Text Assignment: Chapter 2, pages 34-55 (includes end of chapter materials for the entire chapter). The sequence of presentation of topics in the textbook is somewhat different than in these notes. The textbook begins with the basic mechanisms involved in DNA replication, and then adds details, such as how the points at which replication starts are identified and the bidirectional nature of replication. These notes take a more direct chronological approach. However, the total set of topics covered is more-or-less the same in both.

Replication of the genetic material: This is the second of four lectures outlining basic principles of molecular biology and the central dogma that are covered in MCDB 1150. The primary focus of this lecture is on the replication of double-stranded DNA with complete conservation of the base sequences and the genetic information carried in those sequences. In an oversimplified model, the two strands can be visualized as separating, followed by assembly and polymerization of new base-paired complementary strands to give rise to two complete copies of the original double-stranded DNA molecule. However, actual processes that are involved are far more complex, as summarized below.

These notes attempt to move chronologically through the process of DNA synthesis. Except where labeled otherwise, they describe bacterial DNA synthesis. Eukaryotic DNA replication is similar in principle, but differs in a number of details, and is less well understood overall. The textbook also presents a brief summary of alternative forms of DNA replication in diverse other systems that will not be convered in detail in this class. You should be aware of their existence, but you do not need to study them in detail.

Templated replication.

• Sense and antisense strands: The sense strand of a double-stranded DNA molecule has a base sequence similar to that of the RNA that is transcribed from the DNA. The antisense strand (also known as template strand) has a sequence that is the reverse complement of the sense strand (base paired to it with an antiparallel 5' to 3' polarity). (See page 61 of our current textbook for a discussion of sense and antisense strands).

• Semiconservative replication: Discovery of the double helical structure of DNA immediately suggested a mechanism for precise replication -- namely separation of the two strands and templated synthesis of a new copy of each of the missing complementary strands. This model predicted that each new double helix should have one strand of parental DNA and one strand of newly synthesized DNA.

• Messelson-Stahl experiment: Semiconservative replication was demonstrated by starting with bacteria whose DNA was labeled with heavy nitrogen (¹⁵N) and then allowing various amounts of growth to occur in media labeled with normal nitrogen. After one round of replication, all of the DNA had an intermediate density. After two rounds, one half was light and the other half still had an intermediate density. There was no conservation of heavy DNA. However, the data suggested that each of the labelled strands from the original DNA remained intact and separated from its partner in each replication cycle.

• Sister chromatid labeling: The use of special labeling techniques that distinguish between DNA that has or has not incorporated a thymidine analog, 5-bromodeoxyuridine, have provided direct evidence for semiconservative replication of DNA in eukaryotic chromosomes. This can best be seen by examining the two halves of condensed metaphase chromosomes. After one generation of labeling, followed by one more generation of growth in the absence of label, sister chromatids (the two halves of the condensed post-replicative chromosomes) exhibit a pattern in which one of the chromatids is labeled (on one strand of its double-stranded DNA) and the other chromatid is unlabeled. This is fully consistent with one strand of the double-stranded DNA becoming labeled during the first replication cycle and the labeled strand staying intact during the second cycle of replication. (Much later in the semester, we will see that there are occasional breaks and rejoining, leading to sister chromatid exchange, as seen in figure 16-24, but even in that figure there are long segments of DNA that exhibit strictly semiconservative replication)

Initiation of replication

• Origin of Replication: The DNA of a bacterial chromosome is a closed circular structure. DNA replication begins at a specific site (origin of replication) characterized by the presence of repeated 9 base and 13 base nucleotide sequences. The repeating units are referred to as 9-mers and 13-mers. The 13 mers are AT rich, making easier to separate the two strands of the double-stranded DNA. See pages 44-45 and figure 2.26 in the textbook for additional information on the origin of replication.

• DNAa protein: The protein coded by the DNAa gene binds to the repeated 9mers. This forms a tight loop and generates a strain that causes strand separation in the region containing the AT-rich 13-mers (Fig. 2.26 in the textbook).

• Helicase: Enzymes called helicases use energy derived from ATP to further separate the two strands of the DNA double helix.

