Genes and Evolution Chapter 1 and 19 (PDF)

Genes and evolution Darwin recognized role of hereditary variation in evolution (but was unaware of true mechanism of heredity) Genetic variation (product of mutation) is raw material for evolutionary change Natural selection: differential reproduction of individuals with different alle...

Genes and evolution Darwin recognized role of hereditary variation in evolution (but was unaware of true mechanism of heredity) Genetic variation (product of mutation) is raw material for evolutionary change Natural selection: differential reproduction of individuals with different alleles Random genetic drift: change in frequencies of genetic variants resulting from random, non-selective processes Chapter 1: Genetics and the organism © 2002 by Evolution Evolution was an accepted fact among many scholars prior to Darwin Darwin provided a plausible explanation for evolution: natural selection All living organisms are related through descent from common ancestor homologous features have the same developmental origin inherited from a common ancestor analogous features have independent origin similarities of DNA and protein sequences allow inferences about evolutionary origin Chapter 19: Evolutionary genetics © 2002 by Darwinian evolution Principle of variation. Among individual members of a population there is variation in morphology, physiology, and behavior. Principle of heredity. Offspring resemble their parents more than they resemble individuals to which they are unrelated. Principle of selection. Some variants are more successful at surviving and reproducing than other variants in a given environment. Such individuals are naturally selected. Chapter 19: Evolutionary genetics © 2002 by Evolutionary history Phyletic evolution: change within a continuous line of descent Diversification: many different contemporaneous species evolved from common ancestor (branching) Natural selection converts heritable variation among members of a population into heritable differences among populations Chapter 19: Evolutionary genetics © 2002 by Synthesis of evolutionary forces Adaptive evolutionary change is a balance between forces of breeding structure, mutation, migration, and selection Forces that increase or maintain variation within populations prevent differentiation of populations (e.g., migration, mutation, balancing selection) Divergence of populations is a result of forces that make each population homozygous (e.g., inbreeding, founder effect, directional selection) Evolution requires genetic variation in order to occur; direction of change unpredictable Chapter 19: Evolutionary genetics © 2002 by Genetics and human affairs >1000 inherited genetic diseases in humans Cancer is caused by mutation in somatic cells Genetics pertains to social policy debate over role of genetics and IQ genetics of sexual orientation Biotechnology and genetic engineering new pharmaceuticals new varieties of plants and animals concerns over ethics and safety Chapter 1: Genetics and the organism © 2002 by  Cont’d The Structure of Genes and Genomes DNA Chapter 2: Genes and genomes © 2002 Overview Each species has a uniquely fundamental set of genetic information, its genome. The genome is composed of one or more DNA molecules, each organized as a chromosome. The prokaryotic genomes are mostly single circular chromosomes. Eukaryotic genomes consist of one or two sets of linear chromosomes confined to the nucleus. A gene is a segment of DNA that is transcribed into a functional RNA molecule. Introns interrupt many eukaryote genes. Viral genomes consist of either DNA or RNA. Chapter 2: Genes and genomes © 2002 Nature of DNA Transformation (uptake of foreign DNA) in prokaryotes and eukaryotes has repeatedly shown that DNA is hereditary material. DNA is accurately replicated prior to each cell division. DNA encodes proteins needed by the cell. DNA is capable of mutation, providing raw material for evolutionary change. Chapter 2: Genes and genomes © 2002 The DNA nucleotide Building block of DNA (and RNA) Deoxyribose (pentose sugar), with 3’ –OH N base Phosphate (on 5’ carbon) Nitrogenous base N purine 5’ P C adenine O guanine pyrimidine sugar thymine cytosine OH Chapter 2: Genes and genomes 3’ © 2002  The double helix DNA normally consists of two antiparallel polynucleotide chains sugar–phosphate backbone phosphodiester bonds 5’ to 3’ connection complementary base pairs 5’ 3’ A–T G–C 3’ 5’ hydrogen bonds 2 per A – T 5’-AATTGGCCGATC-3’ 3 per G – C 3’-TTAACCGGCTAG-5’ 5’ 3’ chain polarity Major and minor grooves (see model) Chapter 2: Genes and genomes © 2002 DNA Units of measurement base pair (bp) kilobase (1kb) megabase (1Mb) Replication: each strand serves as template for synthesis of complement, using rules of base pairing Information: specified by sequence of nucleotides; may be copied into RNA Mutation: replacement, insertion, deletion of nucleotide results in altered sequence Chapter 2: Genes and genomes © 2002 Structure of genes Gene encodes functional RNA molecule, mostly mRNA (also tRNA, rRNA, etc.) regulatory DNA encoding functional RNA region RNA primary transcript Gene is functional part of chromosome which is transcribed into RNA at the correct time and place in development or cell cycle Gene includes its adjacent regulatory region(s) Chapter 2: Genes and genomes © 2002  Eukaryote introns and exons Intron: noncoding region of gene, excised by processing from primary transcript zero to many per eukaryote gene variable length, may be bulk of gene unknown function Exon: coding region of gene (sequence is included in mature transcript) transcript E1 I1 E2 I2 E3 I3 E4 nuclear processing steps mature transcript Chapter 2: Genes and genomes E1 E2 E3 E4 © 2002 Gene neighborhoods In prokaryotes, genes are often tandemly arranged, with little or no spacer sequences in between In eukaryotes, there is considerable spacer DNA between genes some is repetitive DNA: identical or nearly identical repeated units dispersed tandem much is derived from mobile genetic elements Chapter 2: Genes and genomes © 2002 The nature of genomes Genomics: study of structure and function of genomes Genome size variable, by orders