Chapter 3 Nucleic Acid1. Physical and chemical structure of DNA●Double-stranded helix● Major groove and minor groove● Base pairing● The two strands are antiparallel● G+C content (percent G+C)● Satellite DNASatellite DNA consists of highly repetitive DNA and is so called because repetitions of a short DNA sequence tend to produce a different frequency of the nucleotides adenine, cytosine, guanine and thymine, and thus have a different density from bulk DNA - such that they form a second or 'satellite' band when genomic DNA is separated on a density gradient.2. Alternate DNA structureTwo bases have been extruded from base stacking at the junction. The white line goes from phosphate to phosphate along the chain. O is shown red, N blue, P yellow and C grey.3. Circular and superhelical DNADNA can also form a double-stranded, covalently-closed circle. These circular molecules are often coiled into a superhelix, the formation of which is catalyzed by enzymes called topoisomerases.4. Denaturation of DNADenaturation: A transition from the native to the denatured stateDNA denaturation: also called DNA melting, is the process by which double-stranded DNA unwinds and separates into single-stranded strands through the breaking of hydrogen bonding between the bases.Hyperchromicity / Hyperchromic effect: the striking increase in absorbance of DNA (A260) caused by the denaturation of the double-stranded DNA moleculeMelting temperature (Tm) : the temperature at which half of the DNA strands are in the double-helical state and half are denatured. The melting temperature depends on both the length of the molecule, and the specific nucleotide sequence composition of that molecule.Factors Affecting Tm●G-C content of sample● reagents that increase the solubility of the bases (anything that disrupts H-bonds or base stacking) ● Salt concentration● pH● Length5. RenaturationStrands can be induced to renature (anneal) under proper conditions. Factors to consider:● Temperature● Salt concentration● DNA concentration● TimeRepetitive Sequences●Unique: Single Copy Genes● Slightly repetitive (2-10 copies)● Middle repetitive (10- hundreds)--Clustered--DispersedHighly repetitive (hundreds to millions)--Short sequences in satellite DNA--Sequences of normal length in certain genes that exist in very large numbersC-value ParadoxThere is apparently a lack of association between C-value (the amount of DNA present in the haploid genome of different organisms )and the degree of organismal complexity of various multi-cellular organisms. In 1971, Thomas named this phenomenon, “C-value Paradox”.在每一种生物中其单倍体基因组的DNA总量是特异的，被称为C值 (C Value)。

C值和生物结构或组成的复杂性不一致的现象称为C值悖论(C-value paradox)。

6. HybridizationHybridization: the technique wherein renatured DNA is formed from separate single-stranded samples . Heteroduplexing: renaturation combined with electron microscopy in a procedure allows the localization of common, distinct,and missing sequences in DNA.DNA-RNA hybridization (Northern hybridization): the use of filter hybridization to detect sequence complementarity between a single strand of DNA and an RNA molecule.7. The structure of RNATypes: mRNA, tRNA, rRNADistinctions:- ribose replaces deoxyribose;- U replaces T;- Single-strandedConformation: stem-loop or hairpin8. Hydrolysis of nucleic acidThe phosphodiester bonds of both DNA and RNA can be broken by hydrolysis either chemically or enzymatically.Ribozymes: the RNA enzymes, are able to cleave and form specific phosphodiester bonds in a manner analogous to protein enzymes.Chapter 6 The genetic materialThe Path to the Watson and Crick Model1928, Griffith, transformation in pneumococci（肺炎球菌）1944, Avery, Griffith’s transforming principle was DNA1950, Chargaff, a pattern in the amounts of the four bases1952, Hershey and Chase, DNA is the genetic material1953, Franklin, the x-ray picture of DNAChargaff’s ruleIn the DNA of all species examined, A=T, G=CThe total amount of purines (A+G)=pyrimidines (T+C) in DNAThe ration of (A+T)/(G+C) varies from species to speciesDNA properties and functions1.DNA has the ability to store genetic information, which can be expressed in the cell as need.2.This information can be transmitted to daughter cells with minimal error. (This process requires complexenzymes and repair