• Topoisomerase I: Separation of the two strands of the DNA double helix requires substantial unwinding of the helix (page 37 of textbook). An enzyme known as topoisomerase I (gyrase) relieves twisting strain that is generated by unwinding the double helix. It is believed to act by cutting one of the strands such that the other strand can rotate freely to relieve the strain, and then resealing the strand that has been temporarily cut.

• Keeping the helix open: Single stranded DNA-binding proteins (SSBPs) attach to the single stranded DNA generated by unwinding the double helix and temporarily keep it from reforming double helical structures. .

Patterns of replication

• Replication forks: When the two strands of double helical DNA separate and replication of both strands begins, a forked or Y-shaped structure is formed.

• Bidirectional replication: In bacterial cells, replication starts at a specific origin of replication within the circular DNA molecule and proceeds in both directions away from the origin. This results in the formation of a replication "bubble", which continues to elongate as replication proceeds (pages 45-46). A similar pattern is seen in eukaryotic chromosomes, except that multiple origins are involved (see replicons, below).

• Theta structure in bacteria: As replication proceeds in both directions around the circular chromosomes of bacteria, a structure reminiscent of the Greek letter theta is formed (see page 47 and figure 2.29 of text).

• Replicons: The very long DNA double helices found in eukaryotic chromosomes contain multiple origins of replication, which often initiate bidirectional replication more or less synchronously. Each unit of replication is called a replicon. Replication continues until the replication bubbles fuse to yield fully replicated DNA strands (see figure 2.27 of text).

• Other patterns There are also two alternative strategies for replication of circular DNA that are more complex than the simple bidirectional model. These are the the rolling circle mode (sigma mode) used by some types of viruses, and the D loop mode, used for replication of mitochondrial and chloroplast DNAs. These modes are described on pages 47 - 50 of our textbook, but will not be studied in detail in this course.

5'-to-3' synthesis.

• Abbreviations: In the discussions that follow, the abbreviation dNTP refers to any deoxyribonucleoside triphosphate (dATP, dCTP, dGTP, TTP). Similarly NTP refers to any ribonucleoside triphosphate (ATP, CTP, GTP, UTP).

• Unidirectional addition of nucleotides: Polymerization of DNA, and also of RNA, occurs by condensing a 5'-nucleoside triphosphate (dNTP or NTP) onto the 3' hydroxyl group of another nucleotide, or onto the 3'-end of a growing polynucleotide chain (see figure 2.18, and note that in this figure the template strand has been deliberately drawn with its 5'-end to the right). No mechanism exists for extending the 5'-end of a nucleotide chain.

• Energy for synthesis: Hydrolysis of the dNTP or NTP that is being added provides the energy needed to form a covalent bond. The outer two phosphates of the triphosphate are split off and the innermost phosphate forms an ester linkage with the 3'-hydroxyl group at the end of the pre-existing chain. This results in a phosphodiester bond

RNA priming,

• No new DNA starts: Deoxynucleotides can only be added to 3'-end of a pre-existing strand of DNA (or RNA). There are no enzymes capable of initiating the synthesis of a DNA-templated DNA molecule at the level of a single nucleotide. This makes necessary to use an indirect priming procedure.

• RNA primers: A variety of enzymes are capable of initiating new DNA-templated RNA synthesis (as in transcription). New DNA synthesis is primed with a short segment of RNA that is later removed

• Primase: A separate enzyme in the initiation complex called primase synthesizes a short RNA primer each time that new DNA synthesis begins, including all new starts in the discontinuous pattern of synthesis described below.

Leading and lagging strands

• Unidirectional synthesis of antiparallel DNA: The inability to synthesize new chains in a 3' to 5' direction adds a major complication to the replication of antiparallel double-stranded DNA. At any replication fork, one of the template strands has a 3' to 5' orientation, which is what is needed for synthesis of a new complementary strand in a 5' to 3' direction. Synthesis on that template is primed and starts very quickly. However, the other template strand has a 5' to 3' orientation and is thus unable to support synthesis beginning at the origin and moving away from it in a 3' to 5' direction. Because of this, the 5' to 3' template strand accumulates in a single stranded configuration until there is a sufficient length so that synthesis of its complementary antiparallel strand can be primed and initiated in a 5'-to-3' direction ("backward" toward the origin of replication). The strand whose synthesis begins immediately is called the "leading" strand, and the one whose synthesis is delayed is called the "lagging" strand.