of magnitude number of genes roughly proportional to genome size Plasmids symbiotic DNA molecules, not essential mostly circular in prokaryotes Organellar DNA chloroplast, mitochondrion derived by endosymbiosis from bacterial ancestors Chapter 2: Genes and genomes © 2002 Viral genomes In prokaryotes, viruses are sometimes referred to as bacteriophages. Nonliving particle nucleic acid protein DNA or RNA single-stranded or double-stranded linear or circular Compact genomes with little spacer DNA Chapter 2: Genes and genomes © 2002 Prokaryotic genome Usually circular double helix occupies nucleoid region of cell attached to plasma membrane Genes are close together with little intergenic spacer Operon tandem cluster of coordinately regulated genes transcribed as single mRNA Introns very rare Chapter 2: Genes and genomes © 2002  Eukaryotic nuclear genomes Each species has characteristic chromosome number Genes are segments of nuclear chromosomes Ploidy refers to number of complete sets of chromosomes haploid (1n): one complete set of genes diploid (2n) polyploid (3n) In diploids, chromosomes come in homologous pairs (homologs) In humans, somatic cells have 2n = structurally similar 46 chromosomes. same sequence of genes may contain different alleles Chapter 2: Genes and genomes © 2002 Eukaryotic chromosomes (1) Cytogenetics: microscopic study of chromosomes Considerable difference in size and number of genes Variable centromere position telocentric: centromere at end acrocentric: centromere close to end metacentric: centromere in middle p arm is shortest, q arm is longest Telomere: end of chromosome Nucleolar organizer (rRNA) Chromomere Chapter 2: Genes and genomes © 2002  Eukaryotic chromosomes (2) Heterochromatin densely stained regions of highly compact DNA mostly repetitive sequences Euchromatin: poorly stained, less compact, contains transcribed genes Banding patterns (metaphase chromosomes) differential uptake of dyes G bands, Giemsa stain (A/T rich) R bands, reverse of Giemsa (G/C rich) Polytene chromosomes replicated, unseparated chromosomes present in certain tissues of dipteran insects Chapter 2: Genes and genomes © 2002  Nuclear DNA Highly organized, various degrees of coiling Nucleosome fundamental unit of chromatin DNA wound around histone core (octamer) histones are highly conserved proteins H2A, H2B, H3, H4 10 nm fiber A haploid set of human solenoid, 30 nm fiber chromosomes consists of about 1 meter of DNA. Higher order coiling solenoid loops attach to scaffold scaffold attachments contain topoisomerase II form larger diameter fibers Chapter 2: Genes and genomes © 2002  Comparative genomics Study of similarities and differences among genomes Many genes are shared among all living things or between related groups Study of genes in model organisms provides useful information regarding genes in other organisms Large genome projects produce considerable information computer analysis Chapter 2: Genes and genomes © 2002  Store, retrieve, and translate genetic information Hereditary info. is passed on from One cell to its daughter cells at cell division. One generation of an organism to the next through the organism`s reproductive cells. Stored as genes, the information containing elements that determine the characteristics of a species as a whole and of the individula within it. The informations in genes is copied and transmitted from cell to daughter cell millions of times during the life of a multicelular organism, and it survives the process essentially unchanged. Genetic info. consist primarily of instructions for making proteins. Proteins perform most cellular functions: Building block for cell structure Catalyze chemical reactions Regulate gene expression Cellular communication Cell movement Properties and functions of cells determined largely by its proteins Hereditary info. İs carried on chromosomes, tread like structures in the nucleus of a cell that become visible by light microscope as the cells begin to divide. DNA as carrier of genetic information Deoxyribonucleic acid (DNA) Two polynucleotide chains. Composed of 4 nucleotides subunits. Each of the chains is known as DNA chain or DNA strand. Two chains are held together by hydrogen bonds. Nucleotide: 5 C sugar (deoxyribose) P group (phosphate) N containing base (Adenine, Cytosine, Guanine, or Thymine) Nucleotides are covalently link together in chain througy sugar and phosphates. Sugar phosphate backbone. The way nucleotides subunits are linked gives a DNA strand a chemical polarity.: 5’ phosphate (5’ end) 3’ hydroxyl (3’ end)  Hydrogene bond between bases hold two chain together. Therefore bases are on the inside and sugar phospahate backbone are on outside. A Purine (bulkier, two ring base, A and G) is paired with Pyrimidine (single ring base, C and T). Base pair is similar width Sugar phosphate backbones are equally distanced. Increase packing by two sugar- phosphate backbones wind around each other and form double helix. One comlete turn every ten base pairs. Antiparallel, reverse complement  Genes carry info. that must be copied accurately for transmission to the next generation. DNA encodes info. through base sequence. 4 lettered alphabet Organisms differs from one another because their respective DNA molecules have different nucleotide sequence. DNA molecule must encode proteins. a.a. Sequence = 3D structure = biological function The complete set of information in an organism`s DNA is called genome. It carries info for all proteins and RNA molecules that the organism will ever synthesize. 1 human cell contains 1m doublehelix DNA 24000 P + other info Template polimerisation At cell division, the cell must copy its genome to pass it to both daughter cells.  DNA in Eucaryotic cell is sequestered in a Nuclear envelope is mecanically supported nucleus. (10 % of cell volume). by a internal network filaments called nuclear lamina. Nuclear envelope: formed by two concentric lipid bilayer membrane. The N.E. : allow the many proteins that act on DNA to be concentrated Nuclear pores: transports molecules between nucleus and cytosol. Keep nuclear and cytosolic enzymes seperate Nuclear envelope is direcly connected to E.R. Which extend out to cytoplasm.  