BIOL2010 DNA Replication & Repair PDF

BIOL2010 DNA replication & repair Dr M.L.Bellamy  BIOL2010 DNA replication Lectures 2-7 Dr M.L.Bellamy Overview 1) Composition/structure of DNA 2) DNA synthesis vs DNA replication 3) Problems and solutions in E.coli 4) The 3 steps in replication 5) Differences...

BIOL2010 DNA replication & repair Dr M.L.Bellamy  BIOL2010 DNA replication Lectures 2-7 Dr M.L.Bellamy Overview 1) Composition/structure of DNA 2) DNA synthesis vs DNA replication 3) Problems and solutions in E.coli 4) The 3 steps in replication 5) Differences in eukaryotes & viruses Cells Nucleic acids are the genetic material Bacterial cell Animal cell Plant cell Prokaryote Eukaryotes “Before nucleus” “True nucleus” Cell division Fundamental feature of life → Reproduction in unicellular life → Growth in multicellular life Creating new cells means synthesizing new DNA Binary fission = doubling of DNA Cell division Cell division 4.6x1066 bp Cell division Chromosome 1 2.5x1088 bp Y chromosome 6.2x1077 bp All chromosomes in one cell = 2m of DNA DNA Genome size Bacteriophage 5386 bp Mitochondrial 16,569 bp E.coli 4.6x1066 bp Amoeba dubia Yeast 1.2x10 bp 77 6.7x1011 b.p. Nematode 9.55x1077 bp Cress 1.17x1088 bp Fruit Fly 1.8x1088 bp Mouse 2.7x1099 bp Human 3.1x1099 bp DNA Compositio Polynucleotide 5’ Four nucleotide monomers: Adenosine Cytidine Guanosine Thymidine Four nitrogenous bases: Adenine Phosphate Cytosine (phosphodiester) Guanine Deoxyribose 3’ Thymine DNA Compositio Four nucleotides in DNA PYRIMIDINE O PURINE NH2 NH T N N N O A P Thymidine HO N N P HO O thymine H H H O H Adenosine H OH H H H H adenine OH H NH2 O N N NH C G N O N N NH2 P P HO Cytidine HO O O H H H H Guanosine H H cytosine OH H guanine H OH H H DNA Structure WATSON CRICK DNA Structure DNA Structure Double-helix 3’ Right-handed 5’ Antiparallel Phosphodiester backbone ~10 nt per turn Bases on the inside Hydrogen bonds between bases on 3’ opposite strands 5’ DNA Structure Complementary base-pairing G-C pair: 3 H bonds A-T pair: 2 H bonds →Chargaff’s rule DNA Structure AT and GC base pairs are the same width O H N T N H H O N N O N C N H N O N G N H N N G N O H N N H N H H H H O H N N N N N H O T N H A N N N A N N N H N N G N O N H N DNA Structure DNA Structure “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.’’ Watson & Crick (1953) Nature. 171, 737 DNA Semi-conservative 5’ A C G A A A A A WATSON 5’ A C G A A A A A T G C T T T T T CRICK 3’ T G C T T T T T 3’ DNA Semi-conservative 5’ A C G A A A A A T G C T T T T T 3’ 5’ A C G A A A A A T G C T T T T T 3’ DNA Semi-conservative Meselson & Stahl (1958) E.coli cells grown in a 15N medium E.coli cells transferred NH2 to a 14N medium N N A N N P P P HO O Next generation DNA is 50% 15NH : 50% H 14 N H H OH H Further generations revealed only three types of molecule possible: 100% 15N, 50:50, 100% 14N DNA synthesis PCR Only 1 enzyme required: Taq (DNA Polymerase) + dNTPs (ATP,CTP,GTP,TTP), Template DNA, primers Heat to 95°C (Denaturing) Cool to 55°C (Annealing) Heat to 72°C (Extending) DNA synthesis PCR Only 1 enzyme required: Taq (DNA Polymerase) + dNTPs (ATP,CTP,GTP,TTP), Template DNA, primers G A T T A C A C G G A C G G A dNTP enters Pyrophosphate (P-P) released G A T T A C A C G G A C G G A DNA synthesis Polymerase mechanism Pi + Pi PPi H3O+ DNA synthesis Overview DNA synthesis is simple →addition of a dNTP to a 3’ end Next time: DNA replication is complex