• Half diagrams: At this point, diagrams of bidirectional synthesis begin to get so complicated that most illustrations, including those in our textbook, only show one of the two replication forks that are moving in opposite directions away from the origin of replication. As you study these diagrams, always remember that there are are actually two forks, with the second looking exactly like the one being depicted, but moving in the opposite direction with the top and bottom halves reversed in position due to the antiparallel nature of the DNA double helix. If you turn the page 180 degrees (upside down), you will have an accurate picture of what is happening in the fork moving to the left (everyone draws the single fork moving to the right).

• Discontinuous synthesis -- Okazaki fragments: As synthesis of the leading strand continues, more and more of the single stranded DNA of the lagging strand is unwound. Each time that a sufficient length is reached, synthesis of a new segment of the complementary strand begins. If the replicating DNA is denatured (separated into individual strands) before the newly synthesized pieces of the lagging strand have been ligated together, a number of relatively small fragments of newly synthesized DNA will be recovered, together with the much longer strands produced by continuous synthesis in the leading strand. The small fragments are called "Okazaki fragments", named for the person who first discovered them.

DNA polymerase III

• Templated DNA synthesis: After the RNA primer has reached an adequate length, DNA polymerase III begins synthesis of DNA, which proceeds to completion in the leading strand, and proceeds until the 5'-end of the previous primer is encountered in the lagging strand.

• DNA polymerase III holoenzyme: DNA polymerase III is a highly complex dimeric aggregate, consisting of 20 or more protein subunits (figure 2.21). The alpha subunits perform the actual DNA synthesis, but operate in conjunction with multiple accessory proteins

• Simultaneous synthesis of leading and lagging strands: There is yet another complicating factor in DNA synthesis. It is now generally believed that the leading and lagging strands are synthesized simultaneously by a single dimeric DNA polymerase III complex. This requires formation of a looped structure with the leading and lagging strands so positioned that their synthesis can occur side by side in the same orientation, despite the fact that the newly synthesized chains are growing in opposite directions relative to the overall DNA that is being replicated (figure 2.22). A similar looping also appears to occur in eukaryotic DNA replication (Fig. 2.25).

• Clamping function of beta subunits: As shown in figures 2.21 and 2.22, the beta subunits appear to have a clamping function that keeps the leading and lagging strands appropriately aligned with the catalytically active alpha subunits.

• Replisome complex: The entire DNA-synthesizing complex at each replication fork, which also includes topoisomerase, helicase, and primase, is sometimes referred to as a replisome (see page 42 and figure 2.25).

DNA polymerase I

• Not the primary enzyme for DNA synthesis: DNA polymerase I is so named because it was the first of the DNA polymerase enzymes to be isolated and characterized. Although it is capable of template-directed DNA synthesis, it is now known not to be the enzyme primarily responsible for new DNA synthesis, but it does have other important roles, as described below.

• 5' to 3' exonuclease activity: DNA polymerase I has an unusual 5' to 3' exonuclease activity. This gives it the ability to start at a single-stranded break and progressively remove nucleotides and replace them in a 5' to 3' direction.

• Removal of RNA primer: In the lagging strand, when DNA polymerase III runs into the 5'-end of the previous RNA primer, it is unable to proceed further. Although the new DNA butts up against the primer, it is not covalently joined to it. DNA polymerase III dissociates, leaving a "nick" (a single stranded gap) between the new DNA and the primer. Starting at the nick, DNA polymerase I removes the primer ribonucleotides one at a time, using its 5' to 3' exonuclease activity, and replaces them with deoxyribonucleotides, using its DNA polymerase activity..

• DNA repair: DNA polymerase I is also used to fill in short gaps in DNA, often as a part of a repair process that excises part of a damaged strand and replaces it with new DNA templated from the remaining strand.

