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.

Outline format: Because it is mostly review, this lecture was originally all in outline format. I have attempted to flesh out parts of it to help compensate for the delay in obtaining the textbook, but there has not been enough time to do all of it.

Replication of the genetic material: This is the second of five 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 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.

The notes that follow 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 antiparalled 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, but 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 half are labeled (on one strand of their double-stranded DNA) and the other half are 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.

Initiation of replication

• Origin of Replication: The DNA of a bacterial chromosome is a closed circular structure. Specific sequences at origin(s) of replication (9mers and 13 mers)DNA replication begins at a specific site characterized by the presence of repeated 9-mer and 13-mer structures in the DNA (repeated occurrence of specific 9 base and 13 base sequences -- see pages 44-45 and figure 2.26 in the textbook).

• 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 separationin 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 to reseal 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 0 50 of our textbook, but will not be studied in detail in this course.

5'-to-3' synthesis.

• Unidirectional addition of nucleotides: Polymerization of DNA, and also of RNA, occurs by condensing a 5'-nucleotide triphosphate (dNTP or NTP onto the 3' hydroxyl group of another nucleotide, or of a growing polynucleotide chain. No mechanism exists for extending the 5'-end of a nucleotide chain.

• Energy for synthesis: Hydrolysis of the dNTP or NTP being added provides the energy needed form the phosphodiester bond that links each nucleotide into the growing nucleic acid chain. The outer two phosphates of the triphosphate are split off, the the innermost phosphate forms an ester linkage with the 3'-hydroxyl group at the end of the pre-existing chain.

• Leading and lagging strands: The antiparallel nature of the DNA double helix, together with the inability to synthesize new chains in a 3'-to-5' direction makes it impossible to immediately begin synthesis of a new strand complementary to one of the separated DNA strands at a newly formed replication fork. Synthesis begins immediately in the 5'-to3' direction, but it is necessary to wait until a sufficient amount of the other strand has been converted to single stranded so that "backward" synthesis of its complement can occur in a 5'-to-3' direction. The strand whose synthesis begins immediately is called the "leading" strand, and the one whose synthesis is delayed is called the "lagging" strand.

• 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, another segment begins synthesis of a new complementary strand. If the replicating DNA is denatured (separated into individual strands) before the newly synthesized pieces of the lagging strand have all 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 segment. The small fragments are called "Okazaki fragments", named for the person who first discovered them.

RNA priming,

• Deoxynucleotides can only be added to 3'-end of pre-existing strand
• New synthesis is primed with a short segment of RNA that is later removed
• Primase enzyme adds RNA primer
• Primasome complex (both leading and lagging strands).

DNA polymerase III

• Replisome complex
• Dimer plus many accessory protiens
• Loading function of gamma complex (not discussed in current textbook)
• Clamping function of beta subunits
• dNTP's are added only at 3' ends.
• Concrrent synthesis on leading and lagging strands requires a looped structure both in prokaryotes (Fig. 2.22) and in eukaryotes (Fig. 2.25).

Proofreading

• 3' to 5' exonuclease activity of DNA polymerases III and I.
• Polymerases can remove newly inserted nucleotides and try again for a correct match.

DNA polymerase I.

• Discovered first, but not the primary enzyme for new DNA synthesis
• Probable has a major role in DNA repair.
• 5' to 3' exonuclease activity
• Important role in removal of RNA primer
• Primer ribonucleotides replaced with deoxyribonucleotides

Final steps

• DNA Ligase, joining of fragments.
• Topoisomerase II (cuts and reseals at termination of DNA synthesis).

DNA polymerase II

• Probably involved primarily in repair.

Eukaryotic DNA replication

• Polymerase alpha is currently believed to be responsible for synthesis of the lagging strand and polymerase delta for the leading strand (Figure 2.25).
• Polymerase beta and eta are also nuclear and are generally thought to be involved in repair (Table 2.2).
• Polymerase gamma is mitochondrial.
• Multiple replicons in eukaryotic cells
• Histone synthesis is tightly linked to DNA synthesis with immediate formation of new nucleosomes.

Telomerase

• In a linear chromosome, DNA polymerase cannot replace primer at 5'-end of lagging strand with DNA
• Telomerase uses its own RNA primer to generate special end structures known as telomeres.
• Telomeres have characteristic repeated sequences (TTAGGG in humans)
• Telomeres and telomerase provide a mechanism for maintaining full length ends of chromosomes.

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