* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download APDNA 2015 16
Survey
Document related concepts
DNA sequencing wikipedia , lookup
Zinc finger nuclease wikipedia , lookup
DNA repair protein XRCC4 wikipedia , lookup
DNA profiling wikipedia , lookup
Homologous recombination wikipedia , lookup
Eukaryotic DNA replication wikipedia , lookup
Microsatellite wikipedia , lookup
DNA nanotechnology wikipedia , lookup
United Kingdom National DNA Database wikipedia , lookup
DNA polymerase wikipedia , lookup
DNA replication wikipedia , lookup
Transcript
DNA AND ITS ROLE IN HEREDITY 2006-2007 DNA is the genetic material: a short history - DNA was found in the nucleus by Miescher (1868) • Early in the 20th century, the search for genetic material led to DNA – – – – – – – – T. H. Morgan’s group (1908): genes are on chromosomes Frederick Griffith (1928): experiments on S. pneumoniae Oswald Avery (1944): confirmed Griffith’s experiments Hershey and Chase (1952): DNA is the genetic material Erwin Chargaff (1947): The amount of thymine = adenine Watson and Crick (1953): Structure of DNA Rosalind Franklin (1951): X-Ray Structure of DNA Meselson and Stahl (1958): DNA Replication DNA was found in chromosomes using dyes that bind specifically to DNA. Avery, McCarty & MacLeod injected protein into bacteria - no effect injected DNA into bacteria - transformed harmless bacteria into virulent bacteria • Conclusion – first experimental evidence that DNA was the genetic material Oswald Avery Maclyn McCarty Colin MacLeod Evidence That Viral DNA Can Program Cells • Bacteriophages (or phages), are viruses that infect bacteria T2 Phage Protein coat labeled with 35S T2 bacteriophages are labeled with radioactive isotopes S vs. P Hershey & Chase bacteriophages infect bacterial cells bacterial cells are agitated to remove viral protein coats Which radioactive marker is found inside the cell? Which molecule carries viral genetic info? DNA labeled with 32P 35S radioactivity found in the medium 32P radioactivity found in the bacterial cells DNA Structure Reflects Its Role as the Genetic Material • After identifying DNA as the genetic material, scientists hoped to answer two questions about the structure: 1. How is DNA replicated between cell divisions? 2. How does it direct the synthesis of specific proteins? Structure of DNA • How does the structure of DNA account for its role in genetic inheritance? • Maurice Wilkins and Rosalind Franklin – used X-ray crystallography to study molecular structure Sugar–phosphate backbone 5 end Nitrogenous bases DNA STRUCTURE Thymine (T) Adenine (A) Cytosine (C) Chargaff’s rules state that in any species there is an equal number of A and T bases, and an equal number of G and C bases DNA nucleotide Phosphate Sugar (deoxyribose) 3 end Erwin Chargaff reported (1947) that DNA composition varies from one species to the next. Guanine (G) Fig. 16-5 Watson and Crick 5 end Hydrogen bond 3 end 1 nm 3.4 nm 3 end 0.34 nm 5 end Watson and Crick reasoned that the pairing was specific, dictated by the base structures Purine + purine: too wide Pyrimidine + pyrimidine: too narrow Purine + pyrimidine: width consistent with X-ray data DNA in the Nucleus and in the Cell Cycle DNA Is a Double Helix Base Pairs in DNA Can Interact with Other Molecules Adenine (A) Thymine (T) Guanine (G) Cytosine (C) But how is DNA copied? • Replication of DNA – base pairing suggests that it will allow each side to serve as a template for a new strand “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” — Watson & Crick Models of DNA Replication • Alternative models – become experimental predictions conservative P 1 2 semiconservative dispersive DNA Replication • Semiconservative replication • Each half of the double helix acquires a new mate • Each new DNA molecule, then, is really half old and half new 5 DNA has directionality • Putting the DNA backbone together – refer to the 3 and 5 ends of the DNA • the last trailing carbon PO4 base 5 CH2 O 4 1 C 3 O –O P O O 5 CH2 2 base O 4 1 2 3 OH 3 Each New DNA Strand Grows by the Addition of Nucleotides to Its 3′ End DNA Replication • Large team of enzymes coordinates replication Replication: 1st step • Unwind DNA – helicase enzyme • unwinds part of DNA helix • stabilized by