Mechanism of Replication Step 1 Origins of Replication = Special site(s) on DNA w/Specific sequence of nucleotides where replication beginsOrigins of Replication –Prokaryotic Cells = 1 site (circular DNA) –Eukaryotic Cells = several sites (strands)
Steps 2 - 5 Helicase: (enzyme) unwinds DNA helix forming a “Y” shaped replication fork on DNA Replication occurs in two directions, forming a replication bubble To keep strands separate, DNA binding proteins attach to each strand of DNA Topoisomerases: enzymes that work w/helicase to prevent “knots” during unwinding.
Step 6 - Priming Priming = due to physical limitation of DNA Polymerase, which can only add DNA nucleotides to an existing chain RNA primase – initiates DNA replication at Origin of Replication by adding short segments of RNA nucleotides. Later these RNA segments are replaced by DNA nucleotides by DNA Pol.
Step 7 DNA Pol. = enzyme that elongates new DNA strand by adding proper nucleotides that base- pair with parental DNA template DNA Pol. can only add nucleotides to the 3’ end of new DNA, so replication occurs from a 5’ to 3’ direction Leading vs. Lagging Strand results
LeadingLeading vs. Lagging StrandLagging Strand Leading Strand: strand that can elongate continuously as the replication for progresses Lagging Strand: strand that cannot elongate continuously and moves away from replication fork. Short Okazaki fragments are added from a 5’ to 3’ direction, as replication fork progresses.
Step 10: Fixing Errors DNA Pol. Proofreads as it elongates Special enzymes fix a mismatch nucleotide pairs Excision Repair: –Nuclease: Enzyme that cuts damaged segment –DNA Pol. Fills in gap with new nucleotide
Mutations Thymine Dimers (covalent bonding btwn Thymine bases) –often caused by over- exposure to UV rays DNA buckeling skin cancer results, unless corrected by excision repair Substitutions: incorrect pairing of nucleotides Insertions and Deletions: an extra or missing nucleotide causes “frameshift” mutations (when nucleotides are displaced one position)
Problems with Replication Since DNA Polymerase can only add to a 3’ end of a growing chain, the gap from the initial 5’ end can not be filled Therefore DNA gets shorter and shorter after each round of replication
Solution? Bacteria have circular DNA (not a problem) Ends of some eukaryotic chromosomes have telomeres at the ends (repeating nucleotide sequence that do not code for any genes) Telomeres can get shorter w/o compromising genes Telomerase = enzyme that elongates telomeres since telomeres will shorten
Telomerases are not in most organisms Most multicellular organisms do not have telomerases that elongate telomeres (humans don’t have them) So, telomeres = limiting factor in life span of certain tissues Older individuals typically have shorter telomeres