Genetics - Ch. 9 DNA Replication & Recombination

Although the process of replication includes many components, they can be combined into three major groups:

1. A template consisting of single-stranded DNA 2. Raw materials (substrates) to be assembled into a new nucleotide strand 3. Enzymes and other proteins that "read' the template and assemble the substrates into a DNA molecule

Each active replication fork requires five basic components:

1. Helicase to unwind the DNA 2. Single-strand-binding proteins to protect the single nucleotide strands and prevent secondary structures 3. The topoisomerase gyrase to remove strain ahead of the replication fork 4. Primase to synthesize primers with a 3'-OH group at the beginning of each DNA fragment 5. DNA polymerase to synthesize the leading and lagging nucleotide strands

The Basic Rules of Replication

1. Replication is always semiconservative. 2. Replication begins at sequences called origins. 3. DNA synthesis begins with the synthesis of short segments of RNA called primers 4. The elongation of DNA strands is always in the 5' to 3' direction. 5. New DNA is synthesized from dNTPs; in the polymerization of DNA, two phosphate groups are cleaved from a dNTP and the resulting nucleotide is added to the 3'-OH group of the growing nucleotide strand. 6. Replication is continuous on the leading strand and discontinuous on the lagging strand. 7. New nucleotide strands are complementary and antiparallel to their template strands. 8. Replication occurs at very high rates and is astonishingly accurate, thanks to precise nucleotide selection, proofreading, and mismatch repair.

Despite their differences, all of E. coli's DNA polymerases have 7 actions in common:

1. synthesize any sequence specified by the template strand. 2. synthesize in the 5' to 3' direction by adding nucleotides to a 3'-OH group. 3. use dNTPs to synthesize new DNA. 4. require a 3'-OH group 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. 7. are associated with a number of other proteins.

All DNA polymerases require a nucleotide with a _____________ 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 an existing 3'-OH group to get started.

3'-OH

After DNA polymerase III attaches a DNA nucleotide to the 3'-OH group on the last nucleotide of the RNA primer, each new DNA nucleotide then provides the ___________ group needed for the next DNA nucleotide to be added. This process continues as long as ______________ is available. DNA polymerase I follows DNA polymerase III and, using its 5' to 3' exonuclease activity, removes the RNA ____________. It then uses its 5' to 3' polymerase activity to replace the RNA nucleotides with DNA nucleotides. DNA polymerase I attaches to the first nucleotide to the OH group at the 3' end of the preceding Okazaki fragment and then continues, in the 5' to 3' direction along the nucleotide strand, removing and replacing one at a time the RNA nucleotides of the primer.

3'-OH; template; primer

Characteristics of DNA polymerases I & III that function in replication in E. coli:

5' to 3' polymerase activity: Yes/Yes 3' to 5' exonuclease activity: Yes/Yes 5' to 3' exonuclease acitivity: Yes/No Function: Removes & replaces primers/Elongates DNA

Function of Single-strand-binding proteins

Attach to single-stranded DNA and prevent secondary structures from forming

Function of initiator proteins

Bind to origin and separate strands of DNA to initiate replication

Compare theta and linear eukaryotic replication.

DNA Template: Circular/Linear Breakage of nucleotide strand: No/No Number of replicons: 1/Many Unidirectional or bidirectional: Uni or bidirectional/Bidirectional Products: Two circular molecules/Two linear molecules

The process by which a cell doubles its DNA before division

DNA replication

_________________ replication would always produce hybrid molecules, containing some original and some new DNA, but the proportion of new DNA within the molecules would increase with each replication event.

Dispersive

Function of DNA polymerase III

Elongates a new nucleotide strand from the 3'-OH group provided by the primer

True or False? Replication has been most thoroughly studied and best understood in mammal systems.

False; Replication has been most thoroughly studied and best understood in bacterial systems.

The first E. coli polymerase to be discovered, DNA polymerase ________, also has 5' to 3' polymerase and 3' to 5' exonuclease activities, which allow the enzyme to synthesize DNA and to correct errors. Unlike DNA polymerase III, however, DNA polymerase _________ also possesses 5' to 3' exonuclease activity, which is used to remove the primers laid down by ______________ and replace them with DNA nucleotides by synthesizing in a 5' to 3' direction. The removal and replacement of primers appears to constitute the main function of DNA polymerase _________.

I; I; primase; I

DNA gyrase is a type ________ topoisomerase. In replication, DNA gyrase reduces the ________________ strain (torque) that builds up ahead of the replication fork as a result of unwinding. It reduces torque by making a double-strand break in one segment of the DNA helix, passing another segment of the helix through the break, and then resealing the broken ends of the DNA. This action removes a twist in the DNA and reduces the supercoiling.