single-stranded binding proteins helicase single-stranded binding proteins replication fork Fig. 16-13 Replication: 1st step Primase Single-strand binding proteins 3 Topoisomerase 5 3 5 Helicase 5 RNA primer 3 Fig. 16-13 Replication: 2nd step Primase Single-strand binding proteins 3 Topoisomerase 5 RNA primer 5 3 5 Helicase Primase starts an RNA chain from scratch - adds RNA nucleotides one at a time using the parental DNA as a template - 3 end serves as the starting point for the new DNA strand 3 Replication: 2nd step Build daughter DNA strand add new complementary bases to the 3’ end of the RNA primer DNA polymerase III DNA Polymerase III What is driving polymerization? New strand 5 end Sugar Phosphate Template strand 3 end 5 end 3 end T A T C G C G G C G C T A A Base 3 end DNA polymerase A 3 end C Nucleoside triphosphate 5 end C 5 end Fig. 16-16a Overview Origin of replication Leading strand Lagging strand Lagging strand 2 1 Leading strand Overall directions of replication DNA Replication Animation • To summarize, at the replication fork, the leading stand is copied continuously into the fork from a single primer. • The lagging strand is copied away from the fork in short segments, each requiring a new primer. Fig. 16.16 3 Template strand 5 5 The Lagging Strand: A Closer Look 3 3 Template strand 3 5 5 RNA primer 3 1 3 5 5 DNA Pol III works in the direction away from the replication fork 3 Template strand 3 Okazaki 5 5 RNA primer 3 3 1 5 5 Okazaki fragment 3 1 5 3 5 3 Okazaki 5 5 Template strand 3 RNA primer 3 3 1 5 5 3 5 Okazaki fragment 3 1 5 3 5 2 1 3 5 3 Okazaki 5 5 Template strand 3 RNA primer 3 3 1 5 5 3 1 5 3 5 2 3 3 5 Okazaki fragment 1 3 5 5 3 5 2 1 3 Okazaki 5 5 Template strand 3 RNA primer 3 3 1 5 5 3 1 5 3 5 2 3 3 5 Okazaki fragment 3 5 1 5 3 5 2 1 5 3 1 2 Overall direction of replication 3 5 Chromosome erosion All DNA polymerases can only add to 3 end of an existing DNA strand DNA polymerase I 5 3 3 5 5 growing replication fork 3 DNA polymerase III RNA Loss of bases at 5 ends in every replication chromosomes get shorter with each replication limit to number of cell divisions? 5 3 Telomeres Repeating, non-coding sequences at the end of chromosomes = protective cap limit to ~50 cell divisions 5 3 3 5 growing replication fork 5 3 telomerase 5 Telomerase enzyme extends telomeres can add DNA bases at 5 end different level of activity in different cells high in stem cells & cancers -- Why? TTAAGGG TTAAGGG 3 Fig. 16-20 1 µm Correcting Mistakes more than 130 DNA repair enzymes have been identified in humans • An enzyme detects something wrong in one strand of the DNA and removes it • Then DNA polymerase copies the information in the intact second strand and creates a new stretch of DNA • DNA ligase seals the gap Nobel Prize in Chemistry 2015 Interview Sunburn Damages DNA Nucleotide excision repair Nuclease – a DNA cutting enzyme • In mismatch repair, repair enzymes fix incorrectly paired nucleotides. – A hereditary defect in one of these enzymes is associated with a form of colon cancer. Ghosts of Lectures Past Frederick Griffith The “Transforming Principle” live pathogenic strain of bacteria A. mice die live non-pathogenic strain of bacteria B. mice live heat-killed pathogenic bacteria C. mice live mix heat-killed pathogenic & non-pathogenic bacteria D. mice die Semiconservative replication • Meselson & Stahl – label “parent” nucleotides in DNA strands with heavy nitrogen = 15N – label new nucleotides with lighter isotope = 14N “The Most Beautiful Experiment in Biology” parent 15N/15N 15N parent strands replication 1958 P 1 2 conservative P 1 2 semiconservative dispersive DNA: Count the Carbons! 3 ’ 5’ 3’ 5’ 3’ Okazaki Leading & Lagging strands Limits of DNA polymerase III can only build onto 3 end of an existing DNA strand 5 3 5 3 5 growing replication fork 3 5 5 5 Lagging strand ligase 3 Leading strand 3 Lagging strand 3 Okazaki fragments joined by ligase “spot welder” enzyme 5 3 DNA polymerase III Leading strand continuous synthesis Replacing RNA primers with DNA DNA polymerase I removes sections of RNA primer and replaces with DNA nucleotides DNA polymerase I 5 3 3 5 5 growing replication fork ligase 3 RNA 5 3 But DNA polymerase I still can only build onto 3 end of an existing DNA strand