Proofreading and DNA repair

• Error in textbook: On page 42, 4 lines from the bottom of the second column, "5' --> 3' exonuclease activity" should read "3' --> 5" exonuclease activity."

• (If you have not already done so, please go to the Errors in Textbook web page and correct all of the errors in your textbook so that you will not be studying wrong information).

• Removal of mismatched bases: DNA polymerases III and I both have 3' to 5' exonuclease activity. This allows them to remove a mismatched nucleotide that has just been added to a growing DNA chain and make another attempt to insert the correct nucleotide (figure 2.23). This "proofreading" function helps to reduce the number of mistakes in DNA synthesis that would otherwise result in mutation. There are also a number of other "repair" mechanisms that are used to minimize mutation, as will be discussed in chapter 5 of the text (lecture 7).

• DNA polymerase II: There is yet another prokaryotic DNA polymerase, designated DNA polymerase II, whose main function appears to be to synthesize replacement DNA during DNA repair. We will examine DNA repair in much greater detail in lecture 7.

Final steps in DNA synthesis

• DNA Ligase: After the last ribonucleotide is removed from the primer and replaced with a deoxynucleotide, there is still a nick in the newly synthesized lagging strand. This nick is closed by DNA ligase, which forms a covalent phosphodiester bond between the Okazaki fragments, joining them into a continuous strand of DNA (figure 2.20e). Note that Okazaki fragments accumulate when ligase function is impaired (boxed example 2.4).

• Topoisomerase II: One final enzyme that does not receive much attention in our textbook is topoisomerase II, whose role is to cut and reseal the newly synthesized DNAs as needed so that they can separate from each other (it is easy to visualize two circular genomes linked through each other at the end of replication).

Eukaryotic DNA replication

• Five DNA polymerases: Eukaryotic DNA replication is not as well understood as prokaryotic. However, there are at least five separate DNA polymerases, as described below.

• Separate enzymes for leading and lagging strands: Leading and lagging strands appear to be synthesized simultaneously in eukaryotic cells, but two separate enzymes are involved, rather than a dimer of a single enzyme. Polymerase alpha is currently believed to be responsible for synthesis of the lagging strand and polymerase delta for the leading strand (Figure 2.25).

• DNA repair: Polymerases beta and eta are also nuclear and are generally thought to be involved in repair (Table 2.2).

• Mitochondrial DNA synthesis: As we will see in lecture 35, mitochondria contain an independent DNA genome. Polymerase gamma is believed to be involved in mitochondrial DNA synthesis.

• Replicons: Because of the great length of the DNA molecules in eukaryotic chromosomes, they have multiple origins of replication. Each unit of DNA replication is referred to as a replicon (figure 2.27)

• Histone synthesis: Histones are basic proteins that interact with eukaryotic DNA to form stuctural units known as nucleosomes (figure 10.8). The synthesis of new histones is tightly linked to DNA synthesis with immediate formation of new nucleosomes.

Telomerase

• Chromosome ends cannot be replicated with DNA polymerase: Beause DNA can only be synthesized at the 3'-end of a preesisting DNA or RNA chain, there is no available mechanism for achieving DNA synthesis all of the way to the end of the lagging strand. Even if priming occurs from the extreme end, it is not possible to replace the primer with DNA. Figure 2.33a illustrates a situation where the primer has been removed by a 5' to 3' exonuclease but has not been replaced. If this keeps happening over many rounds of replication, the ends of the chromosomes will gradually experience major shortening.

• Restoration of ends: Telomerase uses its own RNA primer to generate special end structures known as telomeres. Telomeres have a relatively simple repeat structure that is characteristic for each species (TTAGGG in humans). As many repeated units as needed can be added. Telomeres and telomerase thus provide a mechanism for maintaining full length ends on chromosomes.

• Absence of telomerase activity from somatic cells: Telomerase is absent from human somatic cells, but present in germ line cells and in malignant cells. Some investigators believe lack of telomerase may be involved in aging, and that the presence of telomerase may be involved in the unrestrained multiplication of malignant cells. However, both of these ideas remain controversial, and both phenomena are far more complex than just the presence or absence of telomerase.

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