DNA Synthesis DNA Synthesis in General DNA Synthesis in Pro/Eu-karyotes DNA Repair Genetic Rearrangements (Recombination) Reverse Transcriptase (Transposon) A. Strategies for Obtaining Fragments of DNA and Copies of Genes Restriction Fragments DNA produced by reverse transcriptase Chemical Synthesis B. Techniques for Identifying DNA Sequences Probes Gel Electrophoresis Detection of Specific DNA Sequences DNA Sequencing

DNA Techniques A. Strategies for Obtaining Fragments of DNA and Copies of Genes Restriction Fragments DNA produced by reverse transcriptase Chemical Synthesis B. Techniques for Identifying DNA Sequences Probes Gel Electrophoresis Detection of Specific DNA Sequences DNA Sequencing A. Strategies for Obtaining Fragments of DNA and Copies of Genes Restriction Fragments DNA produced by reverse transcriptase Chemical Synthesis B. Techniques for Identifying DNA Sequences Probes Gel Electrophoresis Detection of Specific DNA Sequences DNA Sequencing 2

Figure 11-10 Copyright © 2006 Pearson Prentice Hall, Inc. Antiparallel + Single Direction 5’  3’ How to recognize initiation site? (three different protein) Primer = 뇌관, 도화선 Figure 11-10 Copyright © 2006 Pearson Prentice Hall, Inc.

Assembling a Complementary Chain

DNA synthesis in bacteria involves Five Polymerases, as well as other enzymes DNA polymerase 1 catalyzes DNA synthesis and requires a DNA template and all four dNTPs DNA polymerase is a processive enzyme.

Replication From Multiple Origins

DNA Synthesis Begins at sites that act as replication origins Proceeds from the origins as two replication forks moving in opposite directions

Enzymes of DNA Replication Helicase unwinds the DNA (H-bond) Topo-isomerase (gyrase) unwinds the DNA (공유결합) Primase synthesizes RNA primer (starting point for nucleotide assembly by DNA polymerases) DNA polymerases assemble nucleotides into a chain, remove primers, and fill resulting gaps DNA ligase closes remaining single-chain nicks

Assembling Antiparallel Strands

Two Antiparallel Strands As DNA helix unwinds, one template strand runs in a direction allowing new DNA strand to be made continuously in the direction of unwinding Other template strand is copied in short lengths that run in the direction opposite to unwinding Discontinuous replication produces short lengths, then linked into a continuous strand

Replication process

Template for leading strand Template for lagging strand 1. Helicase (gyrase)  2. primase Unwinding enzyme (helicase H-bonding) Template for leading strand Primase 1 Helicase unwinds the DNA, and primases synthesize short RNA primers. RNA primers Overall direction of replication Template for lagging strand Replication fork Leading strand DNA polymerase RNA primers are used as starting points for the addition of DNA nucleotides by DNA polymerases. 2 RNA DNA DNA Figure 14.12: Steps in DNA replication, including the activities of the helicase, primase, DNA polymerases, and DNA ligase taking part in the process. Primer synthesis, removal, gap filling, and nick sealing occur primarily in the lagging strand. The drawings simplify the process. In reality, the enzymes assemble into a replication machine at the fork, replicating both strands from that position as the template strands fold and pass through it. Lagging strand DNA polymerase

3~4. DNA polymerase  DNA unwinds further, and leading strand synthesis proceeds continuously, while a new primer is synthesized on the lagging strand template and extended by DNA polymerase. 3 DNA polymerase Newly synthesized primer Primer being extended by DNA polymerase Another type of DNA polymerase removes the RNA primer, replacing it with DNA, leaving a nick between the newly synthesized segments. 4 Nick Figure 14.12: Steps in DNA replication, including the activities of the helicase, primase, DNA polymerases, and DNA ligase taking part in the process. Primer synthesis, removal, gap filling, and nick sealing occur primarily in the lagging strand. The drawings simplify the process. In reality, the enzymes assemble into a replication machine at the fork, replicating both strands from that position as the template strands fold and pass through it. DNA polymerase

5. DNA ligase  DNA ligase Nick is closed by DNA ligase. 5 DNA continues to unwind, and the synthesis cycle repeats as before: continuous synthesis of leading strand and synthesis of a new segment to be added to the lagging strand. 6 Lagging strand Leading strand DNA polymerase Figure 14.12: Steps in DNA replication, including the activities of the helicase, primase, DNA polymerases, and DNA ligase taking part in the process. Primer synthesis, removal, gap filling, and nick sealing occur primarily in the lagging strand. The drawings simplify the process. In reality, the enzymes assemble into a replication machine at the fork, replicating both strands from that position as the template strands fold and pass through it. Newly synthesized primer Primer being extended by DNA polymerase

