DNA Replication and Repair Watson and Crick Predicted Semiconservative Replication of DNA • Watson and Crick: "It has not escaped our notice that the specific (base) pairing we have postulated immediately suggests a possible copying mechanism for the genetic material." • The mechanism: Strand separation, followed by copying of each strand. • Each separated strand acts as a template for the synthesis of a new complementary strand. The Semiconservative Model • Matthew Meselson and Franklin Stahl tested semiconservative model • Template DNA labeled with 15N –nucleotides. (more dense than normal DNA) • Fed 14N –nucleotides. (newly synthesized DNA was less dense than template) • Isolated DNA at different times and fractionated DNA on a density gradient • denser/heavier DNA found lower in the gradient. • Less dense/lighter DNA found higher in gradient. Replication is bidirectional • E. coli genome size = 4.6 X 106 bp • Bacteria have circular chromosome with single origin of replication. • Replication rate is ~1000 base pairs per second. • Duplicate chromosome in 38 minutes. • Eukaryotes have larger genomes 3 X 109 bps • Rate of Eukaryote chromosome replication is slower • But because eukaryote chromosomes have multiple origins of replication, it takes about the same amount of time to replicate complete genome. DNA Replication is Semidiscontinuous Okazaki Fragments The Enzymology of DNA Replication • If Watson and Crick were right, then there should be an enzyme that makes DNA copies from a DNA template • In 1957, Arthur Kornberg and colleagues demonstrated the existence of a DNA polymerase • Three DNA polymerases in E. coli - DNA polymerase I – DNA repair and participates in synthesis of lagging strand - DNA polymerase II – DNA repair - DNA polymerase III – major polymerase involved in DNA replication. DNA Polymerase II is a Multisubunit Enzyme DNA Polymerase II Subunit Organization DNA Replication is a Processive Process. • DNA Polymerase remains bound to the replication fork. • Dimer of b-subunit forms ring structure around the growing DNA chains. DNA Polymerase also has proof reading function • The polymerization reactions have an error rate of 1 mistake for every 100,000 base pairs incorporated (1 X 10-5 errors per base) • DNA polymerase has 3’ to 5’ exonuclease function (epsilon-subunit) that recognizes base pair mismatches and removes them. • Therefore proof reading function helps eliminate errors which could lead to detrimental mutations. • However proof reading exonuclease has error rate of 1 mistake for every 100 base pairs (1 X 10-2 errors per base) • Overall error rate is 1 X 10-7 errors per base. Stages of DNA Replication • Initiation • Elongation • Termination Initiation of Replication in E. coli • The replisome consists of: DNAunwinding proteins, the priming complex (primosome) and two equivalents of DNA • polymerase III holoenzyme • Initiation: DnaA protein binds to repeats in ori, initiating strand separation and DnaB, a helicase delivered by DnaC, further unwinds. Primase then binds and constructs the RNA primer Elongation Stage of Replication • Elongation involves DnaB helicase unwinding, SSB binding to keep strands separated. • Primase Complex Synthesizes short RNA primers. • DNA polymerase grinding away on both strands • Topoisomerase II (DNA gyrase) relieves supercoiling that remains DNA Polymerase I/ Ligase Required to Join Okazaki Fragments • DNA polymerase I has 5’ to 3’ exonuclease activity that removes RNA primer. • Also has 5’ to 3’ DNA polymerase activity to fill in the gap. (proofreading 3’-5’ exonuclease activity) • Ligase connects loose ends. Used NAD+ in phosphoryltransfer reaction, not a redox reaction (Page 643) Termination of Replication • Termination occurs at ter region of E. coli chromosome. • ter region rich in Gs and Ts, signals the end of replication. • Terminator utilization substance (Tus) binds to ter region. • Tus prevents replication fork from passing by inhibiting helicase activity. DNA Replication in Eukaryotes • Occurs similarly to what occurs in prokaryotes. • Multiple origins of replication • Replication is slower than in prokaryotes. • 5 different DNA polymerases in Eukaryotes. Eukaryotic DNA Polymerases • • • • Alpha – Primer synthesis and DNA repair Beta – DNA repair Gamma – Mitochondrial DNA replication Delta – Leading and lagging strand synthesis, and DNA repair • Epsilon – Repair and gap filling on lagging strand. PCNA analogous to E. coli bsubunit of E. coli DNA polymerase • Proliferating cell nuclear antigen • Trimeric protein • Sliding clamp structure binds to newly synthesized DNA strand DNA Repair • A fundamental difference from RNA, protein, lipid, etc. • All these others can be replaced, but DNA must be preserved • Cells require a means for repair of missing, altered or incorrect bases, bulges due to insertion or deletion, UV-induced pyrimidine dimers, strand breaks or cross-links • Two principal mechanisms: methods for reversing chemical damage and excision repair. Repair of UV Induced Thymine Dimers General excisionrepair pathway •Excision-repair systems scan DNA duplexes for mismatched bases, excise the mispaired region and replace it Repair of damage resulting from the deamination of cytosine • Deamination of cytosine to uracil is one of most common forms of DNA damage • DNA glycosylases cleave bases at N-glycosidic linkages. Leaving sugar-phosphate backbone. • Endonuclease identifies abscent base and sugar phosphate. • Gap then filled in by DNA polymerase and ligase.