DA Replication Smita Patel UMDJ-Robert Wood Johnson Medical School 1 DA replication DA, the genetic material of the living cell needs to be duplicated every time the cell divides 2 Chemical composition of DA DA (deoxyribose nucleic acid) is a polymer of repeating units of phosphate, sugar, and s 5 -end sugar phosphate s A, T, G, C Cytosine (C) pyrimidines H2 X H dx CH3 Thymine (T) H2 purines H 3 -end X Adenine (A) H2 X 3 Guanine (G) The screen versions of these slides have full details of copyright and acknowledgements 1
Chargaff s rule H2 H CH3 X Adenine (A) dx Thymine (T) Equal number of A and T as well as G and C H2 H A = T & G = C H2 X Guanine (G) X Cytosine (C) How is DA replicated? 4 DA structure Work of four scientists led to the discovery of the DA structure Francis Crick James Watson Maurice WilkinsRosalind Franklin Watson and Crick proposed a double helical DA structure. ature 1953 5 Watson and Crick DA model, 1953 DA structure Watson and Crick s DA double helix model Two DA chains coiled around an axis The strands are coiled in an antiparallel manner The s are facing the inside and the phosphates outside Each in the chain is bonded to a in the opposite chain 6 The screen versions of these slides have full details of copyright and acknowledgements 2
DA structure Adenine pairs with Thymine and Guanine pairs with Cytosine Explains Chargaff s rule of DA pairing Equal number of A and T as well as G and C The double helical structure provided the mechanism of DA replication 7 Base pairing specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material Watson and Crick, 1953 Each DA chain serves as a template for the synthesis of the complementary strand 8 Semiconservative DA replication Meselson and Stahl, 1958 Daughter DAs contain one new (blue) and one old (red) DA chain Parent DA Daughter DAs 9 The screen versions of these slides have full details of copyright and acknowledgements 3
DA polymerase DA polymerase (an enzyme that makes DA) was discovered, Arthur Kornberg, 1958 DA polymerase elongates a primer in the 5-3 direction using dtps as substrates 5 3 (DA chain) n-h + dtp (DA chain) n+1 + PPi The sequence of the newly synthesized DA is dictated by the template strand All known polymerases add dmp to the 3 -H of the ribose of the DA chain 10 Mechanism of DA replication Problem: how are the two DA chains replicated simultaneously? 11 Semidiscontinuous DA replication Reiji kazaki, 1968 ne strand (leading strand) is synthesi zed continuously The complementary strand (lagging strand) is synthesized discontinuousl y kazaki fragments DA is synthesized in fragments that are ligated later on Leading strand DA synthesis Lagging strand DA synthesis 12 The screen versions of these slides have full details of copyright and acknowledgements 4
Minimal requirements Minimally, what do you need to replicate a double helix by the polymerase Template for the polymerase Primer for the polymerase dtps to elongate the primer 13 DA replication is catalyzed by the cooperative action of a number of proteins: DA polymerase: synthesizes the complementar y strands Helicase: separates the strands of the double stranded DA Primase: synthesizes short RA primers for kazaki fragment synthesis Single stranded DA binding protein: activates DA replication Processivity factor DA Polymerase Leading strand RA Primer Primase kazaki fragment SSB Lagging strand 14 What we know The DA replication mechanism is conserved in all living organisms Replication proteins from many organisms have been isolated, purified, and studied individually in great detail DA replication can be reconstituted in a test tube using phage or bacterial proteins The replication proteins coordinate their action, and current and future efforts are toward understanding the nature of this coordination 15 The screen versions of these slides have full details of copyright and acknowledgements 5
DA polymerase structure Klenow fragment of DA polymerase I, Thomas Steitz, 1985 Right hand, thumb, fingers, and palm Structures of DA polymerases are conserv ed Finger domain: template and dtp interactions Palm domain: phosphoryl transfer reaction Thumb domain: processiv ity, translocation llis et al., ature (1985) 313, 762 Klenow fragment 16 Structure of phage T7 DA polymerase in the act of adding dmp Polymerase domain: thumb, finger and palm domains bind DA and catalyze phosphoryl transfer Doublie et al., ature (1998) 391, 251 17 Chemical reaction catalyzed by DA polymerases Attack of the 3 -H of the primer on the alpha-beta phosphate bond of a correctly paired dtp results in the DA chain elongated by one nucleotide and PPi is released primer strand primer strand - adjascent attack as drawn P P - - Mg 2+ : H P - template strand - P template strand H : H : + - P P - - - Mg 2+ 18 The screen versions of these slides have full details of copyright and acknowledgements 6
Metal ions play an essential role in DA synthesis Two metal ion catalysis Both metals play a role in transition state stabilization Metal B is coordinated to the alpha beta phosphate of dtp Metal A activ ates the 3 -H of the primer Doublie et al., ature (1998) 391, 251 19 DA synthesis has to be efficient and accurate DA polymerase has to choose one out of four dtps at each cycle of nucleotide incorporation 5 3 T C T G C T T C G G A C A T C G A G A G T T C A C T G T C T G G A C A T C A G A G T T C (DA chain) n-h + dtp (DA chain) n+1 + PPi 20 Rates of correct and incorrect nucleotide addition C T G 5 3 T C T T C G C G A A T C G A G A G T T C A 0.03-0.7 s -1 300 s -1 E.DA n E.DA n+1 C T G T C T G G A 300 s -1 C A T C A G A G T T C Correct addition G A C C T G T C G G A A 0.03-0.7 s -1 C A T C A G A G T T C Incorrect addition K d,dtp 20 mm 8 mm k pol/k d 2 x 10 7 M -1 s -1 4-90 M -1 s -1 Patel et al., Biochemistry (1991) 30, 511 21 The screen versions of these slides have full details of copyright and acknowledgements 7
