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Transcript
Chapter 12 DNA Synthesis, Mutation, and Repair DNA structure: A-T G–C Antiparallel Occurs during S phase in eukaryotes E. coli Distinguishing between Models of DNA Replication Three different models of how DNA might replicate were proposed based on DNA structure. • Semi-conservative replication (Fig. 12.1a) • Conservative replication (Fig. 12.1b) • Dispersive replication (Fig. 12.1c) Figure 12.1a-c Alternative Hypotheses for DNA Synthesis (a) Hypothesis 1: (b) Hypothesis 2: (c) Hypothesis 3: Semi-conservative replication Conservative replication Dispersive replication Intermediate molecule Distinguishing Between Models of DNA Replication The Meselsohn and Stahl experiment determines which model is correct. • 15N was fed to growing E. coli cells to mark DNA, then cells were switched to 14N. • DNA replication is semi-conservative: new DNA has one 15N strand and one 14N strand. (Fig. 12.2) Figure 12.2a,b Applying Ideas, Question 2 Be certain that you understand the behavior of DNA replication that gives this pattern in eukaryotes too. Apply the three models to this labeling scheme to see what chromosomal labeling patterns would occur. 3H-thymidine non-labeled-thymidine 1. One round of DNA replication in radioactive solution 2. Mitosis 3. One round of DNA replication in nonradioactive solution Figure 12.3a Structure of dNTPs: P What’s happening at the level of the nucleotide? P P 5' CH2 Base O 3' OH Figure 12.3b DNA synthesis reaction: addition of nucleotides at the 3’ end 5' end of strand P P Base CH2 Base CH2 O P O P CH2 O CH2 Base H20 + 3' P P Synthesis reaction P 5' CH2 3' OH P P OH P 1,2 Base O O Base CH2 Base O base addition occurs at the 3’ end base addition occurs at the 3’ end, next one here OH 3' end of strand Cell free in vitro DNA synthesis reactions were used to identify the enzymes involved in DNA replication. Proteins were extracted from E. coli and tested for activity in the cell free system. • Kornberg et al. determine that: • DNA replication requires an enzyme; the first discovered was DNA polymerase I. • DNA replication requires a DNA template. (Fig. 12.4, 12.8) Figure 12.4 TESTING TEMPLATE-DIRECTED SYNTHESIS 1. Isolate single strand of X174 DNA. X174 virus 2. Make copies of X174 DNA in vitro using DNA polymerase I. Normal X174 DNA Synthetic X174 DNA 3. Add synthetic X174 DNA to E.coli cells growing in culture. E. coli Synthetic DNA 4. Observe result: New generation of X174 particles appears. Synthetic X174 DNA is Note: It will turn out that infectious. DNApol I is often Conclusion: DNA polymerase I catalyzes template-directed synthesis involved in DNA repair Figure 12.6 Classic work of John Cairns in early 1960s: photomicrographs of E. coli replicating chromosome Radioactively labeled strand Two labeled strands = newly synthesized Cell free in vitro DNA synthesis reactions were used to identify the enzymes involved in DNA replication. Proteins were extracted from E. coli and tested for activity in the cell free system. • Cairns and de Lucia: DNA polymerase I is not the main replication enzyme. • Kohiyama and Kornberg discover DNA polymerase III, which is the main replication enzyme. Figure 12.7a Characteristics of replication in E. coli: • At the replication fork, DNA polymerase III builds the new strands in the 5’-3’ direction. • New nucleotides are only added to 3’ hydroxyl groups of other nucleotides. (creates a problem) Formation of the leading strand 3' 5' DNA polymerase III 5' Newly synthesized leading strand 3' 5' Replication fork Figure 12.7b Formation of lagging strand 3' 5' Lagging strands 5' 3' 3' DNA polymerase III 5' 3' 5' Okazaki fragments 5' 3' 3' 5' Gap DNA polymerase III beginning synthesis of new fragment Figure 12.8 Completely single stranded 3' 5' G A A T C T G Completely double stranded 3' G A A T C T G C Polymerase inactive 5' C Polymerase inactive C T T A G 5' A C G 3' Single strand as template plus 3' end to start synthesis 3' 5' G A A T C T G C Polymerase active C 5' T T OH 3' The new strands are initiated by adding nucleotides to a short RNA primer because there is no DNA on which to build. (Fig. 12.7) Figure 12.9 (error prone start, remove RNA, remove errors) 2 Topoisomerase nicks DNA to relieve tension from unwinding 3 Pol III synthesizes leading strand 1 Helicase opens helix 4 Primase synthesizes RNA primer (what’s the story here?) 5 6 Pol I excises RNA primer; fills gap 7 Pol III elongates primer; produces Okazaki fragment 1 DNA ligase links Okazaki