Supercoiling Causes Problems for Replication

Several major problems must be solved to accomplish bacterial DNA replication. First, there are the topological problems due to DNA being not only a double helix but also supercoiled. Because the two strands forming a DNA molecule are held together by hydrogen bonding and are twisted around each other to form a double helix, they cannot simply be pulled apart. The higher level supercoiling further complicates the problem of separating the strands. Consequently, before new DNA can be made, first the supercoiling must be unwound. Next the two strands of the parental DNA double helix must be untwisted (see below). In addition, since the vast majority of bacterial chromosomes are circular, it is important to untangle the two new circles of DNA.

The supercoiled bacterial chromosome is circular and two replication forks proceed in opposite directions around the circle (Fig. 5.03). This process is known as semi-conservative replication Mode of DNA replication in which each daughter molecule gets one of the two original strands and one new complementary strand replication fork Region where the enzymes replicating a DNA molecule are bound to untwisted, single stranded DNA replisome Assemblage of proteins (including primase, DNA polymerase, helicase, SSB protein) that replicates DNA template strand Strand of DNA used as a guide for synthesizing a new strand by complementary base pairing

FIGURE 5.02 Semi-conservative Replication and the Replisome

The replication fork is the site of DNA replication and, by definition, includes both the DNA and associated proteins. The assembled proteins, known as the replisome, facilitate the unwinding of the helix and the addition of new nucleotides. The arrows indicate the direction of movement of the replication fork. The synthesis of two DNA helices results from adding a new complementary strand to each one of the separated old strands.

Old strand

Two identical daughter molecules

Replisome (protein)

Replication Fork (DNA + protein)

Parent DNA

Two identical daughter molecules

Replisome (protein)

Replication Fork (DNA + protein)

Parent DNA

Theta Replication

FIGURE 5.03 Theta-Replication

Successive steps in DNA replication are shown for a circular bacterial chromosome. The chromosome (1) begins to replicate using two replication forks (2). Continued replication results in division of the chromosome (3) and its apparent resemblance to 9, the Greek letter, theta (4).

FIGURE 5.03 Theta-Replication

Successive steps in DNA replication are shown for a circular bacterial chromosome. The chromosome (1) begins to replicate using two replication forks (2). Continued replication results in division of the chromosome (3) and its apparent resemblance to 9, the Greek letter, theta (4).

bi-directional replication. A circle observed half way through division looks like the Greek letter theta (9) and so this mode of replication is also called theta-replication.

Supercoiling of DNA has been discussed in detail in Ch. 4. In E. coli, the two type II topoisomerases, DNA gyrase and topoisomerase IV, are involved in DNA replication. As the replication fork proceeds along the DNA, it over-winds the DNA and generates positive supercoils ahead of the replisome. Since the bacterial chromosome is negatively supercoiled, the overwinding introduced by replication is at first cancelled out. However, after replication of about 5 percent of the chromosome, the preexisting negative supercoils would all be used up. For DNA replication to proceed, the over-winding must be removed. DNA gyrase binds to the DNA ahead of the replica-

bi-directional replication Replication that proceeds in two directions from a common origin

DNA gyrase An enzyme that introduces negative supercoils into DNA, a member of the type II topoisomerase family theta-replication Mode of replication in which two replication forks go in opposite directions around a circular molecule of DNA topoisomerase IV A particular topoisomerase involved in DNA replication in bacteria

Properties of DNA Polymerase 107

Dna SupercoilingTheta Mode Replication

FIGURE 5.04 Unwinding of Double Helix and of Supercoils

For the replication fork to proceed, both the double helix and the supercoils must be unwound. Helicase unwinds the double helix and DNA gyrase removes the supercoiling.

FIGURE 5.04 Unwinding of Double Helix and of Supercoils

For the replication fork to proceed, both the double helix and the supercoils must be unwound. Helicase unwinds the double helix and DNA gyrase removes the supercoiling.

Quinolone antibiotics kill bacteria by inhibiting DNA gyrase. This blocks replication of the DNA.

tion fork and introduces negative supercoils that cancel out the positive supercoiling. The net result is that DNA gyrase "removes" supercoiling ahead of the replication fork (Fig. 5.04). Topoisomerase IV may help in this process to some extent, but its main function is disentangling daughter molecules after replication has finished, as described below.

The quinolone antibiotics (e.g., nalidixic acid and ciprofloxacin) inhibit bacterial DNA replication by acting on the type II topoisomerases, in particular DNA gyrase. Inhibited DNA gyrase remains bound to the DNA at one location and blocks movement of the replication fork. The net effect is cell death.