Information on Protein, RNA and info about: When In what type of cells In what quantity each protein is to be made. The genome of eucaryots are divided between set of different chromosomes. Human 3.2 x 109 nucleotide is distributed over 24 different chromosomes. Each chromosomes consist of a single, long linear DNA molecule associated with proteins that fold and pack the fine DNA into a more compact structure. All human cell contains two copies of each chromosome (exp): One from mother One from father The maternal and paternal chromosomes of a pair are called homologous chromosomes. Sex chromosomes are nonhomologous X and Y. 22 pairs common to both male and female.  The display of 46 human chromosomes at mitosis is called the human karyotype. Chromosomal abnormalities can be detected by this method.  Some correlation exist between the complexity of an organism and number of genes in its genome. Bacteria (500) vs. Human (25000) Bacteria and some single cell eucaryotes has consise genome. Chromosomes form many eucaryotes contains, in addition to genes, a large exess of interspersed DNA that dose not carry critical information (junk DNA).  Human genome is x 200 larger than some yeast. Human genome is x 30 smaller than some plants. Human genome is x 200 smaller than some amoeba.  Related species can have different chromosome number There is no simple relation ship between chromosome number, species complexity, and total genome size. How little of human genome codes for proteins Short mobile piece of DNA Human genome have large average gene size (27,000 n). 1,300 n are required to encode a protein of average size. Remaining DNA in a gene conconsists of long streches of noncoding DNA that interrupts short segments of coding DNA. Coding sequences are called exon. Non coding sequences are called intron. Regulatory DNA sequences, responsible for: Turning on and of at proper time Level of expression Type of cell Genome comparison Sequences that have a function are relatively conserved during evolution. Closely similar regions are known as conserved regions. Exons Regulatory DNA sequence 5 % of the human genome consists of “multi-species conserved sequences” 1/3 code for proteins Protein-binding sites that are involved in gene regulation RNA molecules (not translated) Majority unknown Genome comparison Sequences that have a function are relatively conserved during evolution. Closely similar regions are known as conserved regions. Exons We understand much less Regulatory DNA sequence than we had previously 5 % of the human genome consists of “multi-species conserved imagined. sequences” 1/3 code for proteins Protein-binding sites that are involved in gene regulation RNA molecules (not translated) Majority unknown  Large block of our genomes contain these genes in the same order (conserved synteny)  Must replicate, replicated copies must be seperated and partitioned into daughter cells. Through ordered series of stages. (Cell cycle) During interphase ch are replicated, and during mitosis (M Phase) become highly condensed and then are seperated and distributed. Mitotic chromosome Interphase chromosome Cell proliferation Required for both development and homeostasis Stem cells proliferate and differentiate in response to local signals to replace dead cells Apoptosis eliminates cells as a consequence of internal and external signals Cancers develop through accumulation of mutations in a clone of somatic cells Hundreds of mutational events leading to neoplasia (cancer formation) have been identified Chapter 15: Regulation of cell division © 2002 by W. H. Freeman  Cell proliferation and elimination Proliferation is controlled by cell cycle checkpoints Protein kinases and protein phosphatases modulate activities of proteins regulating the cell cycle In multicellular organisms, somatic cells may be eliminated by apoptosis In apoptosis, a cascade of caspase enzyme activity destroys the cell Proliferation and apoptosis pathways are linked at various points involving protein phosphorylation, allosteric interactions, and interaction between protein subunits Chapter 15: Regulation of cell division © 2002 by W. H. Freeman Extracellular signals Cell cycle positive signals: mitogens (growth factors) secreted by paracrine source Cell cycle negative controls: ligands initiate signal cascade that inhibits cell cycle progression Apoptosis positive controls: signal from neighboring cell Apoptosis negative controls: survival factors Chapter 15: Regulation of cell division © 2002 by W. H. Freeman Cancer Aberrant cell cycle control Results from accumulation of mutations in somatic cell clone Differ in many ways from neighboring cells Genetic basis for all cancer cells most carcinogens are also mutagenic some highly penetrant inherited cancer genes many less penetrant susceptibility genes oncogenes transmitted by tumor viruses Chapter 15: Regulation of cell division © 2002 by W. H. Freeman Mutations in cancer cells Two categories Oncogenes, typically dominant Mutated tumor-suppressor genes, typically recessive Sometimes associated with chromosomal abnormalities, e.g., translocation that brings gene under control of another gene’s strong enhancer Cells that loose ability to undergo apoptosis have longer time to accumulate proliferation-promoting mutations Chapter 15: Regulation of cell division © 2002 by W. H. Freeman Oncogenes Proto-oncogenes generally encode proteins that regulate normal cell proliferation or apoptosis Normally encode either positive or negative regulators Accumulate mutations to become oncogenes point mutations alter structure/function loss of protein domains resulting from deletion gene fusions, often resulting from translocations sometimes mutation results in misexpression, with protein expressed in wrong place or time Chapter 15: Regulation of cell division © 2002 by W. H. Freeman  Tumor-suppressor genes May encode either negative regulators of cell cycle or positive regulators of apoptosis Retinoblastoma caused by mutated RB gene p53 (refers to protein with mass of 53 kDa) ~50% of all tumors have mutated form normal p53 is transcription factor that is activated in response to DNA damage