fragments to form continuous strand Laboratory Analysis and Manipulation of DNA Sequences • Polymerase Chain Reaction (PCR): amplifies DNA from primers, produces numerous copies. (Fig. 12.11a,b) Mullis • Dideoxysequencing: a method for determining the exact nucleotide sequence of any DNA. (Fig. 12.12) Figure 12.11a Polymerase Chain Reaction: developed by Kary Mullis Nobel Prize 1993 Primers are required to run PCR: bit of a problem! CCCCATGCTTACAAGCAAGT Primer 5' 3' 3' 5' Primer Region of DNA to be amplified by PCR ATCCTATGGTTGTTTGGATGGGTG Figure 12.11 steps 1-3 POLYMERASE CHAIN REACTION 3'5' 1. Start with a solution containing template DNA, synthesized primers, and an abundant supply of the four dNTPs. 5'3' 3' 5' 2. Denaturation Heating leads to denaturation of the double-stranded DNA. 5' 3'5' 5' 3' 5' 5'3' 3. Primer binding At cooler temperatures, the primers anneal to the template DNA by complementary base pairing. Figure 12.11 steps 4-6 5' 5'3' 3'5' 3'5' 5'3' 5'3' 4. Extension During incubation, DNA polymerase synthesizes complementary DNA strand starting at the primer. 5. Repeat cycle of three steps (2-4) again, doubling the copies of DNA. 6. Repeat cycle again, up to 20-30 times, to produce millions of copies of template DNA. Figure 12.11 steps 1-3 POLYMERASE CHAIN REACTION 3'5' 1. Start with a solution containing template DNA, synthesized primers, and an abundant supply of the four dNTPs. 5'3' 3' 5' 2. Denaturation Heating leads to denaturation of the double-stranded DNA. 5' 3'5' 5' 3' 5' 5'3' 3. Primer binding At cooler temperatures, the primers anneal to the template DNA by complementary base pairing. Figure 12.11 steps 4-6 often used to amplify small DNA samples left at a crime scene 5' 5'3' 3'5' 3'5' 5'3' 5'3' 4. Extension During incubation, DNA polymerase synthesizes complementary DNA strand starting at the primer. 5. Repeat cycle of three steps (2-4) again, doubling the copies of DNA. 1-6 6. Repeat cycle again, up to 20-30 times, to produce millions of copies of template DNA. Laboratory Analysis of DNA Sequences Second basic method used in sequence analysis of DNA • Dideoxysequencing: a method for determining the exact nucleotide sequence of any DNA. (Fig. 12.12) Basis for the human genome initiative (Sequencing the Human Genome) Figure 12.12a,b DNA sequencing: one method is dependent on a dideoxynucleotide pool 1 Figure 12.12c Different-length strands can be lined up by size to determine DNA sequence. 1 Mutation and DNA Repair Mechanisms Mutations are created by chemicals, radiation, errors in meiosis and mistakes in DNA replication. • Mutations can be deleterious, beneficial, or silent. (Fig. 12.15, 12.17) • Mutations in an individual are usually deleterious, may cause disease and death. (Fig. 12.16a,b) • Mutations in a population are a source of genetic diversity that allows evolution to occur. Figure 12.15 Mismatch (about 1/1000 base additions) A A C T G G C Wild type T T G A C C G A A C T G G C A A C T A G C MUTANT 3' 5' A A C T G G C T T G A T C G DNA replication T T G A C C G 5' 3' A A C T G G C T T G A T C G DNA replication A A C T G G C Wild type Parental DNA T T G A C C G T T G A C C G First generation progeny A A C T G G C Wild type T T G A C C G Second generation progeny Figure 12.17 upper General Categories of Mutations Mutation type Insertion Deletion Definition Example Consequence G Addition of any number or nucleotides due to an error in DNA synthesis Removal of any number of nucleotides due to an error in DNA synthesis Original sequence: Mutant sequence: Original sequence: Mutant sequence: ACC CAT GAT GTA ACC CGA GAT TGT A ATA ATA ACC ATA ATA A CAT GAT GTA ACC ATG GTC TA Addition of 1 or 2 bases disrupts reading frame. Usually results in a dysfunctional gene product. Deletion of 1 or 2 bases disrupts reading frame. Usually results in a dysfunctional gene product. Figure 12.17 lower Mutation type Definition Gene duplication Addition of a small chromosome segment due to an error during crossing over at meiosis I. Chromosome inversion Change in a chromosome segment when DNA breaks in two places, flips, and rejoins. Example A B C D A B C D Consequence Genes A B C D A A A B B B C C D D C D Mutant A B D C A B C D A B C D Produces an extra copy of one or more genes. If point mutations occur in extra DNA, it can produce a new product. Changes gene order along chromosome. Other types of chromosome breaks can lead to deletion or addition of chromosome segments. Figure 12.16 Consequences of mutations: Well-studied example: DNA point mutation can