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Chapter 17 DNA Replication, Repair, and Recombination Lectures by Kathleen Fitzpatrick © 2016 Pearson Education, Inc. Simon Fraser University 17.1 DNA Replication  All DNA in the nucleus of a parent cell must be duplicated and carefully distributed to the daughter cells  The division process in...

Chapter 17 DNA Replication, Repair, and Recombination Lectures by Kathleen Fitzpatrick © 2016 Pearson Education, Inc. Simon Fraser University 17.1 DNA Replication  All DNA in the nucleus of a parent cell must be duplicated and carefully distributed to the daughter cells  The division process involves nuclear division (mitosis) and division of the cytoplasm (cytokinesis)  Chromosomes that have duplicated consist of two sister chromatids © 2016 Pearson Education, Inc. Separation of Sister Chromatids  Microtubules of the mitotic spindle separate the sister chromatids  Each chromosome move to opposite poles of the cell  New nuclear envelopes form around the chromosomes DNA Synthesis Occurs During S Phase  During interphase, the amount of nuclear DNA doubles during a specific time named S phase  A time gap called G1 phase separates S phase from the previous M phase and a second gap, G2 phase, separates S phase from the next M phase © 2016 Pearson Education, Inc. DNA Replication Is Semiconservative  One of the two strands of each new DNA molecule was derived from the parent molecule and the other strand was newly synthesized  This is called semiconservative replication © 2016 Pearson Education, Inc. Demonstration of Semiconservative Replication  Matthew Meselson and Franklin Stahl (with Jerome Vinograd) showed that replication is semiconservative by using 14N and 15N to distinguish newly formed DNA strands from old  Bacterial cells were grown in 15N medium for many generations to incorporate heavy nitrogen into their DNA, then transferred to 14N medium  The strands were distinguished by equilibrium density centrifugation © 2016 Pearson Education, Inc. © 2016 Pearson Education, Inc. DNA Replication Is Usually Bidirectional  DNA replication is especially well understood in Escherichia coli  Replication is very similar in prokaryotes and eukaryotes © 2016 Pearson Education, Inc. Replication Forks  Cairns observed replication forks  These are formed where replication begins and then proceeds in bidirectional fashion away from the origin © 2016 Pearson Education, Inc. Bacterial Replication  Replication forks move away from the origin, unwind the DNA, and copy both strands as they proceed  The two copies of the replicating chromosome bind to the plasma membrane at their origins; when replication is complete, the cell divides by binary fission © 2016 Pearson Education, Inc. Eukaryotic DNA Replication  In eukaryotes, replication of linear chromosomes is initiated at multiple sites, creating replication units called replicons  The DNA of a typical chromosome may contain several thousand replicons, each 50,000 to 300,000 bp in length  At each origin of replication, two replication forks synthesize DNA in opposite directions, forming a “replication bubble” © 2016 Pearson Education, Inc. © 2016 Pearson Education, Inc. Replication Initiates at Specialized DNA Elements  DNA replication initiates at origins of replication, where synthesis is initiated by several groups of initiator proteins  This sequence is AT rich and about 245 bp in length  The sequence varies among bacterial species but contains recognizable, similar sequences, called consensus sequences © 2016 Pearson Education, Inc. © 2016 Pearson Education, Inc. Replication Initiation in Bacteria  In E. coli, three enzymes, DnaA, DnaB, and DnaC, bind oriC and initiate replication  DnaA binding to part of the oriC sequence results in unwinding of DNA  To stabilize the single strands of DNA, SSB (single stranded binding protein) binds to the unwound regions  DnaB is a DNA helicase, which unwinds the DNA strands as replication proceeds © 2016 Pearson Education, Inc. © 2016 Pearson Education, Inc. Replication Initiation in Eukaryotes  Origins of replication recruit proteins that initiate the unwinding and replication of DNA  First, a multisubunit protein complex called the origin recognition complex (ORC) binds the replication origin  Next, the minichromosome maintenance (MCM) proteins bind the origin © 2016 Pearson Education, Inc. Eukaryotic Replication  The MCM proteins include several DNA helicases that unwind the double helix; a set of proteins called helicase loaders recruit the MCM