prevents cell cycle progression to allow repair causes severely damaged cell to undergo apoptosis mutated form eliminates apoptotic response, allowing damaged cells to survive, elevating mutation level Chapter 15: Regulation of cell division © 2002 by W. H. Freeman Cancer complexities Different cancers have different phenotypes with respect to rate of proliferation, ability to metastasize, etc. Differences caused by: differences in somatic cell progenitor differences in types and severity of mutations Research from wide variety of areas applicable to search for cures Chapter 15: Regulation of cell division © 2002 by W. H. Freeman  Cell cycle regulation Cell cycle phases G1: period between mitosis and S S: period of DNA replication G2: period between S and mitosis M: mitosis Cyclin transcribed in specific phase of cell cycle unstable, resulting in transient activity Cyclin-dependent protein kinase (CDK) substrate specificity and phosphorylation activity controlled by bound cyclin phosphorylate serine or threonine of target protein Sequential activation of different CDK-cyclin complexes controls cell cycle progression Chapter 15: Regulation of cell division © 2002 by W. H. Freeman Steps of MITOSIS Which phase do the following pictures demonstrate?  No species can afford to allow mutations to accumulate at a high rate in its germ cells. The cells of sexually reproducing organisms: Germ cells: transmits genetic information from parent to offspring Somatic cells: forms body of the organism In order to safeguard each individual somatic cells must also be protected. To Mutate or Not to Mutate Accurate duplication of vast quantity of genetic information in chaotic universe requires high degree of order. DNA replication occures before a cell can produce two genetically identical daughter cells. Maintaining order also requires: Continued surveillance Continued repair. DNA inside cells is repeatedly damaged by: Chemicals, Radiation, Thermal accidents Reactive molecules !!! While the short-term survival of a cell can depend on preventing changes in its DNA, the long term survival of a species requires that DNA sequences be changeable over many generations !!! Cells make greate effort to protect their DNA Occasional changes do occur. Overtime these changes provide genetic variation upon which selective pressures act during the evolution  English Peppered Moth Rest on the speckled lichens on tree trunks. Maintenance of DNA Sequences Occasional genetic changes enhance the long term survival of a species. The survival of the individual demands a high degree of genetic stability. Only rarely do the cell´s DNA maintenance process fail, result in permanent change in the DNA. Such a change is called a mutation. Can destroy an organism if it occures in a vital position. The mutation rate: the rate at which observable changes occur in DNA sequences. ~1 nucleotide change per 109 nucleotides each time that DNA is replicated. Underestimated number  DNA sequences are replicated and maintained with high fidelity. Mutation rate = 1 nucleotide change per 109 nucleotides each time the DNA is replicated. ~ 3x 109 nucleotides in our genome. How many mutations in a replication?  Template based polymerisation. Recognition of each nucleotide in the template strand by a free complementary nucleotide It requires the seperation of the two strands of DNA double helix. Seperation exposes the hydrogen bond donors and acceptor groups on each DNA base for base-pairing with the appropriate incoming free nucleotide, aligning it for its enzyme catalised polymerisation. DNA polymerase: nucleotide polymerising enzyme  Teplate strand guides the formation of the new strand of DNA. Adds deoxyribonucleotide to 3΄-OH end Polimerisation at 5΄-to-3΄ direction. Each deoxyribonucleoside triphosphate must pair with the template to be recognised by DNA polymerase. Template determines the DNA sequence. The reaction is driven by a large, favorable free energy change, caused by the release of pyrophosphate and its subsequent hydrolysis to two inorganic phosphate. Correct positioning of deoxyribonucleoside triphosphate couse conformational change and initiation of reaction. Dissociation of pyrophosphate cause release of fingers. Semiconservative Two daughter cells have a new DNA double helix containing: One original and One new strand DNA is replicated semiconservatively by DNA polymerase  A localised region of replication that moves progressively along the parental DNA double helix. This active region is called replication fork. Multienzyme complex that contains the DNA polymerase synthesises the DNA of both new daughter strands All DNA polymerases can synthesize only in the 5΄-to-3΄ direction.  Short fragments of DNA polimerized in 5΄-to-3΄ direction. Joined together after their synthesis to create long DNA chains. DNA fork has asymmetric structure. Leading strand: DNA daughter strand that is synthesized continuously. Lagging strand: daughter strand that is synthesized discontinuously.  1 mistake every 109 nucleotide copied. Complementary base pairs are not the only one possible. G-T, C-A is also possible. Proofreading mechanisms that acts sequentially to correct any initial mispairings that might have occured. First proofreading step performed by DNA polymerase just before adding a new nucleotide After nucleotide binding, but before the nucleotide is covalently added to the growing chain, the enzyme must undergo conformational change. This can occur more readily with correct than incorrect pairing. Exonucleolytic proofreading: takes place after incorrect nucleotide is covalently added. DNA polymerase enzymes are highly discriminating in the type of DNA chains they will elongate. 3΄-OH end of a primer strand. DNA polimerase cannot add new nucleotide to a DNA molecule with a mismatched nucleotide at 3΄-OH end. DNA polymerase corrects this mistake by seperate catalytic site (3΄-to-5΄ proofreading exonuclease). 