DNA Replication 2007-2008
Watson and Crick 1953 1953 article in Nature
Directionality of DNA You need to number the carbons! u it matters! u 3 refers to the 3 carbon on the sugar u 5 refers to the 5 carbon on the sugar. PO 4 CH 2 4ʹ nucleotide N base O ribose 1ʹ 2ʹ OH
The DNA backbone Putting the DNA backbone together u refer to sugar bonded to the phosphate groups. u The BASES are not the backbone. PO 4 CH 2 4ʹ O C O O P O O CH 2 O 4ʹ 2ʹ base 1ʹ base 1ʹ OH 2ʹ
Anti-parallel strands Nucleotides in DNA backbone are bonded from phosphate to sugar between & carbons u DNA molecule has direction u One strand is 5-3 while the other is 3 to 5 u Complementary strand runs in opposite direction u Strands held together with HYDROGEN BONDS.
Bonding in DNA hydrogen bonds covalent phosphodiester bonds.strong or weak bonds? How do the bonds fit the mechanism for copying DNA?
Base pairing in DNA Purines (2 rings) u adenine (A) u guanine (G) Pyrimidines (1 ring) u thymine (T) u cytosine (C) Pairing u A : T 2 bonds u C : G 3 bonds
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
Copying DNA Replication of DNA u base pairing allows each strand to serve as a template for a new strand u new strand is 1/2 parent template & 1/2 new DNA u This is called SEMI- CONSERVATIVE
DNA Replication Let s meet the team Large team of enzymes coordinates replication Speeds up using multiple origins
Slight differences in Prokaryotes
Replication: 1st step Unwind DNA with TOPOISOMERASE u Helicase enzyme splits apart the DNA helix (break H-bonds) Stabilized each piece using single-stranded binding proteins helicase single-stranded binding proteins replication fork
Replication: 2nd step Build daughter DNA strand u add new complementary bases using.. DNA polymerase III DNA Polymerase III But Where s the We re missing ENERGY something! for the bonding! What?
Energy of Replication Where does energy for bonding usually come from? You remember ATP! Are there other energy ways nucleotides? to get energy You out of bet! it? We come with our own energy! energy energy GTP TTP CTP ATP modified nucleotide And we leave behind a nucleotide! ADP AMP GMP TMP CMP
Energy of Replication The nucleotides arrive as nucleosides u DNA bases with P P P P-P-P = energy for bonding u DNA bases arrive with their own energy source for bonding u bonded by enzyme: DNA polymerase III ATP GTP TTP CTP
Replication Adding bases u can only add nucleotides to end of a growing DNA strand need a starter nucleotide to bond to u strand only grows B.Y.O. ENERGY! The energy rules the process energy DNA Polymerase III energy DNA Polymerase III energy DNA Polymerase III DNA Polymerase III energy
need primer bases to add on to no energy to bond energy û energy energy energy energy ligase energy energy
Okazaki Leading & Lagging strands Limits of DNA polymerase III u can only build onto end of an existing DNA strand Okazaki fragments ligase û Lagging strand Lagging strand u Okazaki fragments u joined by ligase growing replication fork spot welder enzyme DNA polymerase III Leading strand! Leading strand u continuous synthesis
Replication fork / Replication bubble DNA polymerase III leading strand lagging strand growing replication fork lagging strand leading strand leading strand lagging strand growing replication fork
Starting DNA synthesis: RNA primers Limits of DNA polymerase III u can only build onto end of an existing DNA strand growing replication fork DNA polymerase III primase RNA RNA primer u built by primase u serves as starter sequence for DNA polymerase III
Replacing RNA primers with DNA DNA polymerase I u removes sections of RNA primer and replaces with DNA nucleotides DNA polymerase I growing replication fork ligase RNA But DNA polymerase I still can only build onto end of an existing DNA strand
Chromosome erosion All DNA polymerases can only add to end of an existing DNA strand DNA polymerase I Houston, we have a problem! growing replication fork DNA polymerase III RNA Loss of bases at ends in every replication u chromosomes get shorter with each replication u limit to number of cell divisions?
Telomeres Repeating, non-coding sequences at the end of chromosomes = protective cap u limit to ~50 cell divisions growing replication fork telomerase Telomerase u enzyme extends telomeres u can add DNA bases at end u different level of activity in different cells high in stem cells & cancers -- Why? TTAAGGG TTAAGGG TTAAGGG
Replication fork DNA polymerase I DNA polymerase III Okazaki fragments lagging strand 5 ligase 3 5 primase 3 SSB 3 helicase 5 5 3 leading strand direction of replication DNA polymerase III SSB = single-stranded binding proteins
DNA polymerases DNA polymerase III u 1000 bases/second! u main DNA builder DNA polymerase I u 20 bases/second u editing, repair & primer removal DNA polymerase III enzyme Roger Kornberg 2006 Arthur Kornberg 1959
Editing & proofreading DNA 1000 bases/second = lots of typos! DNA polymerase I u proofreads & corrects typos u repairs mismatched bases u removes abnormal bases repairs damage throughout life u reduces error rate from 1 in 10,000 to 1 in 100 million bases
Fast & accurate! It takes E. coli <1 hour to copy 5 million base pairs in its single chromosome u divide to form 2 identical daughter cells Human cell copies its 6 billion bases & divide into daughter cells in only few hours u remarkably accurate u only ~1 error per 100 million bases u ~30 errors per cell cycle
What does it really look like? 1 2 4 3
Any Questions?? 2007-2008