7.1 DNA Structure & Replication Essential Idea: The structure of DNA is ideally suited to its function. DNA is a double helix, consisting of two anti-parallel chains of polynucleotides that are held together by hydrogen bonds between complementary bases on the different strands. This structure allows the double helix to be replicated, with one ‘old’ strand combining together with a new strand in semiconservative replication. And DNA is transcribed into mRNA, which is then translated into a polypeptide. By Darren Aherne DNA’s function is to transmit genetic information from one generation to the next. It is a stable macromolecule that can be replicated with a high degree of fidelity in the enzyme assisted process of replication. These copies are passed on from one generation of cells to the next. Understandings, Applications and Skills Understandings, Applications and Skills 7.1 S1 Analysis of results of the Hershey and Chase experiment providing evidence that DNA is the genetic material. • Until the Hershey Chase experiment, it seemed that protein was the genetic material because it had great variety in structures • Hershey & Chase took advantage of the fact that DNA contains phosphorus, but not sulfur, & protein contains sulfur, but not phosphorus. Viruses have protein coats surrounding DNA. • They grew viruses in two environments- type 1 with radioactive phosphorus, and type 2 with radioactive sulfur. http://highered.mheducation.com/olc/dl/1 20076/bio21.swf 7.1.A1 Rosalind Franklin’s and Maurice Wilkins’ investigation of DNA structure by X-ray diffraction. • When X-rays pass through a substance, they diffract. • Crystals have a regularly repeating pattern, causing X-rays to diffract in a regular pattern. • These patterns allow for measurements & calculations to be made about the structure of molecules. • X pattern: DNA is a helix • Angle of X: Calculate the steepness of the helix • Distance between horizontal bars: Distance between bases is 3.4nm. • Distance between center of X and top of image: .34nm vertically between the repeating units (bases) in the molecule- so 10 bases per turn of the helix. http://www.dnalc.org/view/15874Franklin-s-X-ray.html 7.1.Nature of Science: Making careful observations—Rosalind Franklin’s X-ray diffraction provided crucial evidence that DNA is a double helix. • Rosalind Franklin worked at King’s College in London as a technician doing X-ray crystallography. • She improved the resolution of the cameras used in order to obtain the most detailed images yet of X-ray diffraction of DNA. • These detailed images allowed her to make very exact measurements related to the structure of DNA. • Her work was shared with James Watson without her permission. • Watson and Crick used her measurements to show that the phosphate groups were on the outside of the DNA double helix, and that the nitrogenous bases were more hydrophobic and thus on the inside. • Watson & Crick published the structure of DNA first, without crediting Franklin. They were awarded the Nobel prize. Franklin died of ovarian cancer she developed as a result of her work. Rosalind Franklin http://en.wikipedia.org/wiki/Rosa lind_Franklin 7.1.U2 DNA structure suggested a mechanism for DNA replication. Antiparallel Strands • The two strands have their 5’ and 3’ terminals at opposite ends 3’ – 5’ Linkages • 5’ end of a DNA strand- the deoxyribose is linked to a phosphate • 3’ end of a DNA strand- a hydroxyl (-OH) is attached to the 3’ carbon in a deoxyribose • Nucleotides in a strand are linked by covalent phosphodiester bonds, linking the 3’ of one nucleotide to the phosphate attached to the 5’ on the adjacent nucleotide https://sites.google.com/a/canacad.ac.jp/sl-hl-1-biology-4ferguson/04-dna/7-1-dna-structure-and-replication 7.1.U2 DNA structure suggested a mechanism for DNA replication. Hydrogen bonding • Purines (2 ring bases-Adenine & Guanine) form hydrogen bonds with pyrimidines (1 ring bases- cytosine and thymine). • Replication is semi-conservative- one “new” strand joins with one “old” strand. • Complementary base pairing allows for replication to occur by new bases forming hydrogen bonds with old bases. http://highered.mheducation.com/sites/0072943696/student_view0/chapter3/ animation__dna_replication__quiz_1_.html 7.1.U1 Nucleosomes help to supercoil the DNA. Nucleosomes • DNA in eukaryotes is associated with proteins called histones. • The octomer & DNA combination is attached to an H1 histone, forming a nucleosome. • The nucleosome serves to protect the DNA from damage and to allow long lengths of DNA to be supercoiled Supercoiling • Supercoiling allows the chromosomes to be mobile in mitosis & meiosis. • Supercoiled DNA cannot be transcribed for protein synthesis. DNA is wrapped twice around each nucleosome. http://www.ib.bioninja.com.au/higherlevel/topic-7-nucleic-acids-and/71-dnastructure.html#previous-photo 7.1.S2 Utilization of molecular visualization software to analyse the association between protein and DNA within a nucleosome. Visit: http://www.rcsb.org/pdb/101/motm.do?momID=7 and http://www.rcsb.org/pdb/explore/jmol.do?structureI d=1AOI&bionumber=1 Explore: • Find the two copies of each histone protein by locating their “tails”. • Visualize the positively charged amino acids on the nucleosome core. How do they play a role in the association of the protein core with the negatively charged DNA? 7.1.U3 DNA polymerases can only add nucleotides to the 3’ end of a primer. • DNA Polymerases are enzymes that add new nucleotides to a growing strand of DNA in the process of replication. • DNA polymerase III is responsible for forming covalent bonds between the nucleotides as they are added to polynucleotide. Replication happens in a 5’ 3’ direction. http://www.ib.bioninja.com.au/higherlevel/topic-7-nucleic-acids-and/72-dnareplication.html#previous-photo i-biology.net i-biology.net i-biology.net http://highered.mheducation.