AP Biology Synthesis of DNA June

AP Biology DNA synthesis occurs by the process of replication. During replication, each of the two parental strands of DNA serves as a template for the synthesis of a Complementary strand.

AP Biology Each molecule generated by the replication process contains one intact parental strand and one newly synthesized strand.

AP Biology In eukaryotes, DNA replication occurs during the S phase of the cell cycle The cell divides during the next phase (M), and each daughter cell receives an exact copy of the DNA of the parent cells.

AP Biology Cultured MDCK cells Day 1 Day 3

AP Biology Watson and Crick 1953 article in Nature

AP Biology Double helix structure of DNA “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

AP Biology Directionality of DNA  You need to number the carbons!  it matters! OH CH 2 O PO 4 N base ribose nucleotide This will be IMPORTANT!!

AP Biology The DNA backbone  Putting the DNA backbone together  refer to the 3 and 5 ends of the DNA  the last trailing carbon Sounds trivial, but … this will be IMPORTANT!! OH O 3 PO 4 base CH 2 O base O P O C O –O–O CH

AP Biology Anti-parallel strands  Nucleotides in DNA backbone are bonded from phosphate to sugar between 3 & 5 carbons  DNA molecule has “direction”  complementary strand runs in opposite direction

AP Biology Bonding in DNA ….strong or weak bonds? How do the bonds fit the mechanism for copying DNA? covalent phosphodiester bonds hydrogen bonds

AP Biology Base pairing in DNA  Purines  adenine (A)  guanine (G)  Pyrimidines  thymine (T)  cytosine (C)  Pairing  A : T  2 bonds  C : G  3 bonds

AP Biology

Copying DNA  Replication of DNA  base pairing allows each strand to serve as a template for a new strand  new strand is 1/2 parent template & 1/2 new DNA

AP Biology DNA Replication  Large team of enzymes coordinates replication Let ’ s meet the team …

AP Biology Replication: 1st step  Unwind DNA  helicase enzyme  unwinds part of DNA helix  stabilized by single-stranded binding proteins single-stranded binding proteins replication fork helicase

AP Biology Definitions  Template strands: The old strands which are now separated from each other.  New strands: The growing complementary nucleotide sequences that allow for forming the two double helix structures  Note that replication is direction specific.

AP Biology DNA Elongation  Works only 5’ to 3’  This means that the 5’ end of the nucleotide links on to the 3’ end of the DNA (or RNA primer)  The NEW strand only grows 5’ to 3’ OH O 3 PO 4 base CH 2 O base O P O C O –O–O CH

AP Biology DNA polymerase III RNA primer  built by primase  serves as starter sequence for DNA polymerase III Limits of DNA polymerase III  can only build onto 3 end of an existing DNA strand Starting DNA synthesis: RNA primers growing replication fork primase RNA

AP Biology Limits of DNA polymerase III  can only build onto 3 end of an existing DNA strand Leading & Lagging strands Leading strand Lagging strand Okazaki fragments ligase Okazaki Leading strand  continuous synthesis Lagging strand  Okazaki fragments  joined by ligase  “spot welder” enzyme DNA polymerase III  3 5 growing replication fork

AP Biology Replication fork 3’ 5’ 3’ 5’ 3’ 5’ helicase direction of replication SSB = single-stranded binding proteins primase DNA polymerase III DNA polymerase I ligase Okazaki fragments leading strand lagging strand SSB

AP Biology DNA polymerase III Replication fork / Replication bubble leading strand lagging strand leading strand lagging strand leading strand growing replication fork growing replication fork lagging strand 5 3

AP Biology DNA Polymerase III Replication: 2nd step But … We ’ re missing something! What? Where ’ s the ENERGY for the bonding!  Build daughter DNA strand  add new complementary bases  DNA polymerase III

AP Biology DNA polymerase I  removes sections of RNA primer and replaces with DNA nucleotides But DNA polymerase I still can only build onto 3 end of an existing DNA strand Replacing RNA primers with DNA growing replication fork DNA polymerase I RNA ligase

AP Biology Fast & accurate!  It takes E. coli <1 hour to copy 5 million base pairs in its single chromosome  divide to form 2 identical daughter cells  Human cell copies its 6 billion bases & divide into daughter cells in only few hours  remarkably accurate  only ~1 error per 100 million bases  ~30 errors per cell cycle

AP Biology What does it really look like?

