Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Homologous recombination wikipedia , lookup
DNA profiling wikipedia , lookup
DNA repair protein XRCC4 wikipedia , lookup
DNA nanotechnology wikipedia , lookup
Zinc finger nuclease wikipedia , lookup
Eukaryotic DNA replication wikipedia , lookup
United Kingdom National DNA Database wikipedia , lookup
DNA replication wikipedia , lookup
Microsatellite wikipedia , lookup
DNA polymerase wikipedia , lookup
Plant Molecular and Cellular Biology Lecture 2: Fidelity of DNA Replication Gary Peter Learning Objectives 1. List and explain the mechanisms that insure high-fidelity DNA replication 2. Explain why the fidelity of DNA replication is so important for survival and for recombinant DNA work Importance of High-Fidelity DNA Replication z z z z If 1 mutation occurs per 109 bp Then what's the mutation rate of a 1000 bp gene in a population of 106 bacteria? What’s the frequency of the mutant in the population? Generation 1 z 106 copies of 1000 bp gene or 109 bp to be copied z z z Generation 2 z 2x106 copies of 1000 bp gene or 2x109 bp to be copied z z z 4x106 copies of 1000 bp gene or 4x109 bp to be copied z z z 2 new mutants/ 4x106 cells= 5x10-7 Frequency in pop.=4/(4x106)=1x10-6 Generation 3 z z 1 new mutant/ 2 x106 cells= 5x10-7 Frequency in pop.=1/(2x106)=5x10-7 4 new mutants/ 8x106 cells= 5x10-7 Frequency in pop.=12/(8x106)=1.5x10-6 Mutation rate is constant at 5x10-7 Frequency of mutants in the population increases Consequences of High Mutation Rates z Mutator strain propagated for various cycles 40 cycles = 1000 generations Funchain et al., 2000 Genetics 154: 959-970 What Processes and Pathways are Important for DNA Replication? z Monomer biosynthesis z z z z De novo and salvage pathways for nucleotide biosynthesis Purine and pyrimidine biosynthesis Nucleoside monophosphate conversion to triphosphate Polymer biosynthesis z z z z Template Primer DNA polymerases Other enzymes????? Method for Measuring the in Vitro Fidelity of DNA Polymerases z z z z Why use M13? What does the host need? Can this assay be used to measure the fidelity of all polymerases? What do you expect the results to be? Kunkel, 1985 JBC 260 (9):5787-5796 Results of Assay Kunkel, 1985 JBC 260 (9):5787-5796 Frame shifts and substitutions are similar in frequency Kunkel, 1985 JBC 260 (9):5787-5796 Why M13? z z z z z Facilitates creation of gapped duplex Non essential gene and α-complementation already present Large dsDNA region contains adenine methylation which should help limit the mismatch repair of the mutations arising during in vitro synthesis Easy to score large numbers of plaques Single stranded phage are readily sequenced What does the host strain need? z z The rest of the lac Z coding sequence for the intracistronic complementation to work This is located on the F’ plasmid to insure that susceptibility to infection by M13 phage Can this assay be used to measure the fidelity of all polymerases? z z z Only those polymerases that can use gapped templates Template Primer Fidelity of Various Thermostable DNA Polymerases DNA Polymerase Error Rate x 10-6 (Mutation rate per bp) Accuracy x 105 (Error rate-1)* Pfu 1.3 (0.2) 7.7 Deep Vent 2.7 (0.2) 3.7 Tli (VentR) 2.8 (0.9) 3.6 Taq 8.0 (3.9) 1.3 UlTma 55.3 (2.0) 0.2 *Accuracy is the average number of bases duplicated before an error is made z What does this mean for your recombinant DNA work? The Impact of Error Rate on % of clones with sequence errors z z z Error rate of PfuUltra is 4x10-6 and Phusion is 1.4x10-6, The number of mutations per bp equals the (error rate)*number of doublings Mutation frequency is MF=ER*bp*doublings Size PfuUltra Phusion 1 kb 0.7% 2.4% 5 kb 3.4% 11.9% 10 kb 6.8% 23.8% Impact of Reverse Transcriptase Errors on cDNA Sequence z AccuScript RT z z z ER = 1.6 x 10-5 So for a 1 kb cDNA expect 1.6% of the clones to have an error After 20 doublings (106 amplification) expect Enzyme Error Rate (10-6) % Mutant Clones PfuUltra 0.43 2.5% PicoMaxx 4.0 9.6% What’s the Normal Mutation Rate z z The probability of a mutation occurring due to random chemical decomposition of nucleotides is estimated at 1/10,000 bp The standard fidelity of E. coli DNA replication is 1 error in 1 billion base pairs Four Mechanisms that Control the Fidelity of DNA Replication z z z z Complementary base pairing Conformational change of polymerase which delays addition to a growing chain allowing additional time for the incorrect nucleotide to dissociate Removal of incorrect nucleotides at the 3’ terminus of the growing chain by the 3’ to 5’ exonuclease function of the DNA polymerase Removal of mispaired nucleotides by the mismatch repair system Summary z z The fidelity of DNA replication is due to the structure of DNA, the 3’-5’ proofreading activity of DNA polymerases and DNA repair enzymes DNA replication requires the concerted activity of multiple enzymes which bind together at the replication fork into the repliosome Nucleotide Levels in Cells DNA z z z Amount (mM) RNA Amount (mM) dNMP 43 rNMP 270 dATP 0.18 (0.013) ATP 3.0 (2.8) dGTP 0.12 (0.005) GTP 0.92 (0.48) dCTP 0.07 (0.022) CTP 0.52 (0.21) dTTP 0.08 (0.025) UTP 0.89 (0.48) The supply of deoxyribonucleotides in the E. coli is ~1% of that which is needed for replication In mammalian cells the supply is significantly less Why keep the levels so low? z z Inadequate supplies of dNTPs are lethal Too high a concentration is mutagenic Nucleotide Synthesis SALVAGE Deoxyribonucleotides DE NOVO Deoxyribonucleotides Bases Ribonucleotides Ribonucleotides Ribose, amino acids, CO2, NH3 De Novo Purine Biosynthesis z z Pathway is highly conserved from bacteria to mammals What about plants? First Committed Step in Purine Biosynthesis z First step in de novo purine biosynthesis is catalyzed by 5-phophoribosyl pyrophosphate synthase z PRPP synthase is feedback regulated – inhibitors include IMP, GMP, AMP (end products) Pyrimidine Biosynthesis z In contrast to purine biosynthesis, pyrimidine biosynthesis starts with the formation of the pyrimidine skeleton and then ribose is attached