Chapter 11

Part C - Dideoxynucleotide DNA sequencing The diagram below shows an autoradiograph of a DNA sequencing gel. Type the 5' to 3' sequence of the template strand ("inferred strand") based on the pattern in this gel. Use only capital letters for the sequence.

5' - CAACTGGTCCAT - 3' The shortest fragment generated by the dideoxynucleotide DNA sequencing reaction migrates to the bottom of the gel. That fragment represents the 5' nucleotide of the sequenced strand. Therefore, the 5' to 3' sequence of nucleotides in the sequenced strand can be determined by reading the bands in all lanes from the bottom of the gel to the top: 5'-ATGGACCAGTTG-3', in this example. The template strand is complementary and antiparallel to the sequenced strand: 5'-CAACTGGTCCAT-3', in this example.

During replication, primase adds a DNA primer to RNA.

False.

The data obtained from the Meselson-Stahl experiment after one generation of replication eliminated the dispersive model of DNA replication.

False. The data obtained from the Meselson-Stahl experiment after one generation was consistent with both the semiconservative and the dispersive model of DNA replication. The conservative model of DNA replication was eliminated because it predicted that there would be two bands representing the original DNA at one density and the newly replicated DNA at a different density.

The role of DNA polymerase III In DNA replication in bacteria, the enzyme DNA polymerase III (abbreviated DNA pol III) adds nucleotides to a template strand of DNA. But DNA pol III cannot start a new strand from scratch. Instead, a primer must pair with the template strand, and DNA pol III then adds nucleotides to the primer, complementary to the template strand. Each of the four images below shows a strand of template DNA (dark blue) with an RNA primer (red) to which DNA pol III will add nucleotides.

In the example above, DNA pol III would add an adenine nucleotide to the 3' end of the primer, where the template strand has thymine as the next available base. You can tell which end is the 3' end by the presence of a hydroxyl (-OH) group. The structure of DNA polymerase III is such that it can only add new nucleotides to the 3' end of a primer or growing DNA strand (as shown here). This is because the phosphate group at the 5' end of the new strand and the 3' -OH group on the nucleoside triphosphate will not both fit in the active site of the polymerase. blue = TGCGA

Which enzyme catalyzes the addition of nucleotides to a growing DNA chain?

Polymerase. DNA polymerase catalyzes the addition of nucleotides to a growing DNA chain.

RNA primers are formed by _________ and serve as an initiation point for DNA synthesis on the template.

RNA primase

Which of the following statements about DNA replication is true? A) DNA gyrase unwinds the DNA double helix. B) Single‑strand binding proteins stabilize the open conformation of the unwound DNA. C) Okazaki fragments are DNA fragments synthesized on the leading strand. D) DNA polymerase adds dNTP monomers in the 3′-5′ direction.

Single‑strand binding proteins stabilize the open conformation of the unwound DNA. Once helicase unwinds the double helix, single‑strand binding proteins bind to the open DNA and prevent it from winding together again.

DNA replication occurs in the 5′ to 3′ direction; that is, new nucleoside triphosphates are added to the 3′ end.

True

A characteristic of aging cells is that their telomeres become shorter.

True.

DNA strand replication begins with an RNA primer.

True.

In general, DNA replicates semiconservatively and bidirectionally.

True.

Telomerase is an RNA-containing enzyme that adds telomeric DNA sequences onto the ends of linear chromosomes.

True.

The discontinuous aspect of replication of DNA in vivo is caused by ________.

the 5′ to 3′ polarity restriction

All of the following are differences between eukaryotic and prokaryotic DNA replication EXCEPT __________.

the ability to form a replication fork Both prokaryotes and eukaryotes form replication forks during DNA replication.

DNA polymerase III adds nucleotides ________.

to the 3' end of the RNA primer

Which activity of E. coli DNA polymerase I is responsible for proofreading the newly synthesized DNA?

3' to 5'; exonuclease If the wrong nucleotide is inserted, normal base pairing will not be observed and the base in error will be removed from the newly synthesized strand before subsequent nucleotides are added.

Which enzyme in E. coli is responsible for relieving the tension ahead of the fork that results when the DNA unwinds to form the replication "bubble" or "eye"?

DNA gyrase Gyrase relieves the tension by forming negative supercoils in a reaction that requires energy from the hydrolysis of ATP.

These gaps are generated when ___________ removes the RNA primers.

DNA polymerase I

Which DNA polymerase is mainly responsible for genome replication in E. coli?

DNA polymerase III DNA polymerase III is responsible for the synthesis of the bulk of the DNA in E. coli.

Which of the following statements about DNA replication is true?

DNA synthesis is continuous on the leading strand and discontinuous on the lagging strand. This statement correctly describes the conditions of DNA replication.

An endonuclease is involved in removing bases sequentially from one end of DNA or the other.

False.

