DDNA - DNA & Molecular Genetics What is the...

Info iconThis preview shows page 1. Sign up to view the full content.

View Full Document Right Arrow Icon
This is the end of the preview. Sign up to access the rest of the document.

Unformatted text preview: DNA & Molecular Genetics What is the molecular basis of inheritance? • Early 20th century, most scientists assumed proteins. • 1928 —Frederick Griffith discovered that something from heat-killed pathogenic strain of Streptococcus pneumoniae could “transform ” a nonpathogenic strain to become pathogenic. This pathogenicity was inherited by all subcultures. Molecular Genetics EXPERIMENT Bacteria of the “S (smooth) strain of Streptococcus pneumoniae are pathogenic because they ” have a capsule that protects them from an animal ’s defense system. Bacteria of the “R” (rough) strain lack a capsule and are nonpathogenic. Frederick Griffith injected mice with the two strains as shown below: Living S (control) cells Living R (control) cells Heat-killed (control) S cells Mixture of heat-killed S cells and living R cells RESULTS Mouse dies Mouse healthy Mouse healthy Mouse dies Living S cells are found in blood sample. Figure 16.2 • In 1940s, it was found that the fraction containing DNA extracted from the pathogenic strain was causing the transformation. Feb 16, 2001 What is the molecular basis of inheritance? • 1952 —The Alfred Hershey and Martha Chase experiment EXPERIMENT In their famous 1952 experiment, Alfred Hershey and Martha Chase used radioactive sulfur and phosphorus to trace the fates of the protein and DNA, respectively, of T2 phages that infected bacterial cells. 1 Mixed radioactively labeled phages with bacteria. The phages infected the bacterial cells. Phage 2 Agitated in a blender to 3 Centrifuged the mixture separate phages outside so that bacteria formed the bacteria from the a pellet at the bottom of bacterial cells. the test tube. Radioactive Empty protein protein shell Batch 2: Phages were grown with radioactive phosphorus ( 32 P), which was incorporated into phage DNA (blue). 4 Measured the radioactivity in the pellet and the liquid Radioactivity (phage protein) in liquid Bacterial cell Batch 1: Phages were grown with radioactive sulfur ( 35S), which was incorporated into phage protein (pink). DNA Phage DNA Centrifuge Radioactive DNA Pellet (bacterial cells and contents) Centrifuge Radioactivity (phage DNA) in pellet RESULTS Phage proteins remained outside the bacterial cells during infection, while phage DNA entered the cells. When cultured, bacterial cells with radioactive phage DNA released new phages with some radioactive phosphorus. What is the molecular basis of inheritance? • OK, maybe for viruses and bacteria. But what about in “higher ” organisms? • 1947 —Erwin Chargaff analyzed the base composition of DNA [%A / %T / %C / %G] from a number of different organisms, both prokaryotes and eukaryotes. – Reported that the DNA composition varies among species, but it is very consistent within species. • 1940s/50s —Others also noted that in dividing eukaryotic cells, the amount of DNA in the cells exactly doubled before division, with exactly half of the amount going to each daughter cell. • So by 1950, most biologists conceded that DNA is the most likely molecular agent of inheritance. … Pellet Figure 16.4 CONCLUSION CONCLUSION Griffith concluded that the living R bacteria had been transformed into pathogenic S bacteria by an unknown, heritable substance from the dead S cells. – … But how? Hershey and Chase concluded that DNA, not protein, functions as the T2 phage’s genetic material. Nucleic Acids are polymers of Nucleotide monomers Nitrogen base determines type of nucleotide • • • • Adenine Guanine Cytosine Thymine – DNA only • Uracil – RNA only Heyer 1 DNA & Molecular Genetics Nucleic