AP Biology

energy ATP GTPTTPCTP Energy of Replication Where does energy for bonding usually come from? ADPAMPGMPTMPCMP modified nucleotide energy We come with our own energy! And we leave behind a nucleotide! You remember ATP! Are there other ways to get energy out of it? Are there other energy nucleotides? You bet!

AP Biology Energy of Replication  The nucleotides arrive as nucleosides  DNA bases with P–P–P  P-P-P = energy for bonding  DNA bases arrive with their own energy source for bonding  bonded by enzyme: DNA polymerase III ATPGTPTTPCTP

AP Biology  Adding bases  can only add nucleotides to 3 end of a growing DNA strand  need a “starter” nucleotide to bond to  strand only grows 5  3 DNA Polymerase III DNA Polymerase III DNA Polymerase III DNA Polymerase III energy Replication energy B.Y.O. ENERGY! The energy rules the process 5

AP Biology energy need “primer” bases to add on to energy 3 no energy to bond energy ligase 35 

AP Biology Loss of bases at 5 ends in every replication  chromosomes get shorter with each replication  limit to number of cell divisions? DNA polymerase III All DNA polymerases can only add to 3 end of an existing DNA strand Chromosome erosion growing replication fork DNA polymerase I RNA Houston, we have a problem!

AP Biology Repeating, non-coding sequences at the end of chromosomes = protective cap  limit to ~50 cell divisions Telomerase  enzyme extends telomeres  can add DNA bases at 5 end  different level of activity in different cells  high in stem cells & cancers -- Why? telomerase Telomeres growing replication fork TTAAGGG

AP Biology DNA polymerases  DNA polymerase III  1000 bases/second!  main DNA builder  DNA polymerase I  20 bases/second  editing, repair & primer removal DNA polymerase III enzyme Arthur Kornberg 1959 Roger Kornberg 2006

AP Biology Editing & proofreading DNA  1000 bases/second = lots of typos!  DNA polymerase I  proofreads & corrects typos  repairs mismatched bases  removes abnormal bases  repairs damage throughout life  reduces error rate from 1 in 10,000 to 1 in 100 million bases

AP Biology Any Questions??

AP Biology energy ATP GTPTTPATP Energy of Replication Where does energy for bonding usually come from? ADPAMPGMPTMPAMP modified nucleotide We come with our own energy! And we leave behind a nucleotide! You remember ATP! Are there other ways to get energy out of it?

AP Biology  Adding bases  can only add nucleotides to 3 end of the growing DNA strand  need a primer nucleotide to bond to  strand grows 5  3 DNA Polymerase III Replication energy B.Y.O. ENERGY! The energy rules the process 5

AP Biology no energy to bond 

AP Biology energy ligase

AP Biology Loss of bases at 5 ends in every replication  chromosomes get shorter with each replication  limit to number of cell divisions? DNA polymerase III DNA polymerases can only add to 3 end of an existing DNA strand Chromosome erosion growing replication fork DNA polymerase I Houston, we have a problem!

AP Biology Replication fork 3’ 5’ 3’ 5’ 3’ 5’ direction of replication

AP Biology DNA synthesis in prokaryotes 1.Replication is bidirectional. 2.Replication is semi-conservative.

AP Biology Bidirectional replication of a circular chromosome Replication begins at the point of origin (oriC) and proceeds in both directions at the same time.

AP Biology Unwinding of Parental Strands Topoisomerases: can break phosphodiester bonds and rejoin them relieve the supercoiling of the parental duplex caused by unwinding. DNA gyrase is a major topoisomerase in bacterial cells.

AP Biology 以上です。