Helicase unwinds the DNA helix and SSB protein keeps the strands apart.

Strand Separation Precedes DNA Synthesis

The double helix itself is unwound by the enzyme DNA helicase (Fig. 5.05). The major helicase of E. coli is DnaB protein, which forms hexamers. Helicase does not break the DNA chains; it simply disrupts the hydrogen bonds holding the base pairs together. Energy is needed for this process and helicase cleaves ATP to supply this energy.

The two separated strands of the parental DNA molecule are complementary and so capable of base pairing to each other. In order to manufacture the new strands, the two original strands must be kept apart for use as templates. This is done by single strand binding protein, or SSB protein, which binds to the unpaired single stranded DNA and prevents the two parental strands from re-annealing (Fig. 5.06). In reality, the single stranded region between helicase and the lagging strand is longer than that between helicase and the leading strand, due to the three dimensional arrangement of the replication fork.

One new DNA strand is made continuously. The other is made as a series of fragments that must be linked together later.

Properties of DNA Polymerase

Polymerases are enzymes that join nucleotides together. Bacterial cells contain several DNA polymerases that have different roles both in DNA replication and in DNA repair (see Ch. 14). Further problems in DNA replication stem from the peculiarities of DNA polymerase, the enzyme that is responsible for making new chains of DNA. Firstly, DNA polymerase will only synthesize DNA in a 5'- to 3'- direction. Since the

DNA helicase Enzyme that unwinds double helical DNA DNA polymerase Enzyme that synthesizes DNA polymerase Enzyme that synthesizes nucleic acids single strand binding protein (SSB protein) A protein that keeps separated strands of DNA apart

FIGURE 5.05 Helicase Unwinds the Double Helix

To unwind DNA, helicase first binds to DNA and then cleaves the hydrogen bonds connecting base pairs to separate the strands of the helix.

FIGURE 5.05 Helicase Unwinds the Double Helix

To unwind DNA, helicase first binds to DNA and then cleaves the hydrogen bonds connecting base pairs to separate the strands of the helix.

Binding Dna Strand

FIGURE 5.06 Single Strand Binding Protein Keeps DNA Strands Apart

Soon after DNA helicase breaks the hydrogen bonds holding the DNA strands together, SSB protein binds to the freed strands to keep them from re-annealing.

FIGURE 5.06 Single Strand Binding Protein Keeps DNA Strands Apart

Soon after DNA helicase breaks the hydrogen bonds holding the DNA strands together, SSB protein binds to the freed strands to keep them from re-annealing.

Annealing Helicase

DNA polymerase can only make new DNA in one direction. Even stranger, it cannot start new strands of DNA.

New strands of DNA must be started with short segments of RNA, known as primers.

strands in a double helix are anti-parallel, and since a single replication fork is responsible for duplicating the double helix, this means that one of the new strands can be made continuously, but that the other cannot (Fig. 5.07). The strand that is made in one piece is called the leading strand and the strand that is made discontinuously is the lagging strand.

Secondly, all DNA polymerases lack the ability to initiate a new strand of nucleic acid and can only elongate a pre-existing strand. Consequently, a special mechanism for strand initiation is needed. This involves synthesis of a short RNA primer whenever a new DNA strand is begun. To begin a new strand DNA polymerase uses a short RNA primer made by another enzyme. Unlike DNA polymerases, RNA poly-merases can start new strands. A special RNA polymerase, known as primase (DnaG protein) makes the RNA primers that are responsible for strand initiation during DNA

lagging strand The new strand of DNA which is synthesized in short pieces during replication and then joined later leading strand The new strand of DNA that is synthesized continuously during replication primase Enzyme that starts a new strand of DNA by making an RNA primer RNA polymerase Enzyme that synthesizes RNA

RNA primer Short segment of RNA used to initiate synthesis of a new strand of DNA during replication

Supplying the Precursors for DNA Synthesis 109

Parental DNA

FIGURE 5.07 Continuous and Discontinuous Synthesis of DNA at the Replication Fork

Parental DNA

FIGURE 5.07 Continuous and Discontinuous Synthesis of DNA at the Replication Fork

The protein at the replication fork responsible for DNA synthesis, 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).

Precursors for DNA are made from those for RNA by oxidizing the ribose to deoxyribose.

The fact that all new strands of nucleic acid start with a piece of RNA plus the fact that ribonucleotides are made first supports the idea that RNA came first in evolution. This theory, the RNA world, is discussed further in Chapter 20.

synthesis in bacteria. Although the leading strand only needs to be started once, the lagging strand is made in short sections and a new RNA primer must be inserted each time a new portion is made. DNA polymerase will build new strands of DNA starting from each RNA primer (Fig. 5.08).

