transcribed into RNA, and then it is translated into protein. The proteins do most of the work in the cell. Information does not flow in the other direction. This is a molecular version of the incorrectness of “inheritance of acquired characteristics”. Changes in proteins do not affect the DNA in a systematic manner (although they can cause random changes in DNA. Vocabulary: DNA is replicated to make more DNA, using the enzyme DNA polymerase DNA is transcribed into RNA using RNA polymerase RNA is translated into protein using ribosomes.

Replication Watson and Crick’s famous paper on the structure of DNA ends with this: “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” J.D. Watson and F.H.C. Crick (1953). A Structure for Deoxyribose Nucleic Acid. Nature 171: 737-738. DNA is composed of 2 complementary chains. This means that each DNA molecule has 2 copies of the information built into it. Complementary: an A on one chain always corresponds to a T on the other chain; same with G and C. Each strand can be used as a template to build the second strand.

Semi-Conservative Replication After W+C published the structure of DNA, there were several theories of how replication works. Conservative replication: after a DNA molecule replicates, one daughter molecule has both old strands and the other has both new strands. (INCORRECT) Semi-conservative replication: after replication, both daughter molecules have one old strand and one new strand. (This is what really happens). Dispersive replication: after replication, each strand of both daughter molecules is a mixture of old and new pieces. (INCORRECT) The dispersive model was the result of worrying about how the double helix could unwind during replication.

Meselson-Stahl Experiment Grow E. coli using heavy nitrogen (15N vs. normal 14N). The DNA is denser (more grams per milliliter) than normal DNA. In equilibrium density gradient centrifugation, the high gravitational forces create a density gradient is a cesium chloride solution. DNA then floats at the position where its density matches the density of the CsCl. The experiment consisted of growing E. coli on heavy nitrogen, then switching it to light nitrogen for 1 or more generations. The DNA was then extracted and run through the centrifuge.

Meselson-Stahl Results Start with DNA from E. coli grown on heavy nitrogen, then switch to light nitrogen for 1 generation (about 20 minutes). Result: intermediate band. This eliminates the conservative model, which predicts one heavy band and one light band. Grow a second generation on light nitrogen. Result: one light band and one intermediate band. This eliminates the dispersive model, which predicts only one band, between the light band and the intermediate band seen after 1 generation. Further generations: the intermediate band gets weaker and the light band gets progressively larger as fewer molecules have any of the DNA made with 15N. All of these results are consistent with the semiconservative model: the original heavy strands remain intact, but always paired with a light strand, giving an intermediate density band.

Origin of Replication Replication starts at specific sites on the chromosome: origins of replication (ori). Bacterial genomes have just 1 ori. The ori site defines base pair 1 in the DNA sequences of the chromosome. Eukaryotic chromosomes have many ori sites, and they are used differently in different cell types. The DNA sequence of the ori varies between species, but it is always AT-rich. A-T base pairs have only 2 hydrogen bonds, which makes them easier to separate than G-C base pairs. The origin recognition complex (ORC) binds to the ori sequence to start the replication process. The ORC is a multi-subunit protein that melts the ori DNA (makes it single stranded) and prepares the DNA to start replication.

Bidirectional Replication Replication starts at the ori sequence, then proceeds in both directions. There are multiple origins of replication on eukaryotic chromosomes. Replication moves in both directions from each ori. When two replication bubbles meet, they join into a larger bubble, until the whole chromosome is replicated.

Continuous and Discontinuous Synthesis DNA can only be synthesized from 5’ to 3’, by adding new nucleotides to the 3’ end. This is a problem, because both strands must be synthesized at the replication fork, and one strand will necessarily be synthesized in the opposite direction from the movement of the replication fork. In reality, one strand is synthesized continuously, in the same direction that the replication fork is moving. This is called the leading strand. The other strand is synthesized in short, discontinuous pieces, that are then attached together to form the final DNA strand. This is the lagging strand. Each fragment of the lagging strand is called an Okazaki fragment, and they are synthesized in the opposite direction that the replication fork moves. In eukaryotes, Okazaki fragments are 100-300 bp long. In prokaryotes, Okazaki fragments are 1-2 kbp (kilobase pairs = 1000 bp)

Enzymes in the Replication Complex The replication complex (the replisome) consists of many subunits with different functions. They all move down the DNA as a single unit, replicating both DNA strands simultaneously.