3΄-to-5΄ proofreading exonuclease clips off any unpaired residues. DNA polymerase is self correcting enzyme Strand directed mismatch repair. DNA Polymerase I DNA polymerase I (pol I) is a versatile enzyme with 3 distinct activities DNA polymerase 3’5’ exonuclease 5’3’ exonuclease Mild proteolytic treatment results in 2 polypeptides Klenow fragment Smaller fragment 20-88 5’3’ exonuclease This activity allows pol I to degrade a strand ahead of advancing polymerase Removes and replaces a strand in one pass Basic functions are: Primer removal Nick repair 20-90 Polymerases II and III Pol II activity is not required for DNA replication Pol I appears mostly active in repair Only pol III is required for DNA replication Pol III is the enzyme that replicates bacterial DNA 20-91 The Pol III Holoenzyme Pol III core is composed of 3 subunits: DNA polymerase activity is in the -subunit 3’5’exonuclease activity found in -subunit Not yet clear what is the role of -subunit DNA-dependent ATPase activity is located in the g-complex containing 5 subunits Lastly, b-subunit plus the other 8 comprise the holoenzyme 20-92 Fidelity of Replication Faithful replication is essential to life DNA replication machinery has a built-in proofreading system This system requires priming Only a base-paired nucleotide can serve as a primer for pol III holoenzyme If wrong nucleotide is incorporated accidentally replication stalls until 3’5’ exonuclease of pol III holoenzyme removes it Primers are made of RNA which may help mark them for degradation 20-93 Multiple Eukaryotic DNA Polymerases Mammalian cells contain 5 different DNA polymerases Polymerases d and  appear to participate in replicating both DNA strands Priming DNA synthesis is -subunit role Elongating both strands is done by d- subunit 20-94  Do we need such extensive proofreading mechanisms for RNA polymerisation? De novo  5΄-to-3΄ direction of polymerization is required for exonucleolytic proofreading.  Leading strand only requires a primer sequence at the start of replication. On the lagging side for each Okazaki fragment polimerization requires new fragment. DNA primase: An enzyme produces the base-paired primer strand required by DNA polimerase molecule. It uses ribonucleosite triphosphates to synthesize short RNA primers.  In order to produce continuous DNA chain: A special DNA repair system acts and erase the old RNA primer and replace it with DNA. An Enzyme called DNA ligase joins fragments. Why RNA is removed?  DNA double helixes must be opened up for DNA polymerisation. DNA double helix is very stable at physiological conditions. PCR Temperature Two replication proteins are needed to open tha DNA double helix: DNA Helicase: Uses ATP and propel themselves along a DNA single strand. When encounter a double helix, they continue to move along their strand, thereby prying apart the helix. Single-strand DNA-binding protein (SSB):  Single-strand DNA-binding protein (SSB): Helix-destabilizing protein Binds to exposed single-stranded DNA They do not cover the bases They do not open the DNA helix directly. They aid helicases by stabilising single stranded conformation Prevent forming short hairpins. Single-Strand DNA-Binding Proteins Prokaryotic ssDNA-binding proteins bind much more strongly to ssDNA than to dsDNA Aid helicase action by binding tightly and cooperatively to newly formed ssDNA Keep it from annealing with its partner By coating ssDNA, SSBs protect it from degradation SSBs are essential for prokaryotic DNA replication 20-102  DNA polymerase molecule can synthesise only short string of nucleotide before falling off the DNA template. Sliding clamp protein keeps the polymerase firmly on the DNA. Clamp releases DNA polymerase when runs into double strand Clamp loader protein catalyses loading of sliding clamp protein on DNA  Most of the proteins all held together in a large orderly multienzyme complex. This can be liked to a sewing machine. Composed of protein parts and powered by nucleoside triphosphate hydrolysis  Strand-directed mismatch repair: system detects the potential for distortion in the DNA helix from the misfit between noncomplementary base pairs. Recognition of mismatch Exision of the segment of DNA containing the mismatch from the newly synthesized strand Resynthesis of the exised segment using the old strand as a template. Why and How it need to recognise new strand?  Every 10 nucleotide pair corresponds to a compleate turn about the axis of the parental double helix. Replication fork to move: Chromosome have to rotate, or Formation of a swivel in the DNA double helix by the protein called DNA topoisomerases  Topoisomerase I: produces a transient single- strand break. It can rotate free along one strand Any tension in the DNA helix will drive this rotation Topoisomerase II: Topoisomerases Strand separation of DNA is referred to as “unzipping” DNA is not really like a zipper with straight, parallel sides, actually a helix When 2 strands of DNA separate, rotate around each other Helicase could handle this task alone if DNA were linear, short Closed circular DNA present special problems As DNA unwinds at one site More winding must occur at another site 20-109 Cairns’s Swivel A “swivel” in the DNA duplex called DNA gyrase Allows the DNA strands on either side to rotate to relieve the strain Gyrase belongs to the enzyme class topoisomerase These add transient single- or double-stranded breaks into DNA Serves to permit change in shape or topology 20-110 Topoisomerase Mechanism Enzymes called helicases use ATP energy to separate the two parental DNA strands at replicating fork As helicase unwinds 2 parental strands it introduces a compensating positive supercoiling force Stress of this force must be overcome or DNA will resist progression of replicating fork This stress releasing mechanism is the swivel DNA gyrase acts as swivel b pumping negative supercoils into replicating DNA 20-111  Topoisomerase II: covalently binds to both strands of DNA helix at the same time, making a transient double strand breake in the helix. Breakes one double helix reversebly to create a DNA “gate”. It causes the second nearby double helix to pass through this brake, It then reseals the break and dissociates DNA Damage and Repair DNA can be damaged in many different ways, if left unrepaired this damage can lead to mutation, changes in the base sequence of DNA DNA damage is not the same as mutation though it can lead to mutation