GENA RELICATION

Review of NUCLEIC ACIDS DNA Genetic material In the nucleus Double stranded A,T C,G

RNA Carries information from the nucleus to the site where proteins are made Single stranded A,U C,G

DNA is found in the nucleus Humans have 23 pairs of chromosomes Chromosomes are made of DNA

DNA moleculThe sides of the ladder are sugar and phosphates; the rungs of the ladder are the nitrogen bases A,T,C,G

How DNA is the Master Control

DNA Replication The structure of DNA provided an insight to Watson and Crick for how DNA replicates each strand in a duplex of DNA is complementary to each other, each can form a template when separated. The order of bases on one strand can be used to add in complementary bases and therefore duplicate the pairs of bases exactly. Model for DNA Replication Watson and Cricks model: semiconservative replication

Three models of DNA replication

Experiment which supports the Semiconservative Model Matthew Meselson and Franklin Stahl experiments supported the semiconservative model labeled the nucleotides of the old strands with a heavy isotope of nitrogen (15N) while any new nucleotides would be indicated by a lighter isotope (14N). Replicated strands could be separated by density in a centrifuge. Each model:

Meselson-Stahl experiment

Conclusion: DNA replication follows semiconservative model Conservative and Dispersive models were disproven in their experiment.

DNA Replication More than a dozen enzymes and other proteins participate E. coli can replicate 4.5 x 106 base pairs bp) in less than an hour human cells can replicate 6 x 109 bp in only a few hours DNA replication is very accurate less than 1 error per billion nucleotides!! DNA Replication Start Sites Where does DNA replication start? special sites termed origins of replication single site in bacterial chromosome multiple sites in eukaryotic chromosome Enzymes (helicases) separate the two strands forms a replication bubble other proteins (single strand binding proteins - ssb) bind to keep strands separated Origins of Replication

Enzymes of DNA ReplicationDNA polymerases synthesize DNA by adding a nucleotide that is complementary to the base in the template strand Rate of synthesis Bacteria - 500 nucleotides / sec Human cells - 50 nucleotides / sec Incorporation of a nucleotide

DNA Synthesis leading strand is synthesized continuously lagging strand is synthesized discontinuousl in short segments called Okazaki fragments DNA ligase joins the fragments

Initiating DNA SynthesisAfter separation of the DNA strands DNA Polymerase cannot initiate DNA syn. Needs a 3 OH to add nucleotide to. synthesizing a new chain requires a primer, a short segment of RNA Primase (an RNA Polymerase) adds about 10 nucleotides complementary to template Priming DNA SynthesisNote: RNA primer is removed from DNA by another DNA Polymerase

Bacterial DNA Replication Proteins

Bacterial DNA Replication Proteins Helicase Unwinds parental double helix at replication forks DNA double helix are tightly coupled. High temperature is needed to break them (95oC

ssb proteinbinds to and stabilizes ssDNA Topoisomerase Corrects overwinding ahead of replication forks breaks, swivels, and rejoins DNA strands Primase synthesizes single primer for leading strand synthesizes RNA primer for each lagging strand DNA pol III continuous synthesis of leading strand discontinuous synthesis of lagging strand DNA pol Iremoves primer (RNA) from DNA strand and replaces it with DNA DNA Ligase joins 3 end of fragment with 5 end of adjacent fragment DNA replication Fork

Summary of DNA Replication

Mutation Mutation: A change in the base sequence of the DNA Mutations are changes in the genotype which may or may not affect the phenotype Causes of mutations Spontaneous mutations Occur in the absence of mutation causing agents Due to occasional mistakes in DNA replication Induced mutationsCaused by mutagens, agents such as chemicals and radiation which induce mutations Types of Repair of DNA Bases may be damaged by chemical and/or physical agentsUV light, reactive chemicals, radiation, etc. Some mismatched bases may be missed by proofreading activity of DNA pol Must be corrected to ensure high fidelity of DNA sequence Types of mutation Base substitutions The most common type of mutation A single base pair is replaced by another Frame shift mutations One or more base pairs are inserted or deleted in the DNA Results in a change in the reading of codons mutagen Chemical mutagensExample: Nitrous acid alters adenine such that it pairs with cytosine instead of thymine