Acids are polymers of Nucleotide monomers Nucleic Acids are polymers of Nucleotide monomers 5’-end • Nucleotides: phosphates on 5 ’-carbon of sugar • Nucleic Acids: phosphate links 5 ’-C of sugar to 3’-C of preceding nucleotide sugar • Nucleic Acid Polymer runs 5 ’ to 3 ’ 3’-end DNA double strands are anti-parallel (run in opposite directions) 5’-end 3’-end Double-stranded Single-stranded One strand 5’ to 3’. The other strand 3’ to 5’ DNA is a double helix — two complementary nucleic acid strands Start the Revolution: Discovering the 3-dimensional structure of DNA 1953 — • Rosalind Franklin – used X-ray crystallography to study the molecular structure of the DNA molecule. – concluded that DNA was composed of two anti-parallel sugar-phosphate backbones, with the nitrogenous bases paired in the molecule ’ s interior. Figure 16.6 Franklin ’s X-ray diffraction photograph of DNA • James Watson and Francis Crick – built on Franklin ’s work to complete the model for the “Double Helix ”. … – … and of course got all the credit! Heyer 2 DNA & Molecular Genetics Molecular Genetics • Replication – Precisely copying all the genetic information (DNA) – S-stage of cell cycle – Exact replicas passed to daughter cells • Gene Expression – – – – The key to molecular genetics: complementary base pairing Using a specific bit of the genetic information Make a “working copy” of the needed bit (gene) Take the working copy to the workshop (ribosome) Use the copied instructions to build a specific protein The key to molecular genetics: complementary base pairing H N N N H N Sugar O H CH 3 Each pair = 1 purine + 1 pyrimidine N N N Sugar Thymine (T) H O N N Sugar • C pairs only with G • A pairs only with T Figure 16.8 DNA Structure \ the sequence of bases in the two strands are complimentary to each other (not identical). Heyer N N H H O A=T (2 H-bonds) G≡C (3 H-bonds) N N Complementary base pairing in DNA DNA ’s complementary base sequence H • • O Adenine (A) N N H Guanine (G) Sugar Cytosine (C) DNA Replication Semiconservative • Each strand serves as a template for a new strand. • Each “daughter cell” receives one original template strand + one complementary strand. 3 DNA & Molecular Genetics DNA must be unwound to be read DNA Replication • Hydrogen bonds “ unzip” • Hydrogen bonds reform between new nucleotides Template model for DNA replication • Replication depends upon base pairing • Old strands serve as templates determining the sequence of complimentary new nucleotides DNA replication DNA Replication is Semi-Conservative DNA Semi-Conservative Enzymes of Replication Over a dozen enzymes and other proteins needed for replication •DNA Helicase – Unwinds and separates DNA •DNA Polymerase Template model for DNA replication • Replication depends on base pairing • Old strands serve as templates determining the sequence of complimentary new nucleotides • Both daughter copies have one old and one new strand Elongating a New DNA Strand • Elongation of new DNA at a replication fork is catalyzed by enzymes called DNA polymerases, which add nucleotides to the 3¢ end of a growing strand. New strand Template strand 3¢ end 5¢ end Sugar A Base 3¢ end 5¢ end T A T C G C G G Phosphate C G C T A A P P P P OH C P Pyrophosphate OH 3¢ end C 2P Figure 16.13 Nucleoside 5¢ end triphosphate 5¢ end Energy for synthesis from hydrolysis of PPi from nucleotide triphosphate (NTP). Heyer •DNA Ligase – Joins pieces of DNA together “Replication forks ” Antiparallel Elongation • But — Remember that polymerase only runs from 3’-to-5 ’ along a parental strand, adding nucleotides to the 3 ’end of the elongating strand. • Elongation of the new Leading Strand of DNA along the 3 ’-to-5 ’ arm of the parental template can proceed continuously 5 ’-to-3 ’. DNA Polymerase T – Sequentially adds new nucleotides to 3 ’-end of growing new DNA strand (Runs 3 ’ to 5 ’ along parental strand.) • But elongation of the new Lagging Strand of DNA along the antiparallel 5’-to-3 ’ parental template must proceed in 5 ’-to-3 ’ segments ( Okazaki fragments), and joined ( ligated ) later. ND TRA GS DIN LEA LAG GING STRA ND 4 DNA & Molecular Genetics Synthesis of leading and lagging strands during DNA replication 1 Synthesis of leading and lagging strands during DNA replication 1 DNA pol Ill elongates DNA strands only in the 5¢ Æ 3 ¢ direction. 