DNA damage is a chemical alteration Mutation is a change in a base pair Common examples of DNA damage Base modifications caused by alkylating agents Pyrimidine dimers caused by UV radiation 20-114 Damage Caused by Alkylation of Bases Alkylation is a process where electrophiles: Encounter negative centers Attack them Add carbon-containing groups (alkyl groups) 20-115 Damage Caused by Alkylation of Bases Alkylating agents like ethylmethane sulfonate (EMS) add alkyl groups to bases Some alkylation don’t change base-pairing, innocuous Others cause DNA replication to stall Cytotoxic Lead to mutations if cell attempts to replicate without damage repair Third type change base-pairing properties of a base, so are mutagenic 20-116 Damage Caused by Radiation Ultraviolet rays Comparatively low energy Cause a moderate type of damage Result in formation of pyrimidine dimers Gamma and x-rays Much more energetic Ionize molecules around the DNA Form highly reactive free radicals that attack DNA Alter bases Break strands 20-117 Types of DNA Damage 20-118 Directly Undoing UV DNA Damage UV radiation damage to DNA can be directly repaired by a DNA photolyase Uses energy from near-UV to blue light to break bonds holding 2 pyrimidines together 20-119 Undoing High Energy DNA Damage O6 alkylations on guanine residues can be directly reversed by the suicide enzyme, O6-methylguanine methyltransferase This enzyme accepts the alkyl group onto one of its amino acids 20-120 Excision Repair Percentage of DNA damage products that can be handled by direct reversal is small Most damage involves neither pyrimidine dimers nor O6-alkylguanine Another repair mechanism is required, excision repair is the process that removes most damaged nucleotides Damaged DNA is removed Replaced with fresh DNA Base and nucleotide excision repair are both used 20-121 Base Excision Repair Base excision repair (BER) acts on subtle base damage Begins with DNA glycosylase Extrudes a base in a damaged base pair Clips out the damaged base Leaves an apurinic or apyrimidinic site that attracts DNA repair enzymes DNA repair enzymes Remove the remaining deoxyribose phosphate Replace it with a normal nucleotide 20-122 Base Excision Repair in E. coli DNA polymerase I fills in missing nucleotide in BER Base is removed the AP site remains – apurinic or apyrimidinic AP endonuclease cuts or nicks DNA strand Phosphodiesterase removes the AP sugar phosphate Pol I performs repair synthesis 20-123 Eukaryotic BER DNA polymerase b fills in the missing nucleotide Makes mistakes No proofreading activity APE1 carries out proofreading Repair of 8-oxyguanine sites in DNA is special case BER – 2 ways can occur A can be removed after DNA replication by a specialized adenine DNA glycosylase oxoG will still be paired with C and oxoG removed by another DNA glycoslyase, oxoG repair enzyme 20-124 Nucleotide Excision Repair Nucleotide excision repair typically handles bulky damage that distorts DNA double helix NER in E. coli begins when damaged DNA is clipped by an endonuclease on either side of the lesion, sites 12-13 nt apart Enzyme system catalyzing nucleotide excision repair is excinuclease Allows damaged DNA to be removed as part of resulting 12-13-base oligonucleotide 20-125 Human Global Genome NER 20-126 Double-Strand Break Repair in Eukaryotes dsDNA breaks in eukaryotes are probably most dangerous form of DNA damage These are really broken chromosomes If not repaired lead to cell death In vertebrates can also lead to cancer Eukaryotes deal with dsDNA breaks in 2 ways: Homologous recombination Nonhomologous end-joining Role of chromatin remodeling in dsDNA break repair 20-127 Model for Nonhomologous End-Joining This process requires Ku and DNA-PKcs which bind at DNA ends and lets ends find regions of microhomology 2 DNA-PK complexes phosphorylate each other and activates Catalytic subunit to dissociate DNA helicase activity of Ku to unwind DNA ends Extra flaps of DNA removed, gaps filled, ends permanently ligated 20-128 Mismatch Repair Mismatch repair system recognizes parental strand by methylated A in GATC sequence Corrects mismatch in progeny strand Eukaryotes use part of repair system Rely on different, uncharacterized method to distinguish strands at a mismatch 20-129 Coping with DNA Damage Without Repairing It Direct reversal and excision repair are true repair processes Eliminate defective DNA entirely Cells can cope with damage by skirting around it Not true repair mechanism Better described as damage bypass mechanism 20-130 Recombination Repair The gapped DNA strand across from a damaged strand recombines with normal strand in the other daughter DNA duplex after replication Solves gap problem Leaves original damage unrepaired 20-131 Error-Prone Bypass Induce the SOS response This causes DNA to replicate even though the damaged region cannot be read correctly Result is errors in the newly made DNA 20-132  Homologous Recombination Homologous Recombination is crucial for Accurately repairing double-strand breaks and other types of DNA damage, Can also rearrange DNA sequences. These rearrangements often alter the particular versions of genes present in an individual genome, as well as the timing and the level of their expression. Genetic variation produced by this and other types of genetic recombination is crucial for evolution. In homologous recombination genetic exchange takes place between a pair of homologous DNA sequences DNA sequences similar or identical in nucleotide sequence. Homologous recombination has many uses in the cell, but three are of paramount importance. Accurately repairing double-strand breaks Accidents occur during nearly every round of DNA replication. Many types of events can cause the replication fork to break during the replication process. Consider just one example: a single-strand nick or gap in the parental DNA helix just ahead of a replication fork. Then the fork reaches this Iesion, it falls apart-resulting in one broken and one intact daughter chromosome. However, a series of recombination reactions, which can begin with a strand invasion process that triggers DNA synthesis by DNA polymerase, can flawlessly repair the broken chromosome. Exchange