DNA Replication Lecture Notes PDF

DNA replication http://youtu.be/5qSrmeiWsuc 1  Nucleic acids Some review: i. Monomers:___________________ ii. Polymers (2 types): ____________ iii. Monomers linked by ___________________ bond. iv. Functions: ____________________...

DNA replication http://youtu.be/5qSrmeiWsuc 1  Nucleic acids Some review: i. Monomers:___________________ ii. Polymers (2 types): ____________ iii. Monomers linked by ___________________ bond. iv. Functions: ____________________ 2  Double stranded DNA helix is antiparallel (one strand runs 5’ 3’ and the other 3’ 5’) Sugar –phosphate backbone on outside of DNA helix Nitrogenous base-pairs on inside of helix Fig316-7  DNA replication When is DNA replication happening in the cell cycle? 4  Key enzymes and proteins Helicase: Unwinds DNA strands (breaks hydrogen bonds) DNA polymerase: Synthesizes DNA by catalyzing the formation of phosphodiester bonds between nucleotides Uses parental strand as a template (adds complementary nucleotides) Makes DNA in the 5’  3’ direction (links 5’ phosphate group of new nucleotide to 3’-OH group of growing strand) Reads the template strand from 3’  5’ Primase: An RNA polymerase that synthesizes an RNA primer on the template strand Required because DNA polymerase can only add a nucleotide to a free 3’-OH group Ligase: Glues together nicks in DNA sugar-phosphate backbone (forms phosphodiester bond) Needed after RNA primers are replaced by DNA and for Okazaki fragments Single-stranded binding proteins: Stabilizes single stranded DNA 5 Origins of replication: where replication begins Fig 6 16-12  DNA polymerase Catalyzes phosphodiester bond formation between 5’-phosphate group of incoming nucleotide and the free 3’-OH end of the growing strand Fig7 16-14  DNA replication overview (1) Initiation: Helicases bind to origins of replication and begin unwinding DNA to form a replication bubble (2 replication forks) Single stranded binding proteins bind to the single stranded DNA (2) Elongation: Primase makes a complementary RNA primer (~5-10 nt) DNA polymerase synthesizes a new strand of DNA using the parental strand as a template Fig8 16-15  DNA Replication 5’ 3’ A G T T C T C G G T T A G C C T G T A C T G C A A C C T T T T C A A G A G C C A A T C G G A C A T G A C G T T G G A A A 3’ 5’ Reminders: DNA is double stranded Complementary base-pairing (G-C, A-T) DNA has directionality (5’ and 3’ end) Strands are antiparallel (run in opposite directions) 9  DNA Replication 5’ 3’ A G T T C T C G G T T A G C C T G T A C T G C A A C C T T T T C A A G A G C C A A T C G G A C A T G A C G T T G G A A A 3’ 5’ 10  DNA Replication 5’ 3’ A G T T C T C G G T T A G C C T G T A C T G C A A C C T T T T C A A G A G C C A A T C G G A C A T G A C G T T G G A A A 3’ 5’ 5’ 3’ A G T T C T C G G T T A G C C T G T A C T G C A A C C T T T T C A A G A G C C A A T C G G A C A T G A C G T T G G A A A 3’ 5’ 5’ 3’ A G T T C T C G G T T A G C C T G T A C T G C A A C C T T T T C A A G A G C C A A T C G G A C A T G A C G T T G G A A A 3’ 5’ Strands are separated Original strand is used as a template Complementary strand is synthesized in 5’  3’ direction 11  2 replication forks: DNA replication proceeds in both directions! Leading strand: Continuous DNA synthesis DNA polymerase is synthesizing DNA in the same direction as DNA unwinding One RNA primer Lagging strand: Discontinuous DNA synthesis (Okazaki fragments) DNA polymerase must synthesize DNA in the opposite direction of DNA unwinding Many RNA primers 12  Leading strand 5’  3’ continuous synthesis 1. Helicase unwinds DNA 2. Primase makes RNA primer 3. DNA pol extends from primer 4. DNA pol replaces RNA primer with DNA 5. DNA ligase repairs nick in backbone between replaced primer and rest of new strand 13  Lagging strand 3’  5’ discontinuous synthesis 1. Helicase unwinds DNA Okazaki fragments 2. Primase makes RNA primer 1 3. DNA pol extends from primer 1 4. Primase makes RNA primer 2 5. DNA pol extends from primer 2.... 6. DNA pol replaces RNA primers with DNA 7. DNA ligase repairs nicks in backbone between replaced primers and rest of strand and between Okazaki fragments Fig 14 16-16  End replication problem Removal of RNA primer from beginning of lagging strand leaves a gap and a free 5’-phosphate end Gap cannot be filled by DNA pol because can only add DNA to 3’-OH end Each replication results in shorter DNA! Can result in the deletion of genes Limiting factor in life span of cell Solution? Fig 15 16-20  Eukaryotic DNA is flanked by telomeres Why is this not a problem in prokaryotes? http://www.bluemaize.net/boots/red-boot-laces 16  Telomeres Telomeres are repeats of noncoding nucleotide sequences TTAGGG AATCCC Synthesized by the enzyme telomerase (RNA and protein) Telomerase is only active in germ cells and stem cells (not in somatic cells) Telomeres shorten as cells age! https://www.youtube.com/watch?v=2NS0jBPurWQ 17 Uzbas, F [CC BY-SA 3.0 (http://creativecommons.org/licenses/by-sa/3.0)], via Wikimedia Commons Telomeres can be visualized by fluorescence microscopy Fig 16.2118