3¢ DNA polymerase-Ill elongates DNA strands only in the 5¢Æ3 ¢ direction. 3¢ 5¢ Parental DNA 5¢ 3¢ Okazaki fragments 2 1 3¢ 5¢ DNA pol III 5¢ 3¢ Okazaki fragments 2 other new strand, the lagging strand , must grow in an overall 3 ¢Æ5¢ direction by addition of short segments, Okazaki fragments, that grow 5’Æ3¢ (numbered here in the order they were made). DNA pol III Template strand 3 Leading strand Lagging strand 1 2 Template strand Figure 16.14 DNA ligase Overall direction of replication Lagging Strands 1 Primase joins RNA nucleotides into a primer. • DNA polymerases cannot initiate the synthesis of a polynucleotide. They can only add more nucleotides to the 3¢ end of a present oligo- or poly-nucleotide. • The initial nucleotide strand to start is called a primer. – In cells, the primer is a 5–10-nucleotide RNAoligomer synthesized complementary to the parental strand by the enzyme primase. – In the lab, we can use a synthetic oligonucleotide of either RNA or DNA as a primer to initiate DNA synthesis. • For the leading strand, only one primer is needed. • For the lagging strand, a new primer is needed for each Okazaki fragment. 3¢ 5¢ 3¢ 5¢ Template strand DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment. 2 RNA primer 3¢ 5¢ 3¢ 1 5¢ 3 After reaching the next RNA primer (not shown), DNA pol III falls off. Okazaki fragment 3¢ 3¢ 5¢ 1 5¢ 4 After the second fragment is primed. DNA pol III adds DNA nucleotides until it reaches the first primer and falls off. 5¢ 3¢ 5 3¢ 2 5¢ 1 DNA pol 1 replaces the RNA with DNA, adding to the 3¢ end of fragment 2. 3¢ 6 5¢ 2 3¢ 3¢ 5¢ 1 DNA ligase forms a bond between the newest DNA and the adjacent DNA of fragment 1. 5¢ 7 2 Figure 16.15 Other Proteins That Assist DNA Replication • Helicase, topoisomerase, single-strand binding protein DNA ligase joins Okazaki fragments by forming a bond between their free ends. This results in a continuous strand. 4 Overall direction of replication Primers & DNA Synthesis 3 The other new strand, the lagging strand, must grow in an overall 3¢ Æ 5 ¢ direction by addition of short segments, Okazaki fragments, that grow 5¢ Æ 3 ¢ (numbered here in the order they were made). 3¢ 5¢ 1 3 The Figure 16.14 2 One new strand, the leading strand, can elongate continuously 5 ¢ Æ 3 ¢ as the replication fork progresses. 5¢ Parental DNA One new strand, the leading strand , can elongate continuously 5 ¢Æ3¢ as the replication fork progresses. 2 1 The lagging strand in this region is now complete. 3¢ 5¢ Overall direction of replication Origins of Replication • The replication of a DNA molecule begins at special sites called origins of replication, where the two strands are separated • A bacterial chromosome typically has one replication origin • A eukaryotic chromosome may have hundreds or even thousands of replication origins Origin of replication 1 Replication begins at specific sites where the two parental strands separate and form replication bubbles. Bubble Parental (template) strand Daughter (new) strand 0.25 µm Replication fork 2 The bubbles expand laterally, as DNA replication proceeds in both directions. • Most of the various proteins that participate in DNA replication form a single large complex — The DNA replication “machine ” • The DNA replication machine i s probably stationary during the replication process Heyer 3 Eventually, the replication bubbles fuse, and synthesis of the daughter strands is complete. Two daughter DNA molecules (a) In eukaryotes, DNA replication begins at many sites along the giant DNA molecule of each chromosome. Figure 16.12 a, b (b) In this micrograph, three replication bubbles are visible along the DNA of a cultured Chinese hamster cell (TEM). 