φ= Where φ is fidelity, subscript c, correct product, subscript i is incorrect products Error frequency = 1/φ Fidelity ( kcat / Km) c+ ( kcat / Km) i ( kcat / Km) i = 1 error in 10 6 to 10 7 nucleotides added Wong et al., Biochemistry (1991) 30, 526 22 Correct nucleotide after an incorrect addition is also very slow C T G T C G G A A C A T C A G A G T T C 3 -mismatched 0.01 s -1 C T G T C C G G A A C A T C A G A G T T C correct addition after a mismatch Wong et al., Biochemistry (1991) 30, 526 23 How are errors corrected? Proofreading activity of T7 DA polymerase Exonuclease domain: Increases the accuracy of DA synthesis by excising mismatched nucleotides from the 3 -end Doublie et al., ature (1998) 391, 251 24 The screen versions of these slides have full details of copyright and acknowledgements 8
Fidelity: exonuclease activity Freemont et al., PAS (1988) 85, 8924 The 3 -end of the primer shuttles between the polymerase and exonuclease active sites 25 Fidelity of DA synthesis C T T C G C G A A T C G A G A G 0.03-0.7 s -1 0.01s -1 T C T G T C C G T C G G A A C G G A A T T C C A T C A G A G T T C C A T C A G AG T T C 3 -mismatched correct addition after a mismatch 2 s -1 A C T G T C G G A C A T C A G A G T T C 3 -mismatched excision 300 s -1 Correct nucleotide addition Donlin et al., Biochemistry (1991) 30, 538 verall fidelity: 1 mistake in 10 9 to 10 10 nucleotides added 26 Processivity of the DA polymerase Processivity factor thioredoxin : Increases the efficiency of DA synthesis by helping the DA polymerase stay on track Doublie et al., ature (1998) 391, 251 27 The screen versions of these slides have full details of copyright and acknowledgements 9
Processivity factors Many processiv ity factors are ring-shaped and bind DA in their central channel and help the DA polymerase stay on track Irina Bruck and Mike 'Donnell Genome Biology 2001 28 DA unwinding What you minimally need to replicate a double helix by the polymerase Template for the polymerase 29 DA unwinding: helicase protein Double helical DA strands are separated by the helicase protein helicase Helicase acts at the junction of single and double stranded DA Leading strand DA synthesis Lagging strand DA synthesis 30 The screen versions of these slides have full details of copyright and acknowledgements 10
Activities of the helicase protein Helicase uses the energy of TP hydrolysis to separate the strands of DA TP DP+Pi 31 Ring-shaped helicases replicate the genomes of phages to human Bacteriophage E. col i bacte ri ophage T7 gp4 E. coli bacteriophage T4 gp41 B. subti lis phage SPP1 gene 40 Bacterial E. coli DnaB E. coli RuvB E. coli rho Viral Simi an virus large T antigen Bovi ne papill omavirus E1 Plasmid Archeae Eukaryotic Plasmid-encoded RSF1010 Rep A M. thermoautotrophi cum MCM Human Bloom's Human MCM4,6,7 syndrome helicase Patel & Picha, Annu. Rev. Biochem. (2000) 69, 651 32 Mechanism of double helical DA strand separation Helicase encircles one strand of the DA and excludes the other strand Helicase separates the strands of the double helical DA using force produced through unidirectional translocation - Wedge mechanism Speed of the helicase depends on the stability of the duplex DA and ranges from 2 to 30 bp unwound per second 33 The screen versions of these slides have full details of copyright and acknowledgements 11
Mechanism of ATPase and DA translocation Crystal structure of helicase ring Sequential hydrolysis of ATP and DA translocation Singleton et al., Cell (2000) 101, 589 34 Liao et al., JMB (2005) 350, 452 Helicase functions in conjunction with the DA polymerase DA polymerase increases the speed of the helicase and vice versa Leading strand synthesis Stano et al., ature (2005) 435, 370 35 Synergy between the replication proteins in catalyzing DA replication 2-30 bp/s DA unwinding 0 bp/s 5 Helicase DA polymerase 110 bp/s 36 The screen versions of these slides have full details of copyright and acknowledgements 12
Primers for DA replication Helicase action provides the template for the polymerase Primers for the polymerase provided by the primase 37 Properties of the DA primase Primases recognize specific sequences on single stranded DA Using the specific sequence as a template, the primase synthesizes short RA primers with TPs as substrates The primase remains associated with the newly synthesized short RA primer until the polymerase takes ov er and elongates the primer into an kazaki fragment 38 DA primase protein Primase activity is dependent on the helicase activity that makes single stranded DA ften the primase protein is associated with the helicase Kusakabe et al., EMB J. (1998) 17, 1542 39 The screen versions of these slides have full details of copyright and acknowledgements 13
DA replicase Replication of double stranded DA requires the cooperative action of a number of proteins Processivity factor DA Polymerase Leading strand RA Primer Primase kazaki fragment SSB Lagging strand 40 Many questions remain.. How is leading and lagging strand DA synthesis coordinated? How is the DA primer handed over to the polymerase for kazaki fragment synthesis? What happens when the replication machinery encounters a damage in the DA? How are the mistakes made during replication corrected? Leading strand synthesis 41 42 The screen versions of these slides have full details of copyright and acknowledgements 14