II; torsional

The circular chromosome of E. coli ha a single origin of replication (oriC). _______________ _______________(DnaA in E. coli) 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.

Initiator proteins

Function of DNA ligase

Joins Okazaki fragments by sealing breaks in the sugar-phosphate backbone of newly synthesized DNA

Function of DNA gyrase

Moves ahead of the replication fork, making and resealing breaks in the double-stranded helical DNA to release the torque that builds up as a result of unwinding at the replication fork

The short lengths of DNA produced by the discontinuous replication of the lagging strand are called _____________ ______________, after Reiji Okazaki, who discovered them. In bacterial cells, each Okazaki fragment ranges from about 1000 to 2000 nucleotides in length; in eukaryotic cells, they are about 100 to 200 nucleotides long. These fragments on the lagging strand are linked together to create a continuous new DNA molecule.

Okazaki fragments

Function of DNA polymerase I

Removes RNA primers and replaces them with DNA

Function of DNA primase

Synthesizes a short RNA primer to provide a 3'-OH group for the attachment of DNA nucleotides

Meselson & Stahl's Experiment

To determine which of the three models of replication applied to E. coli cells, Matthew Meselson and Franklin Stahl needed a way to distinguish old and new DNA. They accomplish this by using two isotopes of nitrogen, N-14 (the common form) and N-15 (a rare, heavy form). Meselson and Stahl grew a culture of E. coli in a medium that contained N-15 as the sole nitrogen source; after many generations, all the E. coli cells had N-15 incorporated into all the purine and pyrimidine bases of their DNA. Meselson and Stahl took a sample of these bacteria, switched the rest of the bacteria to a medium that contained only N-14, and then took additional samples of bacteria over the new few cellular generations. In each sample, the bacterial DNA that was synthesized before the change in medium contained N-15 and was relatively heavy, whereas any DNA synthesized after the switch contained N-14 and was relatively light. Meselson and Stahl distinguished between the heavy N-15-laden DNA and the light N-14-laden DNA with the use of equilibrium density gradient centrifugation. In this technique, a centrifuge tube is filled with a heavy salt solution and a substance of unknown density - in this case, DNA fragments. The tube is then spun in a centrifuge at high speeds. After several days of spinning, a gradient of density develops within the tube, with high-density material at the bottom and low-density material at the top. The density of the DNA fragments matches that of the salt: light molecules rise and heavy molecules sink. Meselson and Stahl found that DNA from bacteria grown only on medium containing N-15 produced a single band at the position expected of DNA containing only N-15. DNA from bacteria transferred to the medium with N-14 and allowed one round of replication also produced a single band, but at a position intermediate between that expected of DNA containing only N-15 and that expected of DNA containing only N-14. This result is inconsistent with the conservative replication model, which predicts one heavy band (the original DNA molecules) and one light band (the new DNA molecules). A single band of intermediate density is predicted by both the semiconservative and the dispersive models. To distinguish between the two models, Meselson and Stahl grew the bacteria in medium containing N-14 for a second generation. After a second round of replication in medium with N-14, two bands of equal intensity appeared, one in the intermediate position and the other at the position expected of DNA containing only N-14. All samples taken after additional rounds of replication produced two bands, and the band representing light DNA became progressively stronger. Meselson and Stahl's results were exactly as expected for semiconservative replication and were incompatible with those predicted for both conservative and dispersive replication.

True or False? Errors arise whenever information is copied, and the more times it is copied, the greater the number of potential errors.

True

Function of DNA helicase

Unwinds DNA at replication fork

Each replicon contains an origin of replication. Replication starts at the origin and continues until the entire replicon has been replicated. Bacterial chromosomes have (many/ a single) origin(s) of replication, whereas eukaryotic chromosomes contain (many/a single) origin(s) of replication.

a single; many

If there are two replication forks, one at each end of the replication bubble, the forks proceed outward in both directions in a process called _________________ _________________, simultaneously unwinding and replicating the DNA until they eventually meet.

bidirectional replication

In _________________ replication, the entire double-stranded DNA molecule serves as a template for a whole new molecule of DNA, and the original DNA molecule is fully conserved during replication.

conservative

With ________________ replication, after one round of replication 50% of the molecules would consist entirely of the original DNA and 50% would consist entirely of new DNA. After a second round of replication, 25% of the molecules would consist entirely of the original DNA and 75% would consist entirely of new DNA. With each additional round of replication, the proportion of molecules with new DNA would increase, although the number of molecules with the original DNA would remain constant.