Polymerization of Nucleotides

All nucleic acids, whether DNA or RNA, are synthesized in the 5'- to 3'- direction. Incoming nucleotides are added to the hydroxyl group at the 3'-end of the growing chain. The precursors for DNA synthesis are the deoxyribonucleoside 5'-triphosphates (deoxy-NTPs), dATP, dGTP, dCTP, and dTTP. Proceeding from the deoxyribose outwards, the three phosphate groups are designated the a, b, and g phosphates. Upon polymerization, the high energy bond between the a and b phosphates is cleaved, releasing energy to drive the polymerization. The outermost two phosphates (the band g- phosphates) are released as a molecule of pyrophosphate. A new bond is made between the innermost phosphate (the a-phosphate) of the incoming nucleotide and the 3'-OH of the previous nucleotide at the end of the growing chain (Fig. 5.09).

Supplying the Precursors for DNA Synthesis

The DNA precursors, which contain deoxyribose, are made from the corresponding ribose-containing nucleotides (Fig. 5.10). Reduction of ribose to deoxyribose is catalyzed by the enzyme ribonucleotide reductase. This acts on the diphosphate derivatives (ADP, GDP, CDP, and UDP). A kinase then adds the third phosphate in the case of dADP, dGDP and dCDP, so giving dATP, dGTP and dCTP. The dUDP follows a different route, as DNA does not contain uracil but instead has thymine, the methyl derivative of uracil. Before methylation, dUDP is converted to dUMP by removal of a phosphate. Then thymidylate synthetase adds the methyl group, so converting dUMP to dTMP. Finally, two phosphates are added to give dTTP (Fig. 5.10).

The methyl group of thymine is carried by the tetrahydrofolate (THF) cofactor, which is oxidized to dihydrofolate (DHF) during the reaction. The DHF must be reduced back to THF for DNA synthesis to proceed. The enzyme that does this, dihy-drofolate reductase, is inhibited by the antibiotic trimethoprim in bacteria. Moreover, the synthesis of the precursor to the purines, adenine and guanine, also needs a one-carbon fragment carried by THF. So DNA precursor synthesis is actually blocked at two points by trimethoprim.

Eukaryotic dihydrofolate reductase is inhibited by methotrexate (amethopterin). Since growth of tumors involves rapid cell division and DNA replication by cancer cells, methotrexate is used as an anti-tumor agent. The sulfonamide class of antibiotics inhibits synthesis of the folate cofactor itself. Animals do not make folate, but require it in their diet, so sulfonamides are harmless to human patients in reasonable doses. Massive doses may cause liver and kidney problems.

deoxyribonucleoside 5'-triphosphate (deoxyNTP) Precursor for DNA synthesis consisting of a base, deoxyribose and three phosphate groups dihydrofolate (DHF) Cofactor with a variety of roles including making precursors for DNA and RNA synthesis dihydrofolate reductase Enzyme that converts dihydrofolate back to tetrahydrofolate folate Cofactor involved in carrying one carbon groups in DNA synthesis kinase Enzyme that attaches a phosphate group to another molecule methotrexate (or amethopterin) Anti-cancer drug that inhibits dihydrofolate reductase of animals ribonucleotide reductase Enzyme that reduces ribonucleotides to deoxyribonucleotides sulfonamide Antibiotic that inhibits the synthesis of the folate cofactor tetrahydrofolate (THF) Reduced form of dihydrofolate cofactor that is needed for making precursors for DNA and RNA synthesis thymidylate synthetase Enzyme that adds a methyl group, so converting the uracil of dUMP to thymine trimethoprim Antibiotic that inhibits dihydrofolate reductase of bacteria

Multipl RNA

Multipl RNA

% 41 Smgte

& primer

FIGURE 5.08 Strand Initiation Requires an RNA Primer

DNA poly merase cannot begin a new strand but can only elongate. Therefore DNA replication requires an RNA primer to initiate strand elongation. One RNA primer is needed to start the 5' to 3' leading strand. In contrast, m ultiple RNA primers are needed for the 3' to 5' lagging strand because this is made in short stretches each running 5' to 3'. DNA polymerase will then add nucleotides to the end of each RNA primer. Later, the short RNA primers will be removed and replaced by DNA.

Template strand

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Responses

  • Barbara
    What are the problems that come with the separation of strands required for replication?
    2 years ago
  • salvia
    What are the cause problem of supercoiling for DNA replication?
    8 months ago

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