DNA Replication Md. Habibur Rahaman (HbR) Dept. of Biology & Chemistry North South University

Models of Replication

DNA Replication Is Semiconservative Meselson and Stahl grew E. coli cells for many generations in a medium in which the sole nitrogen source (NH4Cl) contained 15-N, the “heavy” isotope of nitrogen, instead of the normal, more abundant “light” isotope, 14 N. The DNA isolated from these cells had a density about 1% greater than that of normal [14 N]-DNA. Although this is only a small difference, a mixture of heavy [15 N] DNA and light [14 N]DNA can be separated by centrifugation to equilibrium in a cesium chloride density gradient. The E. coli cells grown in the 15 N medium were transferred to a fresh medium containing only the 14 N isotope, where they were allowed to grow until the cell population had just doubled. The DNA isolated from these first-generation cells formed a single band in the CsCl gradient at a position indicating that the double-helical DNA molecules of the daughter cells were hybrids containing one new 14 N strand and one parent 15 N strand. This result argued against conservative replication, an alternative hypothesis in which one progeny DNA molecule would consist of two newly synthesized DNA strands and the other would contain the two parent strands; this would not yield hybrid DNA molecules in the Meselson-Stahl experiment. The semiconservative replication hypothesis was further supported in the next step of the experiment (Fig. c). Cells were again allowed to double in number in the 14 N medium. The isolated DNA product of this second cycle of replication exhibited two bands in the density gradient, one with a density equal to that of light DNA and the other with the density of the hybrid DNA observed after the first cell doubling. Meselson and Stahl Experiment Lehninger, 4th edition

Theta Replication https://www.youtube.com/watch?v=2THQtKHkNhQ

Rolling-Circle Replication Takes place in some viruses and in the F factor (a small circle of extra-chromosomal DNA that controls mating) of E. coli https://www.youtube.com/watch?v=fB1OkshHAVw

Linear Eukaryotic Replication DNA synthesis requires, A single-stranded DNA template Deoxyribonucleoside triphosphates A growing nucleotide strand A group of enzymes and proteins

Direction of Replication

DNA polymerase, always synthesizes DNA in the 5’- to 3’-direction DNA polymerase, always synthesizes DNA in the 5’- to 3’-direction. Therefore one new strand (leading strand) can be made continuously, while the other (lagging strand) must be made discontinuously (i.e., in short segments).

Why Uracil is absent in DNA?  Cytosine  One major problem with using uracil as a base is that cytosine can be deaminated, which converts it into uracil. This is not a rare reaction; it happens around 100 times per cell, per day. This is no major problem when using thymine, as the cell can easily recognize that the uracil doesn't belong there and can repair it by substituting it by a cytosine again. There is an enzyme, uracil DNA glycosylase, that does exactly that; it excises uracil bases from double-stranded DNA. It can safely do that as uracil is not supposed to be present in the DNA and has to be the result of a base modification. Now, if we would use uracil in DNA it would not be so easy to decide how to repair that error. It would prevent the usage of this important repair pathway. Uracil This is not a rare reaction; it happens around 100 times/cell/day Explanation: see on the text box below

The Mechanism of Replication of Bacterial DNA Replication takes place in four stages: Initiation Unwinding elongation termination Before we start, lets learn from the animation: https://www.youtube.com/watch?v=-SCnqmJpfp4

Initiation The circular chromosome of E. coli has a single replication origin (oriC). The minimal sequence required for oriC to function consists of 245 bp that contain several critical sites. Initiator proteins bind to oriC and cause a short section of DNA to unwind. This unwinding allows helicase and other single-strand-binding proteins to attach to the polynucleotide strand.

Unwinding DNA synthesis requires a single stranded template and double-stranded DNA must be unwound before DNA synthesis can take place, the cell relies on several proteins and enzymes to accomplish the unwinding. DNA helicases break the hydrogen bonds that exist between the bases of the two nucleotide strands of a DNA molecule. Helicases cannot initiate the unwinding of double-stranded DNA; the initiator proteins first separate DNA strands at the origin, providing a short stretch of single-stranded DNA to which a helicase binds.

After DNA has been unwound by helicase, the single-stranded nucleotide chains have a tendency to form hydrogen bonds and reanneal (stick back together). To stabilize the single-stranded DNA long enough for replication to take place, single-strand-binding (SSB) proteins attach tightly to the exposed single-stranded DNA SSBs are indifferent to base sequence—they will bind to any single-stranded DNA.

The unwinding process is the enzyme DNA gyrase, a topoisomerase. Topoisomerases control the supercoiling of DNA. In replication, DNA gyrase reduces torsional strain (torque) that builds up ahead of the replication fork as a result of unwinding.

Primers All DNA polymerases require a nucleotide with a 3’-OH group to which a new nucleotide can be added. Because of this requirement, DNA polymerases cannot initiate DNA synthesis on a bare template; rather, they require a primer—an existing 3’-OH group—to get started. How, then, does DNA synthesis begin? An enzyme called primase synthesizes short stretches of nucleotides (primers) to get DNA replication started. Primase synthesizes a short stretch of RNA nucleotides (about 10–12 nucleotides long), which provides a 3’-OH group to which DNA polymerase can attach DNA nucleotides.