mechanisms.)3.DNA possesses both physical and chemical stability so information is not lost over long periods of time (years).4.DNA has the potential for heritable change without major loss of parental information.DNA-genetic material: Double-stranded DNA has evolved as the genetic material because it is especially well-suited for replication, repair, occasional change, and long-time stability.Gene: Genes contain all the information for the synthesis and functioning of cellular components. Transcription: the process of synthesizing RNA molecules from a DNA template.Triplets / codons: the RNA nucleotide sequence is read (on ribosomes) in sequential groups of three bases. Mutation: the process by which a base-sequence changes.The central dogma: DNA makes RNA, makes protein.chapter 7 DNA replication Semiconservative replication of double-stranded DNAUntwisting of highly coiled DNA is required for DNA replicationTopoisomerase Type I :•Work ahead of replicating DNA•Mechanism–Makes a cut in one strand, passes other strand through it. Seals gap.–Result: the DNA is “relaxed” somewhatGyrase--A Type II Topoisomeras e–Introduces negative supercoils–breaks both strands–Section located away from actual cut is then passed through cut site.Initiation of DNA replication•Replicaion initiated at specific sites: Origin of Replication (ori)•Two Types of initiation:–De novo –Synthesis initiated with RNA primers. Most common.–Covalent extension—synthesis of new strand as an extension of an old strand (“Rolling Circle”). Limited to certain viruses.De novo Initiation•Binding to Ori C by DnaA protein•Opens Strands•Replication proceeds bidirectionallyCovalent extension initiation Rolling CircleUnwinding of DNA for replicationHelicase:⏹ Breaks hydrogen bonds and eliminates hydrophobic interactions⏹ Needs energy supplied by ATP⏹ Encoded by the DnaB gene in E.coliSingle-strand DNA binding proteins (SSB):Bind to the exposed strands, coat them and block the re-annealing process.Elongation of newly synthesized strands1.The polymerization reaction and the polymerasesEnzyme: polymerase IIINeeded: substrates, template, primerDirection: 5’→3’2. Correcting mismatched basesThe 5’-3’ exonuclease activity of pol I at a single-strand break (nick) can occur simultaneously with polymerization----nick translation.DNA polymerase III consists of multiple subunits⏹Pol I and pol III are both involved in E.coli DNA replication. Pol III is the major polymerase.⏹ Both poly I and poly III possess a proofreading or editing function (3’-5’ exonuclease activity ).⏹ The 5’-3’ exonuclease activity of pol I at a single-strand break (nick) can occur simultaneouslywith polymerization----nick translation.⏹ DNA polymerase III consists of multiple subunits.⏹ All known polymerases can work only in the 5’-P → 3’-OH direction.Pol I and pol III have some features in common:● 5’-3’ polymerization activityThe four deoxynucleoside 5’-triphosphatesA primer with a free 3’-OHA template● 3’-5’ exonuclease activityAntiparallel DNA strands and discontinuous replication⏹The two strands of DNA is antiparallel and the replication is discontinuous synthesis.⏹ A primer is required for chain initiation and two different enzymes (RNA polymerase and primase) areknown to synthesize primer RNA molecules.⏹ DNA ligase joins precursor fragments and pol I as well as RNase H participates in the removal ofprimer.RNA polymerase: initiation of leading-strand synthesisPrimase: synthesis of primers for lagging-strandPrimosome: helicase/primase complexPol I: removal of the primer and replacement of DNADNA ligase: joining the fragment (gap sealed)The complete DNA replication systemBidirectional replication speeds up DNA synthesisReplication of eukaryotic chromosomes1.Eukaryotes have more and large chromosomes.2.Eukaryotic replication may require as much as 6-8 hours for completion versus the 40 minutes neededby E.coli.3.There are multiple, rather than a single, replication origins along eukaryotic chromosomes. They arespaced about 20 kb apart.4.Eukaryotic DNA replication is at the rate of about 10-100 nucleotides per second as opposed to theprokaryotic rate of about 1500 nucleotides per second.5.At least five types of DNA polymerases have been found in eukaryotic cells.真生物DNA的复制有DNA聚合酶及多种蛋白质因子参与，DNA聚合酶也有多种类型。