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/d l/free/0072437316/120076/bio23.swf::How%20Nucleotides%20are%20Added%20in%2 0DNA%20Replication i-biology.net 7.1.U4 DNA replication is continuous on the leading strand and discontinuous on the lagging strand. 7.1.U5 DNA replication is carried out by a complex system of enzymes. i-biology.net i-biology.net i-biology.net i-biology.net i-biology.net http://highered.mheducation.com/olcweb/cgi/pl uginpop.cgi?it=swf::535::535::/sites/dl/free/0072 437316/120076/micro04.swf::DNA%20Replicatio n%20Fork i-biology.net Watch this animation about the function of DNA Gyrase: http://highered.mheducation.com/sites/007299524 6/student_view0/chapter20/action_of_dna_gyrase. html The Enzymes & Molecules of DNA Replication: DNA Gyrase (topoisomerase) Affects the degree of supercoiling of the bacterial chromosome Helicase: Unwinds DNA & breaks H-bonds between base pairs DNA Polymerase III Attaches nucleotides in a 5’ 3’ direction RNA Primase Leaves RNA primers on the lag strand DNA Polymerase I Removes RNA primers DNA Ligase Attaches Okazaki fragments together by forming covalent bonds between nucleotides Single Stranded Binding Proteins Bonds with single stranded DNA that has been opened by helicase and prevents it from reforming a double helix i-biology.net 7.1.U6 Some regions of DNA do not code for proteins but have other important functions. http://www.dbriers.com/tutorials/wpcontent/uploads/2013/12/Central_Dogma-GODSwikimedia.jpg Genes are coding regions of DNA— they code for a polypeptide. • In humans, only about 1%-2% of DNA codes for a polypeptide. These coding regions are called exons. • The remainder of the DNA is noncoding. • The non-coding regions are typically made up of repetitive sequences of DNA. From Biology Course Companion, Allott, A, Oxford University Press, 2014 Some Functions of Non-Coding Regions of DNA Production of RNA Some regions on DNA function to produce tRNA and rRNA Gene expression Non-coding regions can have an role in regulating the expression of genes by promoting or inhibiting. Telomeres Telomeres are located on the ends of eukaryote chromosomes, they have a protective function because DNA cannot be replicated all the way to the ends, so telomeres prevent loss of important genes. • Non-coding regions within genes are called introns. http://www.webbooks.com/eLibrary/ON/B0/B10/05 MB10.html 7.1.A2 Use of nucleotides containing dideoxyribonucleic acid to stop DNA replication in preparation of samples for base sequencing. Determining the Base Sequence of a DNA Sample • Uses florescence • Many copies of sample DNA & materials needed to replicate it placed in test tubes • Samples include a few dideoxyribonucleic acid nucleotides with different florescence. http://www.yourgenome.org/teacher s/sequencing.shtml • Dideoxyribonucleic acid nucleotides stop replication when incorporated to DNA. • Gel Electrophoresis is used to separate DNA by fragment size. • Images of florescent fragments allow for sequencing based on size. 7.1.A3 Tandem repeats are used in DNA profiling. Variable Number Tandem Repeats Locus: the location of a gene (VNTR): are chromosomal regions on a chromosome (plural: loci) in which a short DNA sequence (such as GC or AGCT) is repeated a variable number of times end-toend at a single location (tandem repeat). Schematic of a VNTR in 4 alleles. Each rectangle represents a repeated series of bases in DNA. http://en.wikipedia.org/wiki/Variable_number_t andem_repeat Watch this video about some applications of VNTRspaternity testing and forensics https://www.youtube.com/watch?v=DbR9xMXuK 7c Where does the DNA for analyzing heredity come from? Paternal heredity: Y-chromosome Maternal heredity: Mitochondrial DNA In this example, Locus A is a tandem repeat of the motif GC: there are four alleles, with two, three, four, or five repeats (A2, A3, A4, and A5, respectively). Locus B is a tandem repeat of the motif AGCT: there are only two alleles, with two or three repeats (B2 and B3, respectively). The example shows a DNA fingerprint that includes both loci simultaneously. Individual #1 is heterozygous at Locus A (A2 / A5) and homozygous at Locus 2 (B2 / B2: note that this genotype gives a single-banded phenotype in the fingerprint). Individual #2 is heterozygous at both loci: (A4 / A3 and B3 / B2) respectively). The two individuals are distinguishable at either locus. Typical fingerprints include a dozen or more VNTR loci.` From: http://www.mun.ca/biology/scarr/V Thanks to these fine folks, and any others that I may have forgotten!