conservative

Initially, three models were proposed for DNA replication:

conservative replication, dispersive replication, semiconservative replication

The raw materials from which new DNA molecules are synthesized are ______________________ ________________ (dNTPs), each consisting of a deoxyribose sugar and a base (a nucleoside) attached to three phosphate groups. In DNA synthesis, nucleotides are added to the 3'-hydroxyl (3'-OH) group of the growing nucleotide strand. The 3'-OH group of the last nucleotide on the strand attacks the 5'-phosphate group of the incoming dNTP. Two phosphate groups are cleaved from the incoming dNTP, and a _________________________ bond is created between the two nucleotides.

deoxyribonucleoside triphosphate; phosphodiester

The other template strand is exposed in the 5' to 3' direction. After a short length of the DNA has been unwound, synthesis must proceed 5' to 3'; that is, in the direction opposite that of unwinding. Because only a short length of DNA needs to be unwound before synthesis on this strand gets started, the replication machinery soon runs out of template. By that time more DNA has unwound, providing new template at the 5' end of the new strand. DNA synthesis must start anew at the replication fork and proceed in the direction opposite that of the movement of the fork until it runs into the previously replicated segment of DNA. This process is repeated again and again, so synthesis of this strand is in short, discontinuous bursts. The newly made strand that undergoes _____________________ __________________ is called the lagging strand

discontinuous replication

In _________________ replication, both nucleotide strands break down (disperse) into fragments, which serve as templates for the synthesis of new DNA fragments, and then somehow reassemble into two complete DNA molecules. Each resulting DNA molecule contains interspersed fragments of old and new DNA; none of the original molecule is conserved.

dispersive

Another protein essential for the unwinding process is the enzyme DNA ____________, a topoisomerase. ____________________ control the supercoiling of DNA. They come in two major types: type ______ topoisomerases alter supercoiling by making single-strand breaks in DNA, while type _______ topoisomerases create double-strand breaks.

gyrase; Topoisomerases; I; II

A DNA ______________ breaks the hydrogen bonds that exist between the bases of the two nucleotide strands of a DNA molecule. Helicase cannot initiate the unwinding of double-stranded DNA; the ____________ proteins first separate DNA strands at the origin, providing a short stretch of single-stranded DNA to which a helicase binds. Helicase binds to the lagging-strand template at each replication fork and moves in the 5' to 3' direction along this strand, thus also moving the replication fork.

helicase; initiator

Replication occurs in four stages:

initiation, unwinding, elongation, and termination

As the DNA unwinds, the template strand that is exposed in the 3' to 5' direction allows the new strand to be synthesized continuously, in the 5' to 3' direction. This new strand, which undergoes continuous replication, is called the ______________ ____________.

leading strand

On the ______________ strand, where DNA synthesis is continuous, a primer is required only at the 5' end of the newly synthesized strand. On the ________________ strand, where replication is discontinuous, a new primer must be generated at the beginning of each Okazaki fragment. Primase forms a complex with helicase at the replication fork and moves along the template of the lagging strand. The single primer on the leading strand is probably synthesized by the primase-helicase complex on the template of the lagging strand of the other replication fork, at the opposite end of the replication bubble.

leading; lagging

DNA _____________________ is a large multiprotein complex that acts as the main workhorse of replication. DNA polymerase ________ synthesizes nucleotide strands by adding new nucleotides to the 3' end of a growing DNA strand. This enzyme has two enzymatic activities. Its 5' to 3' polymerase activity allows it to add new nucleotides in the 5' to 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 with a incorrect base is inserted into the growing DNA strand, DNA polymerase __________ uses its 3' to 5' exonuclease activity to back up and remove the incorrect nucleotide. It then resumes its 5' to 3' polymerase activity. These two functions together allow DNA polymerase III to efficiently and accurately synthesize new DNA molecules.

polymerase III; II

In DNA synthesis, new nucleotides are joined one at a time to the 3' end of the newly synthesized strand. DNA __________________, the enzymes that synthesize DNA, can add nucleotides only to the 3' end of the growing strand (not the 5' end), and so new DNA strands always elongate in the same 5'-to-3' direction (5' -> 3'). Because the two single-stranded DNA templates are _______________ and strand elongation is always 5' to 3', if synthesis on one template proceeds from, say, right to left, then synthesis on the other template must proceed in the opposite direction, from left to right. As DNA unwinds during replication, the antiparallel nature of the two DNA strands means that one template is exposed in the 5' to 3' direction and the other template is exposed in the ______ to ______ direction.

polymerases; antiparallel; 3'; 5'

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

polymerization; E. coli

An enzyme called ____________ synthesizes short stretches (about 10-12 nucleotides long) of RNA nucleotides, or _______________, which provide a 3'-OH group to which DNA polymerases can attach DNA nucleotides. (Because primase is an RNA polymerase, it does not require a preexisting 3'-OH group to start the synthesis of a nucleotide strand.) All DNA molecules initially have short RNA primers embedded within them; these primers are later removed and replaced with DNA nucleotides.