Radiation Ionizing radiation e.g., Xrays and gamma rays Causes the formation of ions that can react with nucleotides (causing base changes) and the deoxyribose-phosphate backbone (causes chromosomes to break). UV radiationInduces formation of covalent bonds between adjacent thymines to form thymine dimers which can not be replicated

Consequences of base substitutions Silent mutation: base change results in no change of the amino acid sequence of the translated protein Silent mutations have no effect on phenotype A result of the fact that multiple codons can code for the same amino acidE.g., AGU and AGC both code for Serine Missense mutation: base change results in the change of an amino acid in the translated protein The amino acid substitution induced by the missense mutation may have no effect on the function of the protein OR It may abolish the activity of the protein or alter its function having an effect on phenotype Example: sickle cell disease in humans is due to a missense mutation in the gene for globin. As a result the shape of red blood cells is altered affecting their movement through capillaries.

Nonsense mutation: base change generates a stop codon in place of that coding for an amino acid Results in production of a truncated protein. Usually results in a non-functional protein

Frameshift mutation: addition or deletion of one or more bases Results in misreading of the codons (changed reading frame) Almost always results in long stretches of altered amino acids and the production of inactive protein

Nucleotide Excision Repair In nucleotide excision repair, a nuclease cuts out a segment of a damaged strand. The gap is filled in by DNA polymerase and ligase.

Penyakit mutasi Xeroderma PigmentosumIndividuals with this genetic disease have defective repair enzymes cant remove thymine dimers caused by UV light very sensitive to sunlight and often get skin cancers

Royal Hemophilia X linked Queen Victoria (Great Britain) was the carrier Gene passed on to many other royal families in Europe Russian, Prussian and Spanish affected But not the British royal family Genetic warfare? Translation: Making Proteins A group of three nucleotides in messenger RNA codes for a certain amino acid to be placed in a protein. Each group of three nucleotides is called a CODON. Stop kodon UAG, UGA, UAAStart kodon AUG DNA Repair Spontaneous DNA damage Pathways to remove DNA damage Damage detection The repair of Double-strand break DNA repair enzymes

Now you know---How DNA is the Master Control

TAC CGA TCGATG GCT AGC transcripsiTAC CGA TCGAUG GCU AGC new RNA translasiTAC CGA TCGAUG GCU AGC met, ala, ser So how do you remember the difference? Replication just makes a repeat copy of DNA Transcription rips a new strand of RNA Translation starts with a new slate- the slate of amino acids to make a protein

How DNA is the Master ControlDNAnucleotidesREPLICATIONREpeats DNA can make copies of itself=REPLICATION

5DNA is the master control of the cell, dictating which RNAs and proteins are made. Every cell in an organism has the same DNA. For that reason, there must be a way to make copies of the DNA in a cell, so all new cells made are identical to the old ones. The process by which copies of DNA are made is called REPLICATION.Meselson-Stahl Experiment supports the Semiconservative ModelEach model: the semi-conservative model, the conservative model, and the dispersive model, made specific predictions on the density of replicated DNA strands.ConservativeSemi-ConservativeDispersiveone heavyone lightboth intermediate

Mutation Generation passed on to daughter DNAs48

Emergency DNA Repair for Double helix break49Now you know---How DNA is the Master ControlDNARNAPROTEINTRANSCRIPTIONTRANSLATIONnucleotidesnucleotidesamino acidsREPLICATIONREpeatsRemember you are translating from the language of nucleotides to the language of amino acids

39Now you know that DNA dictates how other DNA will be made, how RNA will be made and ultimately how proteins are made.