How is DNA Replicated?

Once the structure of DNA was understood, it was possible to investigate how the molecule replicated itself during cell division. In their paper, Watson and Crick allude to this possibility that the molecule suggests certain mechanism of replication © 2012 Pearson Education, Inc.

DNA can be synthesized in a test tube if the following substances are present: Deoxyribonucleoside triphophates, dNTPs of dATP, dCTP, dGTP, dTTP DNA template to direct formation of new molecules DNA polymerase an enzyme to catalyze the polymerization reaction Salts and pH buffer to create proper chemical environment. © 2012 Pearson Education, Inc.

If DNA could be synthesized in a test tube, this indicates that DNA contains all the information needed for its own replication So how does it do this? © 2012 Pearson Education, Inc.

Three modes of DNA replication appear possible: Conservative Original helix is conserved and two newly synthesized strands come together Semiconservative Each replicated DNA molecule consists of one "old" strand and one new strand Dispersive Parental strands are dispersed into two new double helices © 2012 Pearson Education, Inc.

Figure 11-2 Results of one round of replication of DNA for each of the three possible modes by which replication could be accomplished. Figure 11.2 © 2012 Pearson Education, Inc.

An elegant experiment demonstrated that DNA replication is semiconservative Meselson and Stahl (1958), using 15N-labeled E. coli grown in medium containing 14N, demonstrated that DNA replication is semiconservative in prokaryotes each new DNA molecule consists of one old strand and one newly synthesized strand © 2012 Pearson Education, Inc.

The key is the use of “heavy” isotope of nitrogen – 15N The key is the use of “heavy” isotope of nitrogen – 15N. Molecules with this heavy form of N are denser than those containing the more common isotope 14N DNA extracts from the E. coli cultures could be centrifuged to separate the heavy strands from the light strands. © 2012 Pearson Education, Inc.

Figure 11-3 The Meselson–Stahl experiment. © 2012 Pearson Education, Inc.

Figure 11-4 The expected results of two generations of semiconservative replication in the Meselson–Stahl experiment. Figure 11.4 © 2012 Pearson Education, Inc.

The results obtained can only be explained by the semiconservative model. © 2012 Pearson Education, Inc.

Three Attributes of DNA Replication Shared by All Organisms Each strand of the parental DNA molecule remains intact during replication Each parental strand serves as a template for formation of an antiparallel, complementary daughter strand Completion of replication results in the formation of two identical daughter duplexes composed of one parental and one daughter strand 12

2 General Steps to DNA Replication DNA replication in the cell requires many different enzymes and proteins. DNA double helix is unwound to separate two template strands New nucleotides form complementary base pairs with template DNA forming phosphodiester bonds © 2012 Pearson Education, Inc.

Figure 11-7 The chemical reaction catalyzed by DNA polymerase I Figure 11-7 The chemical reaction catalyzed by DNA polymerase I. During each step, a single nucleotide is added to the growing complement of the DNA template, using a nucleoside triphosphate as the substrate. The release of inorganic pyrophosphate drives the reaction energetically. Figure 11.7 © 2012 Pearson Education, Inc.

As the nucleotide is added, the two terminal phosphates are cleaved off, providing a newly exposed 3'-OH group that can participate in the addition of another nucleotide as DNA synthesis proceeds © 2012 Pearson Education, Inc.

Figure 11-8 Demonstration of to synthesis of DNA. © 2012 Pearson Education, Inc.

© 2012 Pearson Education, Inc.

DNA replication starts when a large protein complex (pre-replication complex) binds to a region called origin of replication (ori). In E. coli, DNA is unwound and replication proceeds in both directions, forming two replication forks. © 2012 Pearson Education, Inc.

Figure 11-6 Bidirectional replication of the E. coli chromosome Figure 11-6 Bidirectional replication of the E. coli chromosome. The thin black arrows identify the advancing replication forks. The micrograph is of a bacterial chromosome in the process of replication, comparable to the figure next to it. Figure 11.6 © 2012 Pearson Education, Inc.

In eukaryotes, there are more than one origin of replication along linear chromosomes © 2012 Pearson Education, Inc.

Many Complex Issues Must Be Resolved during DNA Replication © 2012 Pearson Education, Inc.

There are seven key issues that must be resolved during DNA replication: Unwinding of the helix Reducing increased coiling generated during unwinding Synthesis of a primer for initiation Discontinuous synthesis of the second strand Removal of the RNA primers Joining of the gap-filling DNA to the adjacent strand Proofreading © 2012 Pearson Education, Inc.

DnaA binds to the origin of replication and is responsible for the initial steps in unwinding the helix © 2012 Pearson Education, Inc.

Subsequent binding of DnaB and DnaC further opens and destabilizes the helix Proteins such as these, which require the energy normally supplied by the hydrolysis of ATP to break hydrogen bonds and denature the double helix, are called helicases Single-stranded binding proteins (SSBPs) stabilize the open conformation © 2012 Pearson Education, Inc.

Unwinding produces supercoiling that is relieved by DNA gyrase, a member of a larger group of enzymes referred to as DNA topoisomerases Gyrase makes single- or double-stranded cuts to undo the twists and knots created during supercoiling, which are then resealed © 2012 Pearson Education, Inc.

DNA polymerase I removes the primer and replaces it with DNA To elongate a polynucleotide chain, DNA polymerase III requires a primer with a free 3'-hydroxyl group Primase synthesizes an RNA primer that provides the free 3'-hydroxyl required by DNA polymerase III DNA polymerase I removes the primer and replaces it with DNA Priming is a universal phenomenon during initiation of DNA synthesis © 2012 Pearson Education, Inc.