DNA gyrase, often referred to simply as gyrase, is an enzyme that unwinds DNA, so that it can duplicate. Many antibiotics work by attacking bacterial DNA gyrase. DNA gyrase is a type II topoisomerase (EC 5.99.1.3) that introduces negative supercoils (or relaxes positive supercoils) into DNA by looping the template so as to form a crossing, then cutting one of the double helices and passing the other through it before releasing the break, changing the linking number by two in each enzymatic step. This process occurs in prokaryotes (particularly in bacteria), whose single circular DNA is cut by DNA gyrase and the two ends are then twisted around each other to form supercoils. The unique ability of gyrase to introduce negative supercoils into DNA is what allows bacterial DNA to have free negative supercoils. The ability of gyrase to relax positive supercoils comes into play during DNA replication. The right-handed nature of the DNA double helix causes positive supercoils to accumulate ahead of a translocating enzyme, in the case of DNA replication, a DNA polymerase. The ability of gyrase (and topoisomerase IV) to relax positive supercoils allows superhelical tension ahead of the polymerase to be released so that replication can continue. Topo-isomerase

Eukaryotic DNA Replication Four DNA polymerases are involved in replication of DNA, and others are involved in repair processes.

Primers

Telomeres Ends of eukaryotic chromosomes Short sequences repeated hundreds to thousands of times Repeats protect against chromosome shortening during replication

Telomerase Chromosome shortening is prevented in some cell types which have a telomerase enzyme (adds telomere repeats to chromosome ends)

Adding Telomere Repeats before replication

Proofreading and Error Correction Are an Integral Part of DNA Replication All of the DNA polymerases have 3' to 5' exonuclease activity that allows proofreading. 10-3  10-6 (proofreading) 10-9 ~ 10-10 (repair system)

Mechanisms That Correct Replication Errors Proofreading depends on the ability of DNA polymerases to reverse and remove mismatched bases (deoxyribonuclease) DNA polymerse = deoxyribonuclease DNA repair system corrects errors that escape proofreading (after replication)

Proofreading (during replication) If a replication error causes a base to be mispaired, DNA polymerase reverses and removes the most recently added base The enzyme then resumes DNA synthesis in the forward direction

Proofreading Template strand DNA polymerase New strand 1 Enzyme continues activity in the forward direction as DNA 3’ polymerase as long as the most recently added nucleotide is correctly paired. Proofreading New strand Enzyme adds a mispaired nucleotide. 2 Enzyme reverses, acting as a deoxyribonuclease to remove the mispaired nucleotide. 3 Figure 14.16 Proofreading by a DNA polymerase. Enzyme resumes forward activity as a DNA polymerase. 4

DNA Repair Mechanisms DNA polymerase enzymes Recognize distorted regions caused by mispaired base pairs Remove DNA section with mispaired base from the newly synthesized nucleotide chain Resynthesize the section correctly, using original template chain as a guide

Mismatch Repair Template strand Base-pair mismatch New strand Repair enzymes recognize a mispaired base and break one chain of the DNA at the arrows. 1 New strand The enzymes remove several to many bases, including the mismatched base, leaving a gap in the DNA. 2 The gap is filled in by a DNA polymerase using the intact template strand as a guide. 3 Figure 14.17 Repair of mismatched bases in replicated DNA. Nick left after gap filled in The nick left after gap filling is sealed by DNA ligase to complete the repair. 4

DNA Organization in Eukaryotes and Prokaryotes Histones pack eukaryotic DNA at successive levels of organization Many nonhistone proteins have key roles in the regulation of gene expression DNA is organized more simply in prokaryotes than in eukaryotes

Bacterial DNA Organized into loops through interaction with proteins Proteins similar to eukaryotic nonhistones regulate gene activity in prokaryotes

The Bacterial Chromosome Closed, circular molecule of DNA packed into nucleoid (핵양체) region of cell Replication begins from a single origin, proceeds in both directions Plasmids (in many bacteria) replicate independently of the host chromosome

Prokaryotic DNA Replication (no telomere)

Bacterial DNA Replication DNA replication begins at the origin of replication and is bidirectional rather than unidirectional

Eukaryotic Chromosomes Consist of DNA complexed with histone and nonhistone proteins DNA wraps around a nucleosome (two molecules each of histones H2A, H2B, H3, H4) Linker DNA connects adjacent nucleosomes Binding of histone H1 causes nucleosomes to package into a coiled structure (solenoid) Nonhistone proteins help control the expression of individual genes

Chromatin Distributed between: Euchromatin (loosely packed region, genes active in RNA transcription) Heterochromatin (densely packed masses, genes are inactive) Folds and packs to form thick, rodlike chromosomes during nuclear division

Nucleosomes and Chromatin Fiber Solenoid DNA Linker Nucleosome and DNA wound around core of 2 molecules each of H2A, H2B, H3, H4 2 nm Chromosome in metaphase 10-nm chromatin fiber 30-nm chromatin fiber Figure 14.18 Levels of organization in eukaryotic chromatin and chromosomes. Nucleosomes Linkers Chromatin fiber DNA Nucleosomes Chromatin fiber(solenoid) Chromosome