5 DNA & Molecular Genetics A summary of DNA replication Overall direction of replication Lagging Leading strand Origin of replication strand 1 Helicase unwinds the parental double helix. 2 Molecules of singlestrand binding protein stabilize the unwound template strands. Proofreading and Repairing DNA 3 The leading strand is synthesized continuously in the 5¢Æ 3¢ direction by DNA pol III. Lagging strand DNA pol III OVERVIEW Leading strand Leading strand 5¢ Replication fork 3¢ Parental DNA 4 Primase begins synthesis of RNA primer for fifth Okazaki fragment. 5 DNA pol III is completing synthesis of the fourth fragment, when it reaches the RNA primer on the third fragment, it will dissociate, move to the replication fork, and add DNA nucleotides to the 3 ¢ end of the fifth fragment primer. Primase DNA pol III Primer 4 DNA ligase DNA pol I Lagging strand 3 2 1 3¢ • DNA polymerases proofread newly made DNA, replacing any incorrect nucleotides. • In mismatch repair of DNA, repair enzymes correct errors in base pairing. • In nucleotide excision repair, enzymes cut out and replace damaged stretches of DNA. 1 A thymine dimer distorts the DNA molecule. 2 A nuclease enzyme cuts the damaged DNA strand at two points and the damaged section is removed. Nuclease 5¢ DNA polymerase 6 DNA pol I removes the primer from the 5¢ end of the second fragment, replacing it with DNA nucleotides that it adds one by one to the 3 ¢ end of the third fragment. The replacement of the last RNA nucleotide with DNA leaves the sugarphosphate backbone with a free 3 ¢ end. 7 DNA ligase bonds the 3 ¢ end of the second fragment to the 5 ¢ end of the first fragment. DNA ligase Figure 16.17 Figure 16.16 Replicating the Ends of DNA Molecules • The ends of eukaryotic chromosomal DNA get shorter with each round of replication 5¢ End of parental DNA strands Leading strand Lagging strand 3¢ Last fragment Previous fragment RNA primer Lagging strand 3 Repair synthesis by a DNA polymerase fills in the missing nucleotides. 4 DNA ligase seals the Free end of the new DNA To the old DNA, making the strand complete. Replicating the Ends of DNA Molecules • Eukaryotic chromosomal DNA molecules have at their ends nucleotide sequences, called telomeres, that postpone the erosion of genes near the ends of DNA molecules. • In germ cells, an enzyme called telomerase catalyzes the lengthening of telomeres so no genes are lost in gametes. 5¢ 3¢ Primer removed but cannot be replaced with DNA because no 3¢ end available for DNA polymerase Removal of primers and replacement with DNA where a 3 ¢ end is available 5¢ 3¢ Second round of replication 5¢ New leading strand 3¢ New lagging strand 5 ¢ 3¢ Further rounds of replication Figure 16.18 Shorter and shorter daughter molecules • This could limit the number of times DNA could be replicated and cells could divide. Figure 16.19 1 µm Telomeres shorten with age • If the chromosomes of germ cells became shorter in every cell cycle essential genes would eventually be missing from the gametes they produce. • In germ cells, an enzyme called telomerase catalyzes the lengthening of telomeres so no genes are lost in gametes. Telomerase extends DNA ends using its own built-in RNA template • Factor in aging and maximum life span? • Long-term chronic stress increases rate of telomere shortening! Heyer So, why only in germ cells? 6 ...
View Full Document

Ask a homework question - tutors are online