Replication Double helix structure of 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 You need to number the carbons! it matters! Directionality of 4 P 4 CH 2 ribose nucleotide N base 1 H 2 1
The backbone P 4 Putting the backbone together refer to the and ends of the the last trailing carbon base CH 2 4 C P base CH 2 4 2 1 1 H 2 Anti-parallel strands Nucleotides in backbone are bonded from phosphate to sugar between & carbons molecule has direction complementary strand runs in opposite direction Bonding in hydrogen bonds covalent phosphodiester bonds.strong or weak bonds? How do the bonds fit the mechanism for copying? 2
Base pairing in Purines adenine (A) guanine (G) Pyrimidines thymine (T) cytosine (C) Pairing A : T 2 bonds C : G 3 bonds Copying Replication of base pairing allows each strand to serve as a template for a new strand new strand is 1/2 parent template & 1/2 new semi-conservative copy process Replication Large team of enzymes coordinates replication 3
Replication: 1st step Unwind helicase enzyme unwinds part of helix stabilized by single-stranded binding proteins helicase single-stranded binding proteins Replication: 2nd step Build daughter strand add new complementary bases polymerase III But Where s the We re missing ENERGY something! for the bonding! What? Energy of Replication Where does for bonding usually come from? You remember ATP! Are there other ways nucleotides? to get You out of bet! it? We come with our own! ATP GTP TTP CTP modified nucleotide And we leave behind a nucleotide! ADP AMP GMP TMP CMP 4
Energy of Replication The nucleotides arrive as nucleosides bases with P P P P-P-P = for bonding bases arrive with their own source for bonding bonded by enzyme: polymerase III ATP GTP TTP CTP Replication Adding bases can only add nucleotides to end of a strand need a starter nucleotide to bond to strand only grows need primer bases to add on to no to bond ligase 5
Limits of polymerase III Leading & Lagging strands can only build onto end of an existing strand Lagging strand kazaki fragments joined by ligase spot welder enzyme ligase polymerase III kazaki Leading strand continuous synthesis Lagging strand Leading strand Replication fork / Replication bubble polymerase III leading strand lagging strand lagging strand leading strand leading strand lagging strand Starting synthesis: RNA primers Limits of polymerase III can only build onto end of an existing strand polymerase III primase RNA RNA primer built by primase serves as starter sequence for polymerase III 6
polymerase I Replacing RNA primers with removes sections of RNA primer and replaces with nucleotides polymerase I ligase RNA But polymerase I still can only build onto end of an existing strand Chromosome erosion All polymerases can only add to end of an existing strand polymerase I polymerase III RNA Loss of bases at ends in every replication chromosomes get shorter with each replication limit to number of cell divisions? Houston, we have a problem Repeating, non-coding sequences at the end of chromosomes = protective cap limit to ~50 cell divisions Telomeres telomerase Telomerase enzyme extends telomeres can add bases at end different level of activity in different cells high in stem cells & cancers -- Why? TTAAGGG TTAAGGG 7
polymerase I Replication fork polymerase III lagging strand kazaki primase fragments 5 ligase 3 5 SSB 3 helicase 3 5 5 3 leading strand direction of replication polymerase III SSB = single-stranded binding proteins polymerases polymerase III 1000 bases/second! main builder polymerase I 20 bases/second editing, repair & primer removal polymerase III enzyme Thomas Kornberg?? Arthur Kornberg 1959 Editing & proofreading 1000 bases/second = lots of typos! 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 8
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 remarkably accurate only ~1 error per 100 million bases ~30 errors per cell cycle What does it really look like? 1 2 4 3 9