Section 18.1 Genetic information - Replication

Chapter 18: Overview §Successful information-based system involves conservation and transfer §DNA - stable structure that maximizes storage and duplication §RNA - more reactive with numerous roles in protein synthesis and gene expression regulation §Decoding DNA requires DNA-protein interactions §Major and minor grooves facilitate sequence-specific binding §Contact between amino acid residues and edges of bases §DNA-binding proteins - most possess twofold axis of symmetry 1. Helix-turn-helix 2. Helix-loop-helix 3. Leucine zipper 4. Zinc finger §Accurate DNA synthesis; effective DNA repair mechanisms §Variation may also be important for adaptability to environments §Caused by genetic recombination and mutation Section 18.1: Genetic Information: Replication Semiconservative replication §Separation of 2 original strands §Each serves as a template for synthesis of a complementary strand (daughter) §1 original strand/1 new strand §Meselson and Stahl established using radio-labeled 15NH4Cl §Replication factories – specific nuclear or nucleoid compartments where replication takes place §DNA Synthesis in Prokaryotes Figure 18.2 Semiconservative DNA Replication §Helicases - unwind DNA §Primer synthesis – required for initiation §DNA polynucleotide synthesis – synthesis of complementary strand n n Replication ¨ Separation of the two original strands ¨ Synthesis of two new daughter strands using the original strands as templates Semiconservative replication: each daughter strand contains one template strand and one newly synthesized strand ¨ Incorporation of isotopic label as sole nitrogen source (15NH4Cl) ¨ Observed that 15N-DNA has a higher density than 14N-DNA, and the two can be separated by density-gradient ultracentrifugation Section 18.1: Genetic Information: Replication Origin of replication – point where replication will begin Replication forks– Y shaped region of an unwinding DNA double helix undergoing replication Section 18.1: Genetic Information: Replication DNA polymerase reaction 5’ à 3’ direction Nucleotide is attached to 3’read hydroxyl on sugar via nucleophilic attack by OH-. . . . . .on a-phosphate of dNTP (5’ C) Leaving group pyrophosphate(PPi) Energy released by PPi hydrolysis drives overall reaction Figure 18.3 The DNA Polymerase Reaction growth Section 18.1: Genetic Information: Replication §DNA polymerase III (pol III) Figure 18.5 Cross Section of the b2-Clamp of DNA Polymerase III §Catalyzes the nucleophilic attack of the 3′-hydroxyl group onto the aphosphate §Pol III holoenzyme - at least 10 subunits §Core polymerase is formed of three subunits: a, e, and t §b-protein (sliding clamp) is two subunits and forms a donut-shaped ring around the template DNA §g-complex (clamp loader) transfers sliding clamp Section 18.1: Genetic Information: Replication §Replisome - two pol III holoenzymes, primosome and DNA unwinding proteins §DNA polymerase I - 3 catalytic activities §5’ à 3’ exonuclease activity §5’ à 3’ template directed polymerase activity §3’ à 5’ exonulease activity §DNA polymerase II, IV, V - DNA repair enzymes known as translesion repair enzymes §Part of the SOS response - prevent cell death §DNA ligase catalyzes the formation of the phosphodiester bond between adjoining nucleotides §DNA topoisomerases – control supercoiling, relieve torque Section 18.1: Genetic Information: Replication §Type I topoisomerases produce transient single-strand breaks §Type II topoisomerases produce transient double-strand breaks §DNA gyrase—in prokaryotes helps separate replication products & create negative (-) supercoils §E. coli – high ATP/ADP ratio; DnaA, begins at origin of replication, oriC §Bidirectional from oriC: 5’ -> 3’ & 3’ -> 5’ §Each replication fork: §helicases – unwind double helix §Replisome – molecular machine that carries out DNA replication Figure 18.6 Replication of Prokaryotic DNA §Replicon - One origin of replication unit and regulatory sequences Section 18.1: Genetic Information: Replication Figure 18.8 DNA Replication at a Replication Fork §DNA synthesis - 5′à3′ direction §DNA polmerase reaction – nucleotide attaches to 5’ end §Incoming nucleotide (5’-triP nucleophile) adds to 3’-hydroxyl on sugar on end of chain §3’-hydroxyl grp attacks P adjacent to sugar being added §Elimination of pyrophosphate, formation of phosphodiester bond §leading strand – continuously synthesized §lagging strand –semi-continuous §Okazaki fragments – short 5’->3’ segments; joined by DNA ligase Section 18.1: Genetic Information: Replication §DNA-binding domain §DnaA proteins bind within oriC (DnaA boxes) – yellow beads §DnaA-DNA complex forms; requires ATP (pink beads) & histone-like protein (HU); complex opens §DnaB helicase (orange beads) binds to DNA; helix is unwound & replication fork moves forward §Topoisomerases relieve torque ahead of the replisome §Single Stranded Binding proteins (SSB)– binds to single strand DNA to keep strands apart (purple) Section 18.1: Genetic Information: Replication Figure 18.10 E. coli DNA Replication Model DNA synthesis requires: § All 4 deoxyribonuleotide triphosphates & Mg2+ (ATP, CTP, TTP, GTP) § RNA primer required for pol III to initiate DNA synthesis § § § All 4 ribonucleoside triphosphates (ATP, CTP, UTP<, GTP) Leading strand, only a single primer is required Lagging strand, a primer is required for each Okazaki fragment § Pol III synthesizes at the 3′ end of the primer § § RNA primers are removed by pol I DNA ligase then joins Okazaki fragments § Tandem operation of pol III complexes requires lagging strand to be looped around replisome Section 18.1: Genetic Information: Replication §DNA replication takes place only once each generation in each cell §Errors in replication (mutations) occur spontaneously only once in every 109 to 1010 base pairs §Can be lethal to organisms §Proofreading - DNA pol I and III, and postreplication repair mechanisms §15 eukaryotic DNA polymerases; 3 (a, b, e) in nuclear replication §e corrects a errors §b is a nuclear repair polymerase Section 18.1: Genetic Information: Replication Figure 18.11 Role of Tus in DNA Replication Termination in E. coli §Replication ends - replication forks meet at the other side of the circular chromosome at the termination site (ter region) ü 6 termination sequences (orange arrows) ü Replication forks become ‘trapped’ in ter region §DNA-binding protein tus - binds to the ter causing replication arrest Section 18.1: Genetic Information: Replication DNA Synthesis in Eukaryotes §15 DNA Polymerases §Three (a, d, and e) are involved in nuclear DNA replication §Pol g replicates and repairs mitochondrial DNA §Polymerases b, z and h function in nuclear DNA repair Figure 18.12 The Eukaryotic Cell Cycle §Timing of replication—limited to S phase in cell cycle §Replication rate – slower 50 bp per second per replication fork due to complex chromatin structure §Replicons—compresses replication of large genomes into short periods §Okazaki fragments are from 100 to 200 nucleotides long Section 18.1: Genetic Information: Replication Eukaryotic Replication Process – begins with assembly of pre-initiation replication complex (preRC) § Preinitiation replication complex (preRC) – initiates replication § § § Licensed preRC -active initiation complex § § § Figure 18.14 Formation of a Preinitiation Replication Complex origin replication complex (ORC) binds to DNA initiation region (origin) § Recruits Cdc6 & CdrI MCM complex (helicase) is recruited § Allows replication proteins to load onto replication fork Requires pol a/primase, pol e, and accessory proteins Cell cycle regulating kinases then phosphorylate and activate Replication licensing factors (RLFs) proteins bind to ORC, complete preRC structure Section 18.1: Genetic Information: Replication §Replication protein A (RPA)- stablizes newly separated strands §Primase – synthesizes RNA primers §Pol a/Primase extends primers by short DNA strand (~20nt) §Replication factor C (RFC) - clamp loader §Controls attachment of polymerase d and e to continue process §Replication continues until replicons meet and fuse §3’ end of lagging strand – no space for new RNA primer §End of chromosome susceptible to nuclease digestion Section 18.1: Genetic Information: Replication §Telemeres– mini-satellite sequences at end of linear chromosome §Telomerase – overcomes susceptibility to nuclease digestion §Ribonucleoprotein with reverse transcriptase activity §RNA base sequence complementary to the TG-rich sequence of telomeres §Uses sequence to synthesize a single-stranded DNA to extend the 3′ strand §Telomere end-binding proteins (TEBPs) –binds to GT rich telomere sequences §Telomere repeat-binding factors (TRFs)– secure 3’ end §During normal human aging - telomeres shorten §Once reduced to a critical length - chromosome replication cannot occur;causes cell death §90% of all cancers have hyperactive telomerase Section 18.1: Genetic Information: Replication Average natural mutation – 1/100,000 genes/generation Direct Repairs – photoreactivation repair of thymine dimers Figure 18.18 Photoreactivation Repair of Thymine Dimers § Occurs in bacteria, archaea, protozoa fungi, plants, & animals (not humans § DNA damage repaired without removal of nucleotides § DNA ligase – repairs breaks in phosphodiester linkages § § Pyrimidine dimers - restored to original monomeric structure Hydrogen bond - UUsing photoreactivating enzyme and visible light Section 18.1: Genetic Information: Replication Figure 18.19 Base Excision Repair Single Strand Repairs – uses complementary, undamaged strand as template §Base excision repair removes then replaces individual nucleotides whose bases have undergone damage §DNA glycosylase cleaves N-glycosidic linkage between damaged base and deoxyribose of nucleotide §Nucleases – remove purines or pyrimidines (apurinic or apyrimidinic sites) §DNA polymerase & DNA ligase – repair gap §pol I in bacteria; DNA polymerase b in mammals Section 18.1: Genetic Information: Replication Nucleotide excision repair - removal of bulky (2-30 nt) lesions; gap is filled §Global genomic repair §excision enzymes recognize distortion rather than base sequence §E. coli - excision nuclease cuts DNA §removes 12 to 13 nt ssDNA sequence containing the lesion §pol I and DNA ligase – repair gap Figure 18.20 Excision Repair of a Thymine Dimer in E. coli Section 18.1: Genetic Information: Replication §Transcription coupled repair - strand