All DNA molecules initially have short RNA primers imbedded within them; these primers are later removed and replaced by DNA nucleotides. On the leading strand, where DNA synthesis is continuous, a primer is required only at the 5’ end of the newly synthesized strand. On the lagging strand, where replication is discontinuous, a new primer must be generated at the beginning of each Okazaki fragment Fig. from Mol Bio_Clark

SSB Proteins Okazaki Fragments ATP 1 Polymerase III 2 3 Lagging strand base pairs 5’ 3’ SSB Proteins Polymerase III Lagging strand Okazaki Fragments 1 Helicase + Initiator Proteins ATP 2 3 RNA primer replaced by polymerase I & gap is sealed by ligase RNA Primer primase Polymerase III 5’  3’ Leading strand

Model of replication in E. coli

Elongation After DNA is unwound and a primer has been added, DNA polymerases elongate the polynucleotide strand by catalyzing DNA polymerization. The best-studied polymerases are those of E. coli, which has at least five different DNA polymerases. Two of them, DNA polymerase I and DNA polymerase III, carry out DNA synthesis associated with replication; the other three have specialized functions in DNA repair

DNA Polymerase III DNA polymerase III is a large multiprotein complex that acts as the main workhorse of replication. DNA polymerase III synthesizes nucleotide strands by adding new nucleotides to the 3’ end of growing DNA molecules. This enzyme has two enzymatic activities: Its 5’ to 3’ polymerase activity allows it to add new nucleotides in the 5’ 3’ direction. Its 3’ to 5’ exonuclease activity allows it to remove nucleotides in the 3’ to 5’ direction, enabling it to correct errors. If a nucleotide having an incorrect base is inserted into the growing DNA molecule, DNA polymerase III uses its 3’ to 5’ exonuclease activity to back up and remove the incorrect nucleotide. It then resumes its 5’ to 3’ polymerase activity.

DNA Polymerase III 2 DNA Pol III enzymes, each comprising α, ε and θ subunits. α subunit has the polymerase activity 5'→3' ε subunit has 3'→5' exonuclease activity. θ subunit stimulates the ε subunit's proofreading. Fig. from Mol Bio_Clark

All of E. coli’s DNA polymerases: synthesize any sequence specified by the template strand; synthesize in the 5’ to 3’ direction by adding nucleotides to a 3’-OH group; use dNTPs to synthesize new DNA; require a primer to initiate synthesis; 5. catalyze the formation of a phosphodiester bond by joining the 5’ phosphate group of the incoming nucleotide to the 3’-OH group of the preceding nucleotide on the growing strand, cleaving off two phosphates in the process; 6. produce newly synthesized strands that are complementary and antiparallel to the template strands; and 7. are associated with a number of other proteins.

DNA Polymerase-I and Ligase DNA polymerase I follows DNA polymerase III and, using its 5’ to 3’ exonuclease activity, removes the RNA primer. It then uses its 5’ to 3’ polymerase activity to replace the RNA nucleotides with DNA nucleotides. After polymerase I has replaced the last nucleotide of the RNA primer with a DNA nucleotide, a nick remains in the sugar–phosphate backbone of the new DNA strand. The term “nick” refers to a break in the nucleic acid backbone with no missing nucleotides. This nick is sealed by the enzyme DNA ligase, which catalyzes the formation of a phosphodiester bond without adding another nucleotide to the strand

DNA Polymerase-I and Ligase Fig. from Mol Bio_Clark

Enzymes and Other Proteins Required for DNA Replication

The Replication Fork The synthesis of both strands takes place simultaneously, two units of DNA polymerase III must be present at the replication fork, one for each strand. In one model of the replication process, the two units of DNA polymerase III are connected, and the lagging-strand template loops around so that, as the DNA polymerase III complex moves along the helix, the two antiparallel strands can undergo 5’ to 3’ replication simultaneously.

In summary, each active replication fork requires five basic components: Helicase to unwind the DNA, Single-strand-binding proteins to keep the nucleotide strands separate long enough to allow replication, The topoisomerase, i.e., gyrase to remove strain ahead of the replication fork, Primase to synthesize primers with a 3’-OH group at the beginning of each DNA fragment, and DNA polymerase to synthesize the leading and lagging nucleotide strands.

Termination In some DNA molecules, replication is terminated whenever two replication forks meet. In others, specific termination sequences block further replication. A termination protein, called Tus in E. coli, binds to these sequences. Tus blocks the movement of helicase, thus stalling the replication fork and preventing further DNA replication.

The Fidelity of DNA Replication Overall, replication results in an error rate of less than one mistake per billion nucleotides. How is this incredible accuracy achieved? No single process could produce this level of accuracy; a series of processes are required, each catching errors missed by the preceding ones

Thank you! You are instructed to follow the slides to get the sequence of study and read relevant materials Reading notes provided on web Chapter: 5, from Mol Bio_Clark NB: Multiple diagrams are provided for better understanding. You can use anyone, which is more comfortable for you.