proteins  At this point, all the DNA-bound proteins make up the pre-replication complex, and the DNA is “licensed” for replication © 2016 Pearson Education, Inc. Replicons Are Not All Fired at the Same Time  Certain clusters of replicons replicate early during S phase, whereas others replicate later  Active genes are replicated early during S phase, whereas inactive genes are replicated later © 2016 Pearson Education, Inc. DNA Polymerases Catalyze the Elongation of DNA Chains  DNA polymerase is an enzyme that can copy DNA molecules  Incoming nucleotides are added to the 3′ hydroxyl end of the growing DNA chain, so elongation occurs in the 5′ to 3′ direction © 2016 Pearson Education, Inc. DNA Is Synthesized as Discontinuous Segments That Are Joined Together by DNA Ligase  DNA is synthesized in the 5′ to 3′ direction, but the two strands of the double helix are oriented in opposite directions  One strand (the lagging strand) is synthesized in discontinuous fragments called Okazaki fragments  The other (leading) strand is synthesized as a continuous chain © 2016 Pearson Education, Inc. © 2016 Pearson Education, Inc. In Bacteria, Proofreading Is Performed by the 3′→ 5′ Exonuclease Activity of DNA Polymerase  About 1 of every 100,000 nucleotides incorporated during DNA replication is incorrect  Such mistakes are usually fixed by a proofreading mechanism  Almost all DNA polymerases have a 3′ → 5′ exonuclease activity © 2016 Pearson Education, Inc. Proofreading  Exonucleases degrade nucleic acids from the ends of the molecules  Endonucleases make internal cuts in nucleic acid molecules  The exonuclease activity of DNA polymerase allows it to remove incorrectly base-paired nucleotides and incorporate the correct base © 2016 Pearson Education, Inc. © 2016 Pearson Education, Inc. RNA Primers Initiate DNA Replication  DNA polymerase can add nucleotides only to the 3′ end of an existing nucleotide chain  Researchers observations 1. Okazaki fragments usually have short stretches of RNA at their 5′ ends 2. DNA polymerase can add nucleotides to RNA chains as well as DNA chains 3. Cells contain primase 4. Primase is able to initiate RNA strands without a preexisting chain to add to © 2016 Pearson Education, Inc. © 2016 Pearson Education, Inc. Eukaryote DNA Primase  Eukaryotic primase is not as closely associated with unwinding proteins but is tightly bound to DNA polymerase α  The term primosome is not used © 2016 Pearson Education, Inc. The Process of DNA Synthesis  Once the RNA primer is made, DNA polymerase III (or DNA polymerase α, followed by δ or ε, in eukaryotes) adds deoxynucleotides to the 3′ end of the primer  For the leading strand, just one primer is needed, but the lagging strand needs a series of primers to initiate each Okazaki fragment  When the DNA chain reaches the next Okazaki fragment, the RNA is degraded and replaced with DNA; adjacent fragments are joined together by DNA ligase © 2016 Pearson Education, Inc. The DNA Double Helix Must Be Locally Unwound During Replication  During DNA replication, the two strands of the double helix must unwind at each replication fork  Three classes of proteins facilitate the unwinding: DNA helicases, topoisomerases, and singlestranded DNA binding proteins © 2016 Pearson Education, Inc. Helicases  DNA helicases are responsible for unwinding the DNA, using energy from ATP hydrolysis  The DNA double helix is unwound ahead of the replication fork, the helicases breaking the hydrogen bonds as they go © 2016 Pearson Education, Inc. Single-Stranded DNA Binding Protein  Once strand separation has begun, molecules of SSB (single-stranded DNA binding protein) move in quickly and attach to the exposed single strands  They keep the DNA unwound and accessible to the replication machinery © 2016 Pearson Education, Inc. Topoisomerases  The unwinding of the helix would create too much supercoiling if not for topoisomerases  These enzymes create swivel points in the DNA molecule by making and then quickly sealing double-stranded or singlestranded breaks © 2016 Pearson Education, Inc. DNA Unwinding and DNA Synthesis Are Coordinated on Both Strands Via the Replisome  Starting at the origin of replication, the machinery at the replication fork adds proteins required for synthesizing DNA  These are DNA helicase, DNA gyrase, SSB, primase, DNA polymerase, and DNA ligase  The proteins involved in replication are closely associated in a large complex called a replisome © 2016 Pearson Education, Inc. The Replisome  The activity and movement of the