being actively transcribed §Damage is recognized when RNA polymerase is stalled §Mfd (transcription-repair coupling factor) displaces polymerase and recruits UvrA2B to initiate damage removal §Mismatch repair (MMR) - corrects helix distorting base mispairings resulting from proofreading errors or replication slippage §Key feature is the capacity to distinguish between old and newly synthesized strands §Methylation of parent strand results in hemimethylated daughter strands Section 18.1: Genetic Information: Replication Double-strand breaks (DSBs) - can result in a lethal breakdown of chromosomes §Caused by radiation, ROS, DNA damaging agents, or as result of replication errors §Repaired by two mechanisms: non-homologous end joining (NHEJ) and homologous recombination §NHEJ is error prone because there is no requirement for sequence homology §Recombination will be explained next Section 18.1: Genetic Information: Replication DNA Recombination – principle source of genetic variations that make evolution possible § Rearrangement of DNA sequences by exchanging segments from different molecules §Two types of recombination: §General recombination occurs between homologous DNA molecules (most common during meiosis) §Site-specific recombination—the exchange of sequences only requires short regions of DNA homology (e.g., transposition) Section 18.1: Genetic Information: Replication General Recombination §Holliday model (1964) Figure 18.21: The Holliday Model 1. Two homologous DNA molecules become paired 2. Two of the DNA strands, one in each molecule, are cleaved 3. Two nicked strand segments cross over, Holliday intermediate 4. DNA ligase seals the cut ends 5. Branch migration leads totransfer of a segment of DNA from one homologue to the other 6. Second series of strand cuts occurs 7. DNA polymerase fills gaps, DNA ligase seals cut strands Section 18.1: Genetic Information: Replication §Messelson-Radding model 1. One strand of two homologous DNA molecules is nicked 2. Extension causes displacement of strand on other side of nick 3. D-loop is cleaved, invading strand is ligated to newly created 3′-end of the homologous strand 4. 3′-end of newly synthesized strand & the 5′-end of a homologous strand are ligated forming a Holliday junction 5. Branch migration may occur 6. Strand nicks and Holliday junction resolution result in a crossover or non-crossover product Section 18.1: Genetic Information: Replication Double-strand break repair model 1. Endonuclease induces a DSB in one pair of homologous chromosomes 2. Exonucleases degrade the 5′-ends leaving 3′-end tails 3. One 3′-end tail invades the homologous DNA molecule and forms a D-loop 4. DNA polymerase extends the invading and noninvading 3′-tail 5. Branch migration and end ligation result in Holliday junction formation 6. Resolution of 2 Holliday structures can give noncrossover or crossover products §DSBR occurs in mitosis, without crossover products §Synthesis-dependent strand annealing model explains this phenomena Section 18.1: Genetic Information: Replication §Bacterial Recombination is involved in several forms of intermicrobial DNA transfer: 1. Transformation - naked DNA molecules enters the cell through small holes in the cell wall 2. Transduction - bacteriophage inadvertently carries bacterial DNA to a recipient cell 3. Conjugation - unconventional sexual mating involving passing DNA from a donor cell through a sex pilus to a recipient cell Section 18.1: Genetic Information: Replication §Eukaryotic Recombination occurs during first phase of meiosis to ensure accurate homologous chromosome pairing and crossing over §Similar to prokaryotic recombination but has a larger number of proteins because of the more complex genomes §Rad52 is believed to be the initial sensor of DSBs §MRN complex – creates scaffold stabilizing DNA ends at DSBs §Recruits & activates ATM – regulates damage response §Rad51, BRCA1, and BRCA2 are involved in DSB repair Section 18.1: Genetic Information: Replication Site Specific Recombination and Transposition - short segments of homologous DNA called attachment (att) sites or insertional (IS) elements §Recombination can lead to insertions, deletions, inversions, and translocations §Integration of bacteriophage l DNA into E. coli chromosome requires homologous att sites in phage and bacterial genomes §A viral recombinase (l integrase) and a bacterial gene product (integration host factor) are also required for l prophage formation §Results in insertion of l genome into bacterial chromosome Section 18.1: Genetic Information: Replication Transposition – move DNA sequence from one site to another in genome §Nonreplicative transposition -doublestranded cut in the donor DNA §Spliced into the staggered ssDNA cut ends of the target site §DNA repair system fills the gaps in target DNA §Transposons – “jumping genes” Figure 18.27 Nonreplicative Transposition Section 18.1: Genetic Information: Replication Figure 18.28 Replicative Transposition Replicative transposition transfer of one strand of donor DNA to target position §Duplication of the transposon rather than insertion at the new site §Involves an intermediate, co-integrate, resolvase (catalyzes the site-specific recombination)