Central dogma of life DNA REPLICATION BY DR LAMIA KANDIL

DNA Replication It is the process of DNA synthesis using parent DNA strands as template. At each cell division, a cell must copy its genome with extraordinar accuracy. It aims at formation of a copy of the parent DNA molecule for the daughter cell

Semiconservative style of replication Because each parental strand serves as the template for one new strand, each of the daughter DNA double helices ends up with one of the original (old) strands plus one strand that is completely new . This style of replication is said to be semiconservative

Which of the following best describes semiconservative replication? A. The translation of a DNA molecule into a complementary strand of RNA. B. A DNA molecule consists of one parental strand and one new strand. C. The number of DNA molecules is doubled with every other replication. D. The replication of DNA never takes place with 100% accuracy. E. The replication of DNA takes place at a defined period in the cell cycle.

The final product of DNA replication is: A. mRNA, tRNA, and rRNA molecules. B. a wide variety of proteins. C. DNA fragments. D. two DNA molecules, each of which contains one new and one old DNA strand. E. the enzymes needed for further processes, such as DNA polymerase.

DNA Synthesis Begins at Replication Origins The DNA double helix is normally very stable: the two DNA strands are locked together firmly by the large numbers of hydrogen bonds between the bases on both strands .

The process of DNA synthesis is begun by initiator proteins that bind to specific DNA sequences called replication origins. bacterial genome, which is typically contained in a circular DNA molecule of several million nucleotide pairs, has a single replication origin. The human genome, which is very much larger, has approximately 10,000 such origins—an average of 220 origins per chromosome. Beginning DNA replication at many places at once greatly shortens the time a cell needs to copy its entire genome

Once an initiator protein binds to DNA at a replication origin and locally opens up the double helix, it attracts a group of proteins that carry out DNA replication. These proteins form a replication machine, in which each protein carries out a specific function. DNA molecules in the process of being replicated contain Y-shaped junctions called replication forks.

A DNA double helix is opened at replication origins

Steps of DNA replication

2.

5.DNA topoisomerase ( DNA GYRASE) DNA on the other side of the fork gets wound more tightly. This excess twisting in front of the replication fork creates tension in the DNA which makes unwinding the double helix increasingly difficult and delays the forward movement of the replication machinery . Cells use proteins called DNA topoisomerases to relieve this tension. These enzymes produce transient nicks in the DNA backbone which temporarily release the tension; they then reseal the nick before falling off the DNA

How can the polymerase begin a completely new DNA strand? 3.primase. Enzyme that synthesizes the RNA primer which is Short Lengths of RNA acting as Primers for DNA Synthesis It makes a short length of a closely related type of nucleic acid RNA (ribonucleic acid) using the DNA strand as a template. This short length of RNA, about 10 nucleotides long, is base-paired to the template strand and provides a base-paired 3ʹ end as a starting point for DNA polymerase.

4.DNA polymerase catalyzes the addition of nucleotides to the 3ʹ end of a growing DNA strand, using one of the original parental DNA strands as a template. A new DNA strand is synthesized in the 5ʹ–to–3ʹ direction

Does the cell have two types of DNA polymerase, one for each direction? NO All DNA polymerases add new subunits only to the 3ʹ end of a DNA strand . As a result, a new DNA chain can be synthesized only in a 5ʹ-to-3ʹ direction. The DNA strand that appears to grow in the incorrect 3ʹ-to-5ʹ direction is actually made discontinuously, in successive, separate, small pieces with the DNA polymerase moving backward with respect to the direction of replication-fork movement so that each new DNA fragment can be polymerized in the 5ʹ-to-3ʹ direction

LAGGING STRAND FROM 5’ TO 3’ REPLICATION FORK DIRECTION LAGGING STRAND FROM 5’ TO 3’

The resulting small DNA pieces called Okazaki fragments after the biochemists who discovered them are later joined together to form a continuous new strand. The DNA strand that is made discontinuously in this way is called the lagging strand, because the backstitching imparts a slight delay to its synthesis; the other strand, which is synthesized continuously, is called the leading strand