bits of genetic information between two different chromosomes to create new combinations of DNA sequences in each chromosome. The potential evolutionary benefit of this type of gene mixing is that it creates an array of new perhaps beneficial, combinations of genes. Chromosomal segregation DNA Base-Pairing Guides Homologous Recombination The hallmark of homologous recombination is that it takes place only between DNA duplexes that have extensive regions of sequence similarity (homology). Two DNA duplexes that are undergoing homologous recombination "sample" each other's DNA sequence by engaging in extensive base-pairing between a single strand from one DNA duplex and the complementary single strand from the other. The match need not be perfect, but it must be very close. In its simplest form, this type of base-pairing interaction can be mimicked in a test tube by allowing a DNA double helix to re-form from its separated single strands. This process, called DNA renaturation or hybridization, occurs when a rare random collision complementary nucleotide sequences on two matching DNA single strands, allowing the formation of a short stretch of double helix between them. This relatively slow helix nucleation step is followed by a very rapid "zippering" step, as the region of double helix is extended to maximize the number of base-pairing interactions. Formation of a new double helix in this way requires that the annealing strands be in an open, unfolded conformation. A Protein enables a DNA ss and DNA dh pairing Extensive base-pair interactions cannot occur between two intact DNA double helices The DNA hybridization can begin only after a DNA strand from one DNA helix is freed from pairing with its complementary strand. This makes its nucleotide available for pairing with a second DNA helix. The single-strand at the 3' DNA end is acted upon by several specialized proteins that direct it to invade a homologous DNA duplex. Like a single-strand DNA binding protein, the RecA type of protein binds tightly and in long cooperative clusters to single-stranded DNA forming a nucleoprotein filament. Because each RecA monomer has more than one DNA-binding site, a RecA filament can hold a single strand and a double helix together. In the first step, the RecA protein intertwines the DNA single strand and the DNA duplex in a sequence-independent manner. Next, the DNA single strand "searches" the duplex for homologous sequences. Once a homologous sequence has been located, a strand invasion occurs: the single strand displaces one strand of the duplex as it forms conventional base pairs with the other strand. The result is a heteroduplex- a region of DNA double helix formed by the pairing of two DNA strands that were initially part of two different DNA molecules.  Branch Migration Once a strand invasion reaction has occurred, the point of strand exchange (the "branch point") can move through a process called branch migration. In this reaction, an unpaired region of one of the single strands displaces a paired region of the other single strand, moving the branch point without changing the total number of DNA base pairs. Specialized DNA helicases can catalyze unidirectional branch migration, readily producing a region of heteroduplex DNA that can be thousands of base pairs long.  In most cells, recombinationmediated double- strand break repair occurs only after the cell has replicated its DNA, when one nearby daughter DNA duplex can serve as the template for repair of the other. Cells carefully regulate homologous recombination Although homologous recombination neatly solves the problem of accurately repairing double-strand breaks and other types of DNA damage, it does present some dangers to the cell and therefore must be tightly regulated. For example, the DNA sequence in one chromosomal homolog can be rendered non-functional by "repairing" it using the other chromosomal homolog as the template. A loss of heterozygosity of this type is frequently a critical step in the formation of cancers, and cells have poorly understood mechanisms to minimize it. Although relatively rare in normal cells, loss of heterozygosity can be viewed as an unfortunate side effect of the versatility of homologous recombination. All eukaryotic cells, prevent recombination-based "repair" in the absence of DNA damage. Enzymes that catalyze recombination: Synthesized at high level Converge on the site of damage Repair Factories This mobilisation is tightly controlled Ex. Brca1 and Brca2 mutated in breast cancer Too much and too little homologous recombination can lead to cancer: Loss of heterozygosity Increased mutation rate caused by ineffective DNA repair Holiday Junction Homologous recombination as a means to generate DNA molecules of novel sequence. During this process a special DNA intermediate often forms that contains four DNA strands shared between two DNA helices. In this key intermediate, known as a Holliday junction, or cross-strand exchange, two DNA strands switch partners between two double helices. The Holliday junction can adopt multiple conformations, and a special set of recombination proteins binds to, and thereby stabilizes, the open, symmetric isomer. The four-stranded DNA structures produced by homologous recombination are only transiently present in cells. The strands connecting the two helices in a Holliday junction must cut, a process referred to as resolution.  Extensive homologous recombination occurs as an integral part of the process whereby chromosomes are parcelled out to germ cells during meiosis. Both chromosome crossing over and gene conversion result from these recombination events, producing hybrid chromosomes that contain genetic information from both the maternal and paternal homologs.  