primase, primers

In theta replication, double-stranded DNA begins to unwind at the origin of replication, producing single nucleotide strands that then serve as templates on which new DNA can be synthesized. The unwinding of the double helix generates a loop, termed a ___________________ _____________.

replication bubble

Typical eukaryotic replicons are from 20,000 to 300,000 base pairs in length. At each origin of replication, the DNA unwinds and produces a _______________ _______________. Replication occurs on both strands at each end of the bubble, with the two replication forks spreading outward. Eventually, the replication forks of adjacent replicons run into each other, and the replicons fuse to form long stretches of newly synthesized DNA. The replication and fusion of all the replicons lead to two identical DNA molecules.

replication bubble

Unwinding occurs at one or both ends of the replication bubble, making it progressively larger. DNA replication on both of the template strands is simultaneous with unwinding. The point of unwinding, where the two single strand separate from the double-stranded DNA helix, is called a __________________ ____________.

replication fork

In some DNA molecules, replication is terminated whenever two _______________ __________ meet. In others, specific termination sequences (called _____ sites) block further replication. A termination protein, called _______ in E. coli, binds to these sequences, creating a _______-_______ complex that blocks the movement of helicase, thus stalling the replication fork and preventing further DNA replication. Each Tus-Ter complex blocks a replication fork moving in one direction, but not the other.

replication forks; Ter; Tus; Tus-Ter

A segment of DNA that undergoes replication is called a

replicon

Because of the __________________ nature of DNA replication, a double-stranded DNA molecule must unwind to expose the bases that act as a template for the assembly of new polynucleotide strands, which will be complementary and antiparallel to the template strands.

semiconservative

In __________________ replication, the two nucleotide strands unwind, and each serves as a template for a new DNA molecule.

semiconservative

With __________________________ replication, one round of replication would produce two hybrid molecules, each consisting of half original DNA and half new DNA. After a second round of replication, half the molecules would be hybrid, and the other half would consist of new DNA only. Additional rounds of replication would produce more and more molecules consisting entirely of new DNA, and a few hybrid molecules would persist.

semiconservative

Watson & Crick's 3D DNA structure implied that the complementary nature of the two nucleotide strands in a DNA molecule suggested that during replication, each strand can serve as a template for the synthesis of a new strand. The specificity of base pairing (A-T and C-G) implied only one sequence of bases can be specified by each template, and so the two DNA molecules built on the pair of templates will be identical with the original. Called the _____________________ ____________________ process, because each of the original nucleotide strands remains intact (conserved), despite no longer being combined in the same molecule; the original DNA molecule is half (semi) conserved during replication.

semiconservative replication

Circular DNA molecules that undergo theta replication have a _________________ origin of replication. Because of the limited size of these DNA molecules, replication starting from one origin can traverse the entire chromosome in a reasonable amount of time.

single

After DNA has been unwound by helicase, ______________________________________________ (SSBs) attach tightly to the exposed single-stranded nucleotide chains and prevent the formation of secondary structures that interfere with replication. Unlike many DNA-binding proteins, SSBs are indifferent to base sequence: they will bind to any single-stranded DNA. SSBs form tetramers (groups of four); each tetramer covers from 35 to 65 nucleotides.

single-strand-binding proteins

DNA synthesis does not happen _________________. Rather, it requires a number of enzymes and proteins that function in a coordinated manner.

spontaneously

A common type of replication that occurs in circular DNA is called ____________ replication, because it generates a structure that resembles the Greek letter theta.

theta

The large linear chromosomes in eukaryotic cells, however, contain far too much DNA to be replicated speedily from a single origin. The replication of eukaryotic chromosomes is initiated at _________________ of origins.

thousands

If a single replication fork is present, it proceeds around the entire circle to produce ________ complete circular DNA molecules, each consisting of one old and one new nucleotide strand.

two

Because the synthesis of both strands takes place simultaneously, _________ 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; the ___________________ template loops around so that it is position for 5' to 3' replication. In this way, the DNA polymerase III complex is able to carry out 5' to 3' replication simultaneously on both templates, even though they run in opposite directions. After about 1000 bp of new DNA has been synthesized, DNA polymerase III releases the lagging-strand template, and a new loop forms. _________________ synthesizes a new primer on the lagging strand, and DNA polymerase III then synthesizes a new Okazaki fragment.

two; lagging-strand; Primase

Because DNA synthesis requires a single-stranded template, and therefore double-stranded DNA must be ________________ before DNA synthesis can occur, the cell relies on several proteins and enzymes to accomplish the unwinding.

unwound