In what way does an exposed 3′−OH group participate in strand elongation?

It directly participates in the formation of a covalent bond with the nucleotide being added.

Okazaki fragments are short DNA fragments synthesized in a __________ manner.

discontinuous

DNA polymerase I is thought to add nucleotides ________.

in the place of the primer RNA after it is removed

DNA ligase forms ________ bonds in gaps between DNA Okazaki fragments.

phosphodiester

Which of the following terms accurately describes the replication of DNA in vivo?

semidiscontinuous

DNA Replication DNA replication is the mechanism by which DNA is copied. It is highly accurate in both bacteria and eukaryotes and requires a variety of DNA polymerases and other accessory proteins. In this tutorial you will learn how DNA is replicated and understand the roles of the proteins involved in the process. You will also learn how the sequence of a DNA molecule is determined using the dideoxynucleotide DNA sequencing method. Part A - The mechanism of DNA replication The diagram below shows a double-stranded DNA molecule (parental duplex). Drag the correct labels to the appropriate locations in the diagram to show the composition of the daughter duplexes after one and two cycles of DNA replication. In the labels, the original parental DNA is blue and the DNA synthesized during replication is red.

1st cycle: even mix of red and blue (2) 2nd cycle: even mix of blue and red (4) During DNA replication, each strand in the parental duplex serves as the template for the production of a daughter strand by complementary base pairing. Therefore, one cycle of replication will produce two daughter duplexes, each with one parental strand and one newly synthesized strand. During a second cycle of replication, all four strands in the two duplexes will serve as templates, resulting in four duplexes (eight strands of DNA).

Synthesis of the lagging strand In contrast to the leading strand, the lagging strand is synthesized as a series of segments called Okazaki fragments. The diagram below illustrates a lagging strand with the replication fork off-screen to the right. Fragment A is the most recently synthesized Okazaki fragment. Fragment B will be synthesized next in the space between primers A and B.

2) pol III moves 5' to 3', adding DNA nucleotides to primer B 3) pol I binds to 5' end of primer A 4) pol I replaces primer A with DNA 5) DNA ligase links fragments A and B Synthesis of the lagging strand is accomplished through the repetition of the following steps. Step 1: A new fragment begins with DNA polymerase III binding to the 3' end of the most recently produced RNA primer, primer B in this case, which is closest to the replication fork. DNA pol III then adds DNA nucleotides in the 5' to 3' direction until it encounters the previous RNA primer, primer A. Step 2: DNA pol III falls off and is replaced by DNA pol I. Starting at the 5' end of primer A, DNA pol I removes each RNA nucleotide and replaces it with the corresponding DNA nucleotide. (DNA pol I adds the nucleotides to the 3' end of fragment B.) When it encounters the 5' end of fragment A, DNA pol I falls off, leaving a gap in the sugar-phosphate backbone between fragments A and B. Step 3: DNA ligase closes the gap between fragments A and B.

Part B - Processes occurring at a bacterial replication fork The diagram below shows a bacterial replication fork and its principal proteins. Drag the labels to their appropriate locations in the diagram to describe the name or function of each structure. Use pink labels for the pink targets and blue labels for the blue targets.

A) Breaks hydrogen bonds, unwinding DNA double helix. B) Synthesizes RNA primers on leading and lagging strands. C) Replaces RNA primers with DNA nucleotides. D) Catalyzes phosphodiester bond formation, joining DNA fragments. E) lagging strand F) Leading Strand G) Relaxes supercoiled DNA. H) Coats single-stranded DNA, preventing duplex formation. I) Synthesizes DNA 5' to 3' on leading and lagging strands. uring replication, DNA synthesis occurs in the 5′ to 3′ direction along both template strands. On one template strand, synthesis proceeds continuously toward the replication fork, generating the leading strand. On the other template strand, DNA is synthesized away from the replication fork in segments called Okazaki fragments, generating the lagging strand. Several proteins are involved in DNA replication, including the following: Helicase breaks the hydrogen bonds between the parental DNA strands and unwinds the double helix. Single-stranded binding proteins bind to the single strands of DNA, preventing them from reannealing and allowing synthesis to occur on both strands. DNA polymerase III synthesizes the new strands, but it requires an existing 3′ hydroxyl (—OH) group to add nucleotides. Primase creates short RNA primers, initiating DNA synthesis on both template strands. DNA polymerase I removes the RNA primers and replaces them with DNA nucleotides. On the lagging strand, DNA ligase joins Okazaki fragments by forming phosphodiester bonds between them, thus completing DNA replication.

Which of the following statements is true regarding Okazaki fragments?

They are short fragments of DNA synthesized from RNA primers on the lagging strand. Okazaki fragments are short sequences synthesized in the lagging strand because DNA polymerase can synthesize only from 5' to 3' and the DNA strands are antiparallel.