Homologous recombination in meiosis starts with a bold stroke: a specialized protein breaks both strands of the DNA double helix in one of the recombining chromosomes. Like a topoisomerase, the reaction of protein with DNA leaves the protein covalently bound to the broken DNA. A specialized nuclease then rapidly processes the ends bound by Spoll, removing the protein and leaving protruding 3'single-strand ends. At this point, a series of strand invasions and branch migrations take place that frequently produce an intermediate consisting of two closely spaced Holliday junctions, often called a double Holliday junction. There are two different ways to resolve the double Holliday intermediate. Noncrossover, the original pairs of crossing strands are cut at both Holliday junctions in the same way, which causes the two original helices to separate from one another in a form unaltered except for the region between the two junctions. Crossover, the portions of each chromosome upstream and downstream from the two Holliday junctions are swapped, creating two chromosomes that have crossed over. How cells read the genome: From DNA to Protein  Much of the DNA-encoded information present in genomes specifies the linear order—the sequence—of amino acids for every protein the organism makes. The amino acid sequence in turn dictates how each protein folds to give a molecule with a distinctive shape and chemistry. Additional information encoded in the DNA of the genome specifies exactly when in the life of an organism and in which cell types each gene is to be expressed into protein. Since proteins are the main constituents of cells, the decoding of the genome determines not only the size, shape, biochemical properties, and behavior of cells, but also the distinctive features of each species on Earth. One might have predicted that the information present in genomes would be arranged in an orderly fashion. The genomes of most multicellular organisms are surprisingly disorderly. Small bits of coding DNA (that is, DNA that codes for protein) are interspersed with large blocks of seemingly meaningless DNA. Some sections of the genome contain many genes and others lack genes altogether. Proteins that work closely with one another in the cell often have their genes located on different chromosomes. Adjacent genes typically encode proteins that have little to do with each other in the cell. Decoding genomes is therefore no simple matter. Even with the aid of powerful computers, it is still difficult for researchers to locate definitively the beginning and end of genes in the DNA sequences of complex genomes, much less to predict when each gene is expressed in the life of the organism. Although the DNA sequence of the human genome is known, it will probably take at least a decade to identify every gene and determine the precise amino acid sequence of the protein it produces. Yet the cells in our body do this thousands of times a second.  The DNA in genomes does not direct protein synthesis itself, but instead uses RNA as an intermediary. When the cell needs a particular protein, the nucleotide sequence of the appropriate portion of the immensely long DNA molecule in a chromosome is first copied into RNA (a process called transcription). It is these RNA copies of segments of the DNA that are used directly as templates to direct the synthesis of the protein (a process called translation). The flow of genetic information in cells is therefore from DNA to RNA to protein All cells, from bacteria to humans, express their genetic information in this way—a principle so fundamental that it is termed the central dogma of molecular biology. Despite the universality of the central dogma, there are important variations in the way in which information flows from DNA to protein. Principal among these is that RNA transcripts in eucaryotic cells are subject to a series of processing steps in the nucleus, before they are permitted to exit from the nucleus and be translated into protein. Ex. RNA splicing These processing steps can critically change the “meaning” of an RNA molecule and are therefore crucial for understanding how eucaryotic cells read their genomes. For many genes RNA is the final product. Like proteins, many of these RNAs fold into precise three-dimensional structures that have structural, catalytic, and regulatory roles in the cell.  DNA to RNA Transcription Transcription and translation are the means by which cells read out, or express, the genetic instructions in their genes. Because many identical RNA copies can be made from the same gene, and each RNA molecule can direct the synthesis of many identical protein molecules, cells can synthesize a large amount of protein rapidly when necessary. But each gene can also be transcribed and translated with a different efficiency, allowing the cell to make vast quantities of some proteins and tiny quantities of others. Moreover, a cell can change (or regulate) the expression of each of its genes according to the needs of the moment—most commonly by controlling the production of its RNA.  The first step a cell takes in reading out a needed part of its genetic instructions is to copy a particular portion of its DNA nucleotide sequence—a gene—into an RNA nucleotide sequence. The information in RNA, although copied into another chemical form, is still written in essentially the same language as it is in DNA—the language of a nucleotide sequence. Hence the name transcription. Like DNA, RNA is a linear polymer made of four different types of nucleotide subunits linked together by phosphodiester bonds. It differs from DNA chemically in two respects: (1) the nucleotides in RNA are ribonucleotides— that is, they contain the sugar ribose (hence the name ribonucleic acid) rather than deoxyribose; (2) although, like DNA, RNA contains the bases adenine (A), guanine (G), and cytosine (C), it contains the base uracil (U) instead of the thymine (T) in DNA. Since U, like T, can base-pair by hydrogenbonding with A the complementary base-pairing properties described for DNA in Chapters 4 and 5 apply also to RNA (in RNA, G pairs with C, and A pairs with U). We also find other types of base pairs in RNA: for example, G occasionally pairs with U. Genome (Genomics: data mining, sequence assignment) Transcription regulation, alternative splicing, Transcripts (RT PCR, micro-array analysis) Translation Proteome (Mass-spectrometry, Proteomics) Folding, sorting, post-transcriptional modification, targeting, complex assembly, co-factors, activation, Functional traits (Phenotype, function, physiology, biochemistry, biophysics)