DNA Polymerase Reaction The enzyme that replicates the DNA molecule is DNA polymerase. DNA polymerase is one part of the replication complex. Discovered by Arthur Kornberg in 1956. DNA polymerase catalyzes the synthesis of the second strand of a DNA molecule. To start, it needs: Single stranded DNA template molecule Primer: a short piece of DNA or RNA base-paired with a region of the template dNTPs: the 4 deoxy nucleoside triphosphates dATP, dCTP, dGTP and dTTP, which are the raw materials for the new DNA strand. The basic reaction: The 2 phosphate groups on the end of the dNTP molecule (the gamma (γ) and beta (β) phosphates) are removed, and the phosphate next to the sugar is attached to the 3’ OH group of the growing DNA chain. The removal of the phosphates provides some of the energy needed to drive the reaction: -3.5 kcal/mole The gamma and beta phosphates are removed as a unit, called pyrophosphate (PPi). The enzyme pyrophosphatase breaks pyrophosphate down to 3 phosphates. This reaction as a free energy of -7 kcal/mol. Added to the free energy of removing pyrophosphate from the dNTP, the addition of a nucleotide to a growing DNA chain has a free energy of -10.5 kcal/mol, which is enough to make the reaction irreversible.

More DNA Polymerase The DNA polymerase reaction is processive: it starts from the 3’ end of the primer and adds new nucleotides, one at a time, until it reaches the end of the template. You now have a complete double stranded DNA molecule. Each nucleotide added is complementary to the nucleotide on the template strand: A paired with T, and G paired with C.

Multiple Forms of DNA Polymerase

Primers and the Lagging Strand A peculiarity of DNA synthesis is that DNA polymerase must attach new bases to the 3’ end of a pre-existing nucleic acid chain. All DNA synthesis starts at a short double-stranded region. In the cell, short (10-13 bases) pieces of RNA, called primers are paired with the DNA bases to create to the short double stranded regions that DNA synthesis builds. The RNA primers are synthesized by an enzyme called primase, and they are removed by DNA polymerase during the synthesis of the next Okazaki fragment. After DNA polymerase has created the new DNA strand by building on the RNA primer, the RNA is degraded by the DNA polymerase extending the next Okazaki fragment.

Avoiding DNA Tangles The DNA double helix needs to be unwound before it can be replicated. DNA helicase breaks the hydrogen bonds between the DNA strands and allows them to separate. Video: https://www.youtube.com/watch?v=bePPQpoVUpM Problem: As you unwind the DNA, you build up tension in the remaining DNA. This tension is relieved by topisomerase. Topoisomerase cuts one strand, allows the other strand to relax, then re-ligates the cut strand. Another form of topisomerase allows one DNA double helix to pass through another one, by cutting and then re-ligating both strands. Topoisomerase video: https://www.youtube.com/watch?v=EYGrElVyHnU&feature=related A third protein important here: single strand binding protein (SSB), which binds to single stranded DNA and keeps it single stranded by preventing base pairing.

Primer Removal and Nick Sealing On the lagging strand, a new RNA primer is needed every few hundred bases. DNA polymerase has a second enzymatic activity: it can function as a 5’ → 3’ exonuclease. That is, as the polymerase moves down the template strand, it removes any nucleic acids ahead of it. This usually means the RNA primer from the previous Okazaki fragment. After DNA polymerase finishes, the two Okazaki fragments are separated by a nick in the DNA: the phosphodiester linkage isn’t complete. The nick is removed, joining the two fragments covalently, by the enzyme DNA ligase.

DNA Polymerase is Self-Correcting Recall that tautomeric shifts in the hydrogens of the bases can cause mispairing: A-C or G-T. Tautomeric shifts occur rapidly and frequently. DNA polymerase compensates for this problem by checking a newly added nucleotide before adding the next one. If the new nucleotide doesn’t form a proper base pair, DNA polymerase backs up and removes it, then tries again. This is the proofreading function of DNA polymerase, also called 3’5’ exonuclease activity. DNA polymerase has 3 enzymatic activities: 1. 5’→3’ polymerase 2. 5’→3’ exonuclease (primer removal) 3. 3’→5’ exonuclease (proofreading)

β Sliding Clamp DNA polymerase is held to the DNA by the β sliding clamp. This mechanism helps prevent replication errors by increasing the processivity of DNA polymerase: the number of nucleotides it can add to a growing chain before falling off. The clamp consists of 2 identical protein subunits that surround the DNA double helix and also bind to the DNA polymerase protein. The clamp is assembled onto the DNA using ATP energy by a clamp loader protein that is also part of the replication complex.

Telomeres The ends of eukaryotic chromosomes are a problem: that RNA primer at the very 5’ end gets degraded, leaving a short single stranded region at the end. And note that the next time this DNA replicates, one strand is shorter than the other. This leads to a gradual shortening of the chromosome, which is thought to be a major factor in aging and mortality. Solution: telomeres, which are special repeated DNA sequences added by the enzyme telomerase. If they get lost in replication, telomerase adds them back. Telomerase is an RNA/protein hybrid structure.