The replication bubble and antiparallel elongation DNA replication always begins at an origin of replication. In bacteria, there is a single origin of replication on the circular chromosome, as shown in the image here. Beginning at the origin of replication, the two parental strands (dark blue) separate, forming a replication bubble. At each end of the replication bubble is a replication fork where the parental strands are unwound and new daughter strands (light blue) are synthesized. Movement of the replication forks away from the origin expands the replication bubble until two identical chromosomes are ultimately produced.

a) --> b) --> c) <-- d) <-- DNA polymerase III can only add nucleotides to the 3' end of a new DNA strand. Because the two parental DNA strands of a double helix are antiparallel (go from 3' to 5' in opposite directions), the direction that DNA pol III moves on each strand emerging from a single replication fork must also be opposite. For example, in the replication fork on the left, the new strand on top is being synthesized from 5' to 3', and therefore DNA pol III moves away from the replication fork. Similarly, the new strand on the bottom of that same replication fork is being synthesized from 5' to 3'. But because the bottom parental strand is running in the opposite direction of the top parental strand, DNA pol III moves toward the replication fork. In summary, at a single replication fork, one strand is synthesized away from the replication fork, and one strand is synthesized toward the replication fork. When you look at both replication forks, note that a single new strand is built in the same direction on both sides of the replication bubble.

DNA Replication (1 of 2): DNA Structure and Replication Machinery (BioFlix tutorial) Part A - The chemical structure of DNA and its nucleotides The DNA double helix is composed of two strands of DNA; each strand is a polymer of DNA nucleotides. Each nucleotide consists of a sugar, a phosphate group, and one of four nitrogenous bases. The structure and orientation of the two strands are important to understanding DNA replication. Drag the labels to their appropriate locations on the diagram below. Use only the pink labels for the pink targets, and the blue labels for the blue targets. Labels can be used once, more than once, or not at all.

a) 5' end b) hydrogen bond c) 3' end d) deoxyribose sugar e) nitrogenous base f) phosphate group g) 3' end h) 5' end The DNA double helix is constructed from two strands of DNA, each with a sugar-phosphate backbone and nitrogenous bases that form hydrogen bonds, holding the two strands together. Each DNA strand has two unique ends. The 3' end has a hydroxyl (-OH) group on the deoxyribose sugar, whereas the 5' end has a phosphate group. In the double helix, the two strands are antiparallel, that is, they run in opposite directions such that the 3' end of one strand is adjacent to the 5' end of the other strand.

Rank the primers in the order they were produced. If two primers were produced at the same time, overlap them.

a-h; b-g; c-f; d-e

As DNA replication continues and the replication bubble expands, the parental double helix is unwound and separated into its two component strands. This unwinding and separating of the DNA requires three different types of proteins: helicase, topoisomerase, and single-strand binding proteins.

helicase: binds at the replication fork, breaks H-bonds between bases topoisomerase: binds ahead of the replication fork, breaks covalent bonds in DNA backbone single-strand binding protein: prevents H-bonds between bases, binds after the replication fork At each replication fork, helicase moves along the parental DNA, separating the two strands by breaking the hydrogen bonds between the base pairs. (This makes the two parental DNA strands available to the DNA polymerases for replication.) As soon as the base pairs separate at the replication fork, single-strand binding proteins attach to the separated strands and prevent the parental strands from rejoining. As helicase separates the two parental strands, the parental DNA ahead of the replication fork becomes more tightly coiled. To relieve strain ahead of the replication fork, topoisomerase breaks a covalent bond in the sugar-phosphate backbone of one of the two parental strands. Breaking this bond allows the DNA to swivel around the corresponding bond in the other strand and relieves the strain caused by the unwinding of the DNA at the helicase.

Okazaki fragments appear on the ___________ strand during DNA replication.

lagging

Match each phrase to the appropriate definition depending on whether it describes the synthesis of the leading strand, the synthesis of the lagging strand, or the synthesis of both strands.

leading strand: made continuously, only one primer needed, daughter strand elongates toward replication fork lagging strand: made in segments, multiple primers needed, daughter strand elongates away from the replication fork both strands: synthesized 5' to 3' Because DNA polymerase III can only add nucleotides to the 3' end of a new DNA strand and because the two parental DNA strands are antiparallel, synthesis of the leading strand differs from synthesis of the lagging strand. The leading strand is made continuously from a single RNA primer located at the origin of replication. DNA pol III adds nucleotides to the 3' end of the leading strand so that it elongates toward the replication fork. In contrast, the lagging strand is made in segments, each with its own RNA primer. DNA pol III adds nucleotides to the 3' end of the lagging strand so that it elongates away from the replication fork. In the image below, you can see that on one side of the origin of replication, a new strand is synthesized as the leading strand, and on the other side of the origin of replication, that same new strand is synthesized as the lagging strand. The leading and lagging strands built on the same template strand will eventually be joined, forming a continuous daughter strand.