lead to a different amino acid sequence. Phenotype Start of coding sequence CAC DNA sequence GTG GTG CAC GAC CTG TGA ACT GGA CCT CTC GAG CTC GAG Normal Amino acid sequence Valine CAC DNA sequence GTG Histidine GTG CAC Leucine GAC CTG Threonine Glutamic Proline Glutamic acid acid TGA ACT GGA CCT CAC GTG Normal red blood cells CTC GAG Mutant Amino acid sequence Histidine Valine Threonine Leucine Proline Valine Glutamic acid Sickled red blood cells Mutation and DNA Repair Mechanisms DNA Repair Mechanisms • DNA polymerase I proofreads and corrects point mutations during replication. • Other excision repair systems scan newly formed DNA and correct remaining mutations. (Fig. 12.13a,b) • Repair enzymes identify the correct template strand by its methyl groups. (Fig. 12.14a,b) • Defects in repair system enzymes are implicated in a variety of cancers. (Fig. 12.18a-c) Figure 12.13 Mismatched bases. 3' T G T C C A C A G G A 5' T C G C G 5' OH 3' But, how does the “system” know which is the correct sequence and which is the mutant sequence? Polymerase III can 3' repair mismatches. 5' 1 5' T G T C C A C A G G A T C G C Figure 12.14b METHYLATION-DIRECTED MISMATCHED BASE REPAIR Mismatch Answer 1. Where a mismatch occurs, the correct base is located on the methylated strand: the incorrect base occurs on the unmethylated strand. 2. Enzymes detect mismatch and nick unmethylated strand. 3. DNA polymerase I excises nucleotides on unmethylated strand. 4. DNA polymerase I fills in gap in 5' 3' direction. 5. DNA ligase links new and old nucleotides. Repaired Mismatch Figure 12.18a Interesting and common: UV-induced thymine dimers caused DNA to kink P CH2 DNA strand with adjacent P thymine bases CH P N O N Thymine H H O N Thymine CH3 H O O N N O UV light Kink P N 2 CH2 O CH3 O H P H O O CH2 O H O N H P Thymine dimer CH3 H N O CH3 Case Study: Xeroderma Pigmentosum quoted from http://www.mssc.edu/biology/B305/GTS/ws99/xero/xero.html by Sherrie Smith (underlines by HN) “Clinical Signs of the Disorder Xeroderma Pigmentosum is a very rare genetic defect. It is caused by a defect in ultraviolet radiation induced DNA repair mechanisms, and is characterized by severe sensitivity to all sources of ultraviolet radiation, especially sunlight. There are less than one thousand known cases of XP worldwide. XP sufferers are grouped according to the capacity of their body to repair DNA. Groups A, C, D, and Variant make up over 90% of all cases. Group A has the lowest level of DNA repair and the most severe symptoms. There is a wide range of symptoms: blindness and deafness, blistering or freckling on minimal sun exposure, developmental disabilities, dwarfism and hypergonadism, increased skin and eye cancers, and mental retardation. There is no cure. DNA damage is cumulative and irreversible. Treatment is limited to avoidance of exposure to UV radiation by staying indoors with sunlight blocked out, and the use of protective clothing, sunscreens, and eyeglasses. It’s also very important to avoid other known carcinogens.1 Inheritance Pattern Xeroderma Pigmentosum is a rare human autosomal recessive disease.2 The first symptoms usually occur between one and two years of age. Children will have a history of severe burns on small amounts of sunlight exposure. Others have numerous freckle-like spots on sun-exposed body parts. Later symptoms include premature aging, skin cancers, eye problems, and neurologic abnormalities. Mechanism of the Disease Process Tests have shown that normal skin fibroblasts can repair ultraviolet radiation damage to DNA by inserting new bases into DNA, XP sufferers lack this capacity , or have a much-reduced capacity for repair. In a study of Japanese patients, they were found to have a splice site mutation where reduced amounts of mRNA of reduced size was found on Northern Blot Anaylsis.” Figure 12.18b Nature of XP cellular responses Vulnerability of cells to UV light damage Percentage of cells surviving 100 Normal individuals 10 Individuals with XP 1 Dose of UV light Figure 12.18c 60 50 (counts per minute) Amount of radioactive thymidine incorporated Ability of cells to repair damage DNA damage repaired by normal individuals 40 30 20 10 No DNA repair in XP individuals 0 Dose of UV light Laboratory Applications of DNA Sequences DNA sequence analysis is used to compare genes within and between species to determine function and evolutionary relatedness. Use for endangered species restoration. Used in numerous forensic applications: http://www.ornl.gov/hgmis/elsi/forensics.html#5 1