FQ

DNA Replication and Repair

Essential Questions What does it mean: “to clone”? What is the polymerase chain reaction (PCR)? Is it possible to make directed changes in the heredity of an organism?

DNA Replication DNA replication yields two daughter DNA duplexes identical to the parental DNA molecule.

How Do Scientists Determine the Primary Structure of Nucleic Acids? DNA is a double-helical molecule Each strand of the helix must be copied in complementary fashion by DNA polymerase Each strand is a template for copying DNA polymerase requires template and primer Primer: an oligonucleotide that pairs with the end of the template molecule to form dsDNA DNA polymerases add nucleotides in 5'-3' direction

DNA replication Separation, Base pair

The Chemistry of DNA replication

DNA Synthesis by DNA polymerase

What Is the Polymerase Chain Reaction (PCR)? What if you don't have enough DNA for colony hybridization or Southern blots? The small sample of DNA serves as template for DNA polymerase Make complementary primers Add primers in more than 1000-fold excess Heat to make ssDNA, then cool Run DNA polymerase (usually Taq) Repeat heating, cooling, polymerase cycle

Virtually Any DNA Sequence Can Be Cloned Nuclease cleavage at a restriction site linearizes the circular plasmid so that a foreign DNA fragment can be inserted. Recombinant plasmids are hybrid DNA molecules consisting of plasmid DNA sequences plus inserted DNA elements (pink here). Such hybrid molecules are called chimeric plasmids. Figure 12.2 An EcoRI restriction fragment of foreign DNA can be inserted into a plasmid.

Plasmids Are Very Useful in Cloning Genes Plamids are naturally occurring extrachromosomal DNA Plasmids are circular dsDNA Plasmids can be cleaved by restriction enzymes, leaving sticky ends Artificial plasmids can be constructed by linking new DNA fragments to the sticky ends of plasmid These recombinant molecules can be autonomously replicated, and hence propagated

Cloning Vectors Cloning vectors are plasmids that can be modified to carry new genes Plasmids useful as cloning vectors must have – a replicator (origin of replication) – a selectable marker (antibiotic resistance gene) – a cloning site (site where insertion of foreign DNA will not disrupt replication or inactivate essential markers)

DNA Maintenance Mutation rate are extremely low 1 mutation out of 10 9 nucleotides per generation

DNA Repair Spontaneous DNA damage Pathways to remove DNA damage Damage detection The repair of Double-strand break DNA repair enzymes

Spontaneous Alterations of nucleotides Red: oxidative damage; blue: hydrolytic attack; green: uncontrolled methylation

Thymine dimer

Emergency DNA Repair for Double helix break

DNA Repair I

DNA Repair II

DNA Repair Spontaneous DNA damage: spontaneous alteration of bases, depurination and deamination, thymine dimer Pathways to remove DNA damage: base excision repair, nucleotide excision repair Damage detection: base flipping The repair of Double-strand break: nonhomolous end joining, homologous end joining DNA repair enzymes: heat shock proteins