Figure 11-10 The initiation of DNA synthesis Figure 11-10 The initiation of DNA synthesis. A complementary RNA primer is first synthesized, to which DNA is added. All synthesis is in the to direction. Eventually, the RNA primer is replaced with DNA under the direction of DNA polymerase I. Figure 11.10 © 2012 Pearson Education, Inc.

The opposite lagging strand undergoes discontinuous DNA synthesis As the replication fork moves, only one strand can serve as a template for continuous DNA synthesis—the leading strand The opposite lagging strand undergoes discontinuous DNA synthesis © 2012 Pearson Education, Inc.

Figure 11-11 Opposite polarity of DNA synthesis along the two strands, necessary because the two strands of DNA run antiparallel to one another and DNA polymerase III synthesizes only in one direction ( to ). On the lagging strand, synthesis must be discontinuous, resulting in the production of Okazaki fragments. On the leading strand, synthesis is continuous. RNA primers are used to initiate synthesis on both strands. Figure 11.11 © 2012 Pearson Education, Inc.

The lagging strand is synthesized as Okazaki fragments, each with an RNA primer DNA polymerase I removes the primers on the lagging strand, and the fragments are joined by DNA ligase © 2012 Pearson Education, Inc.

A model of DNA Replication DNA synthesis at a single replication fork involves DNA polymerase III single-stranded binding proteins DNA gyrase DNA helicase RNA primers © 2012 Pearson Education, Inc.

Figure 11-13 Summary of DNA synthesis at a single replication fork Figure 11-13 Summary of DNA synthesis at a single replication fork. Various enzymes and proteins essential to the process are shown. Figure 11.13 © 2012 Pearson Education, Inc.

Checkpoint In the figure shown, the leading strand grows continuously _______ at its _______ end. backward; 5′ forward; 5′ backward; 3′ forward; 3′ Answer: d

Checkpoint Strand A Strand B 5’ 3’ Strand B In the figure shown, what best describes Strand B? It is the lagging strand. It is the leading strand. It is the Okazaki strand. There is not enough information given. Answer: b

Eukaryotic DNA Replication Is Similar to Replication in Prokaryotes, but Is More Complex © 2012 Pearson Education, Inc.

Eukaryotic DNA replication shares many features with replication in bacteria Double-stranded DNA unwound at replication origins Replication forks are formed Bidirectional snythesis creates leading and lagging strands Eukaryotic polymerases require four deoxyribonucleoside triphosphates, a template, and a primer © 2012 Pearson Education, Inc.

This makes DNA replication more complex in eukaryotes than in bacteria However, eukaryotic DNA replication is more complex due to several features of eukaryotic DNA: There is more DNA than prokaryotic cells The chromosomes are linear The DNA is complexed with proteins This makes DNA replication more complex in eukaryotes than in bacteria © 2012 Pearson Education, Inc.

Three DNA polymerases are involved in replication of nuclear DNA One involves mitochondrial DNA replication Others are involved in repair processes © 2012 Pearson Education, Inc.

Visit Blackboard content and review the media file “ DNA Replication” Stop and view: Visit Blackboard content and review the media file “ DNA Replication” This is a very helpful animation of the steps of replication. © 2012 Pearson Education, Inc.

The Ends of Linear Chromosome Are Problematic during Replication © 2012 Pearson Education, Inc.

Telomeres at the ends of linear chromosomes consist of long stretches of short repeating sequences and preserve the integrity and stability of chromosomes © 2012 Pearson Education, Inc.

Lagging strand synthesis at the end of the chromosome is a problem because once the RNA primer is removed, there is no free 3'-hydroxyl group from which to elongate © 2012 Pearson Education, Inc.

Telomerase directs synthesis of the telomere repeat sequence to fill the gap This enzyme is a ribonucleoprotein with an RNA that serves as the template for the synthesis of its DNA complement © 2012 Pearson Education, Inc.

In most eukaryotic somatic cells, telomerase is not active With each successive cell division, telomeres shorten and erode, causing further cell division to stop Malignant cells maintain telomerase activity and are immortalized © 2012 Pearson Education, Inc.

Telomere length is important for chromosome stability, cell longevity, and reproductive success Telomerase is active in germ-line cells and some stem cells in eukaryotes Differentiated somatic cells and cells in culture have virtually no telomerase activity; such cells have limited life spans (30 to 50 cell divisions) © 2012 Pearson Education, Inc.

Telomerase is normally turned off in somatic cells Reactivation of telomerase can lead to aging cells that continue to proliferate, a feature of many types of cancer TERT reactivation is one of the most common mutations in cancers of all types © 2012 Pearson Education, Inc.

Errors in DNA repaired DNA must be faithfully maintained, yet replication is not perfectly accurate and DNA is subject to damage by chemicals and other environmental agents. Cells have three mechanisms for DNA repair: Proofreading corrects errors in replication as DNA polymerase makes them Mismatch repair scans DNA immediately after replication and corrects any base pairing Excision repair removes abnormal bases that have formed because of chemical damage and replaces them with functional ones. © 2012 Pearson Education, Inc.

Homework Assignment Page 289 in your textbook: Telomeres: Key to Immortality. Under “Your Turn” complete questions #4 and submit to your Box Folder See mastering genetics for homework problems. Mastering assignment has been set as tutorial homework. There is no deductions for wrong answers. Both are due by Monday, March 3, at 9:00 PM © 2012 Pearson Education, Inc.