replisome is powered by nucleoside triphosphate hydrolysis  As the replisome moves along the DNA, it must accommodate the fact that DNA is being produced on both leading and lagging strands © 2016 Pearson Education, Inc. © 2016 Pearson Education, Inc. Eukaryotes Disassemble and Reassemble Nucleosomes as Replication Proceeds  Eukaryotes have much of the same replication machinery found in prokaryotes  For example, a DNA clamp protein acts along with DNA polymerase; one of these is called proliferating nuclear cell antigen (PCNA)  PCNA is a clamp protein for DNA polymerase δ © 2016 Pearson Education, Inc. Replication Factories and Chromatin Remodeling  Studies addressing how many origins of replication can be coordinated suggest that immobile structures called replication factories synthesize DNA as chromatin fibers are fed through them  Unfolding chromatin fibers ahead of the replication fork is facilitated by chromatin remodeling proteins that loosen nucleosome packing © 2016 Pearson Education, Inc. Telomeres Solve the DNA End-Replication Problem  Linear DNA molecules have a problem in completing DNA replication on the lagging strand because primers are required  Each round of replication would end with the loss of some nucleotides from the ends of each linear molecule  Eukaryotes solve this problems with telomeres, highly repeated sequences at the ends of chromosomes © 2016 Pearson Education, Inc. Telomeres and Telomerase  Human telomeres have 100 to 1500 copies of TTAGGG at the ends of chromosomes  Ensure that the cell will not lose DNA during replication  A polymerase called telomerase catalyze the reaction © 2016 Pearson Education, Inc. © 2016 Pearson Education, Inc. Protecting Chromosome Ends  After telomeres are lengthened by telomerase, telomere capping proteins bind to the exposed 3′ end to protect from degradation  In many eukaryotes, the 3′ ends of the DNA also loop back and base-pair with the opposite strand to form a protective closed loop  In multicellular organisms, telomerase function is restricted to germ cells and a few other types of actively proliferating cells © 2016 Pearson Education, Inc. 17.2 DNA Damage and Repair  DNA must be accurately passed on to daughter cells  In addition to ensuring that replication is faithful, this also means that DNA alterations must be repaired  DNA alterations, or mutations, can arise spontaneously or through exposure to environmental agents © 2016 Pearson Education, Inc. Mutations Can Occur Spontaneously During Replication  During DNA replication, some types of mutations occur through 1. Spontaneous mispairing of bases 2. Slippage during replication 3. Spontaneous damage to individual bases © 2016 Pearson Education, Inc. DNA Tautomers  Mispairing of DNA nucleotides due to presence of tautomers is the most common form of spontaneous replication error  Tautomers are rare, alternate resonance structures of nitrogenous bases © 2016 Pearson Education, Inc. Trinucleotide Repeats  Spontaneous replication errors can occur in regions with repetitive DNA  One example involves trinucleotide repeats, which are susceptible to strand slippage  In this process, DNA polymerase replicates a short stretch of DNA twice © 2016 Pearson Education, Inc. © 2016 Pearson Education, Inc. Depurination and Deamination  Another reaction that can occur spontaneously involves chemical modification of bases  Depurination, the loss of a purine base, and deamination, the removal of a base’s amino group, are the most common  A human cell may undergo thousands of depurinations each day, and about 100 deaminations  Failure to repair these can lead to base changes in the DNA sequence © 2016 Pearson Education, Inc. © 2016 Pearson Education, Inc. Mutagens Can Induce Mutations  DNA damage can be caused by mutation-causing agents, mutagens  Environmental mutagens fall into two categories: chemicals and radiation  Mutation can also be induced by mobile genetic elements, such as found in viruses, or transposable elements (transposons) © 2016 Pearson Education, Inc. DNA Damage by Chemical Mutagens  Base analogues resemble nitrogenous bases and are incorporated into DNA  Base-modifying agents react chemically with DNA bases to alter their structures, forming DNA adducts  Intercalating agents insert themselves between adjacent bases, distorting DNA structure © 2016 Pearson Education, Inc. Base Analogues  Base analogues, structurally similar to one of the DNA nucleotides, can be incorporated into a DNA molecule during replication  An example is 5-bromodeoxyuridine (BrdU) with pairing properties similar to thymine  Wherever it is incorporated into DNA, when the DNA is replicated, an A is incorporated into the new strand © 2016 Pearson Education, Inc. Base-Modifying Agents  Several mutagens act by chemically modifying a base that will then mispair at the next replication  Ethyl methansulfonate (EMS) adds ethyl groups to bases, while nitrosoguanidine adds methyl groups  Nitrous acid (HNO2) dramatically increases the likelihood of deamination  Other agents add bulky DNA adducts to DNA  Aflatoxin B1 attaches to guanine, leading to depurination © 2016 Pearson Education, Inc. © 2016 Pearson Education, Inc. Intercalating Agents  Intercalating agents (such as proflavin, acridine orange, benzo(a)pyrene) insert into the DNA double helix  They alter the shape of the double helix, leading to small nicks in the DNA  When repaired, there may be additions or deletions of nucleotides  Ethidium bromide is a common fluorescent dye (and intercalating agent) used for gel electrophoresis of DNA © 2016 Pearson Education, Inc. Radiation  Ultraviolet radiation alters DNA by triggering pyrimidine dimer formation—covalent bonds between adjacent pyrimidine bases  X-rays and related types of radiation, called ionizing radiation, remove electrons from molecules and generate highly reactive intermediates that damage DNA © 2016 Pearson Education, Inc. © 2016 Pearson Education, Inc. DNA Repair Systems Correct Many Kinds of DNA Damage  A variety of mechanisms have evolved for DNA repair  The strategies depend on how severe the damage is and whether or not the cell is undergoing division © 2016 Pearson Education, Inc. Light-Dependent Repair  Pyrimidine dimers can be directly repaired in a lightdependent process called photoactive repair  It depends on the enzyme photolyase, which catalyzes breakage of bonds between thymine dimers  The energy for this repair is provided by visible light © 2016 Pearson Education, Inc. Base Excision Repair  Excision repair pathways are classified into two types: base excision repair and nucleotide excision repair  Base excision repair corrects single damaged bases © 2016 Pearson Education, Inc. Nucleotide Excision Repair  Nucleotide excision repair (NER) uses proteins that detect distortions in the DNA helix and recruit NER endonuclease that cuts the DNA backbone on either side of the lesion  Helicase unwinds the DNA between the nicks and frees it from the DNA © 2016 Pearson Education, Inc. Mismatch Repair  Errors remaining after DNA replication are repaired by excision repair, in which abnormal nucleotides are removed and replaced  E. coli has nearly 100 genes that code for proteins involved in this process  Excision repair works by a basic three-step process © 2016 Pearson Education, Inc. Methylation in Mismatch Repair  DNA methylation does not occur immediately after DNA replication  Therefore, mismatch repair systems can distinguish the original DNA (methylated) from the newly made strand (unmethylated)  The incorrect nucleotide in the newly made strand is excised and replaced © 2016 Pearson Education, Inc. Double-Strand Break Repair  Double-strand breaks cleave DNA into two fragments  It is difficult for the repair system to identify and rejoin the correct broken ends without loss of nucleotides  Two pathways are used: nonhomologous endjoining and homologous recombination © 2016 Pearson Education, Inc. Nonhomologous End-Joining  Nonhomologous end-joining uses a set of proteins that bind to ends of broken DNA fragments and join them together  This is error-prone because nucleotides can be lost from the broken ends, and there is no way to ensure the correct DNA fragments are joined © 2016 Pearson Education, Inc. © 2016 Pearson Education, Inc. Homologous Recombination  Homologous recombination involves the process of crossing over, genetic exchange between DNA molecules with extensive sequence similarity  If the DNA molecule from one chromosome is broken, the homologue is available as a template to guide accurate repair © 2016 Pearson Education, Inc. Process of Homologous Recombination  The break in the DNA is detected and the ends trimmed  As with SDSA, strand invasion occurs, but involving both strands  DNA synthesis fills in DNA on both strands using the intact pieces of DNA as a template  The result is a Holliday junction, a crossed structure, which is resolved to generate two separate stands of repaired DNA © 2016 Pearson Education, Inc. © 2016 Pearson Education, Inc.

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