LEADING STRAND

To produce a continuous new DNA strand from the many separate pieces of nucleic acid made on the lagging strand, three additional enzymes are needed: These act quickly to, replace it with DNA, and join the DNA fragments together. 1.Nuclease degrades the RNA primer 2.DNA polymerase called a repair polymerase then replaces this RNA with DNA (using the end of the adjacent Okazaki fragment as a primer) 3. DNA ligase joins the 5ʹ-phosphate end of one DNA fragment to the adjacent 3ʹ-hydroxyl end of the next

Lagging Strand

Which of the following nucleotide sequences represents the complement to the DNA strand 5´ – AGATCCG- 3´? A. 5´ – AGATCCG- 3´ B. 3´ – AGATCCG- 5´ C. 5´ – CTCGAAT- 3´ D. 3´ – CTCGAAT- 5´ E. 3´ – TCTAGGC- 5´ Two chains of DNA must run in ____________ direction(s) and must be ____________ if they are to bond with each other. A. the same; uncomplementary B. opposite; uncomplementary C. parallel; uncomplementary D. parallel; complementary E. antiparallel; complementary

DNA Polymerase Is Self-correcting DNA polymerase is so accurate that it makes only about one error in every 107 nucleotide pairs it copies. This error rate is much lower than can be explained simply by the accuracy of complementary base-pairing

Proof reading when DNA polymerase makes a rare mistake and adds the wrong nucleotide. It can correct the error through an activity called proofreading. Proofreading takes place at the same time as DNA synthesis..

Before the enzyme adds the next nucleotide to a growing DNA strand, it checks whether the previously added nucleotide is correctly base-paired to the template strand. If so, the polymerase adds the next nucleotide; if not, the polymerase clips off the mispaired nucleotide and tries again. This proofreading is carried out by a nuclease that cleaves the phosphodiester backbone. Proof reading must proceed only in the 5ʹ-to-3ʹ direction

Questions

The enzyme responsible for initiating the unwinding of double-stranded DNA (eliminating supercoiling) by nicking a single strand of the DNA molecule is : • Gyrase  • Ligase  • Helicase  • Topoisomerase Suppose a mutation occurs in a cell such that normal Okazaki fragments were created during DNA replication but were not linked together into a continuous strand. The gene for which enzyme would you predict was altered by this mutation? A. DNA polymerase C. DNA helicase E. DNA ligase B. RNA primase D. ssDNA binding protein

G

The segments labeled F are responsible for: A. linking short DNA segments. B. synthesizing the leading strand. C. forming the replication fork. D. initiating DNA synthesis. E. unwinding the DNA double helix. The enzyme represented by the letter D is responsible for: B. synthesizing the leading strand. C. forming the replication fork. D. forming nucleosomes. E. unwinding the DNA double helix. The structures represented by the letter E are called: A. leading fragments B. Okazaki fragments. C. replication forks. D. nucleosomes. E. DNA polymerases.

The job of a ligase enzyme is to a. unwind the DNA. b. build a short sequence of RNA using the DNA as a template. c. form covalent bonds between DNA nucleotides. d. add DNA nucleotides to an existing strand. What would happen to DNA replication if the helicase enzyme did not function? a. Replication could occur, but there would be errors. b. The new DNA strand would not be held together by covalent bonds. c. Replication would occur in a single direction. d. Replication would not occur at all.

A replication fork: A. is only seen in prokaryotic chromosomes. B. is only seen in bacterial cells. C. is a Y-shaped structure where both DNA strands are replicated simultaneously. D. is a site where one DNA strand serves as a template, but the other strand is not replicated. E. is created by the action of the enzyme RNA polymerase. In replication, once the DNA strands have been separated, reformation of the double helix is prevented by: A. DNA helicase enzyme. B. single-strand binding proteins. C. DNA polymerases. D. ATP. E. GTP

Which of the following adds new nucleotides to a growing DNA chain? A. DNA polymerase B. DNA helicase C. RNA primer D. primase E. RNA polymerase