DNA Replication – Process Lecture 17 1

Forms of DNA Helices 2 DNA Replication Template DNA (parent DNA) Replication of parent DNA can theoretically generate 2 possible conformations of daughter DNA S Phase M Phase

Forms of DNA Helices 3 DNA Replication Design an experiment to differentiate these two possibilities DNA is unique among biomolecules due to very high concentration of Phosphorus Grow cells in P32 enhanced media 32 P labeled DNA Single cell cycle in unenhanced media

Forms of DNA Helices 4 DNA Replication How do bacteria go about replicating DNA? Linearize DNA for replication? Keep DNA circular? The experiment: Grow cells in 3 H-thymidine enhanced media

Forms of DNA Helices 5 DNA Replication Where does replication start and is it uni or bidirectional? Heavy staining on both sides of the replication eye indicates that replication is almost always bidirectional in bacteria. Bacterial replication also starts at the same spot indicating a single origin or replication

Forms of DNA Helices 6 Dealing with DNA Superstructure What enzyme is capable of modifying DNA topology? Topoisomerase (DNA Gyrase) Parent DNA must be unwound at the replication fork For this to occur at biological rates (1000 nt per second for E. coli), genomic DNA must twist at a rate of 100 rps. E. coli’s genome is circular, so this is not possible!

Forms of DNA Helices 7 Semi-discontinuous Replication DNA’s 2 strands are replicated at the same time DNA Polymerase synthesize DNA in the 5’  3’ direction So how does the other strand elongate? 5’ 3’ The ‘Lagging Strand’ is synthesized as small fragments (1000 – 2000 nuclotides in bacteria or nt in eukaryotes) called Okazaki fragments. Okazaki fragments are combined by a DNA Ligase

Forms of DNA Helices 8 DNA Replication How will a dNTP be added to the existing chain? How might the reaction be activated?

Forms of DNA Helices 9 Requirement for priming This model of DNA replication requires a 3’ OH for strand elongation Nucleophile 3’ 5’ Analysis of Okazaki fragments indicated that the 5’ end was always composed of RNA 1  60 nucleotides in length These will have to be replaced at some point

Forms of DNA Helices 10 Enzyme Requirement for Replication DNA Polymerase Catalyze the DNA chain elongation using the ‘parent’ strand as a template RNA Primer Synthesis RNA Polymerase 460 kDa (E. coli) catalyzes primer synthesis for leading strand only Primase 60 kDa Responsible for priming Okazaki fragment Works synergistically with RNA Polymerase to prime leading strand Topoisomerase (Gyrase) Unwind supercoiled template DNA Energy dependent process SSB Binds to ssDNA and prevents reannealing Ligase Join Okazaki fragments

Forms of DNA Helices 11 Formation of the Replication Fork 1.4 DnaA proteins bind to the oriC region (recognizes 9 nucleotide segments) 1.Additional DnaA monomers bind forming a histone like complex of tightly wound DNA 2.Localized melting of a 13bp repeats 2.DnaB (6 subunit helicase) binds and unwinds the DNA 1.Topoisomerase functions downstream to relieve stress 3.Single Strand DNA binding proteins prevent the ssDNA from reannealing

Forms of DNA Helices 12 Formation of the Replication Fork DnaA helical filament – 8 monomers per turn 178 Å dsDNA wraps around the filament This increase Writhing number? Enables some untwisting? L = W + t

Forms of DNA Helices 13 The Big Picture (in E.coli) Topoisomerase: relieves topological strain Helicase - unwinds dsDNA at replication fork Primase: synthesizes RNA primers for lagging strand DNA Polymerase III : elongate primed DNA from 5’  3’ 3’  5’ exonuclease activity limits errors Pol III elongates lagging strand until strained. It then releases the template and rebinds at a new primed location. SSB: keeps ssDNA from reannealing Pol I: closes any gaps in lagging strand and replaces RNA primer Ligase: seals off backbone nicks

Forms of DNA Helices 14 DNA Polymerase What do we need in a DNA polymerase? Template DNA binding site Single Strand or Double Strand? Room for growing dsDNA dNTP binding site Mechanism to differentiate between the 4 possible dNTPs Self Correcting?

Forms of DNA Helices E. coli DNA Polymerase I Palm Domain ~22 Å x 30 Å Ideal shape to bind B-DNA Lined with basic amino acids Thumb Guides newly formed DNA Responsible for processivity Pol I 3’  5’ and 5’  3’ exonuclease activity Small N-terminal domain contains the 5’  3’ exonuclease activity Completely independent from active site or 3’  5 site Fingers Contains dNTP binding site Close in on dNTP/Growing Strand once successful Watson- Crick base pairing made

Forms of DNA Helices 16 Taq DNA Polymerase Projected path of ssDNA template 5’ Thumb folds down over elongating dsDNA preventing it from escaping Initial inspection of the groove between the thumb and fingers suggests the DNA would follow this cleft. This is NOT the case! Active Site Growing Strand Template 3 base pair closest to active site are A-Form DNA

Projected path of ssDNA template 5’Active Site Growing Strand Template Forms of DNA Helices Taq DNA Polymerase Thumb folds down over elongating dsDNA preventing it from escaping Initial inspection of the groove between the thumb and fingers suggests the DNA would follow this cleft. This is NOT the case!

Forms of DNA Helices 18 Taq DNA Polymerase Projected path of ssDNA template dNTP Binding Site – allows for rapid sampling of the dNTPs Thumb Knuckles?

Forms of DNA Helices 19 Taq DNA Polymerase Growing Strand Template Watson-Crick Base Pair between Template and incoming dNTP at active Mg situated for activation of 3’OH DNA polymerases apparently select incoming dNTP based on SHAPE of Watson-Crick base pair (Purine-Pyrimidine) 5’ end of growing strand is 2’-3’-dideoxy CTP Only one combination will provide most favorable pair

Forms of DNA Helices 20 3’  5’ Exonuclease The structure of E. coli Pol I has been solved with DNA arranged in the 3’  5’ exonuclease active site Growing strand peels away from active site The base pair closest to the polymerization active site is significantly weakened Exonuclease site (Zn 2+ activated mechanism) Which bond will be cleaved? NEED 3’-hydroxyl for elongation

Forms of DNA Helices 21 Model for Proofreading in DNA Polymerases dNTPs are in rapid equilibrium at active site When reaction occurs, proper base pair will form a strong tight bonding pair When an error occurs, the base pair is not as tight, resulting in increased favorability for the growing chain to be positioned at the 3’  5’ exonuclease site A-form DNA of polymerization active site makes this process easier (less tightly wrapped and less stable helix)

Forms of DNA Helices 22 5’  3’ Exonuclease Pol I from Thermus aquiticus lacks 3’  5’ exonuclease activity N-terminal domain contains 5’  3’ exonuclease Taq polymerase can translate a single nick in DNA by a mechanism that involves both the polymerase active site and the 5’  3’ exonuclease active site

Forms of DNA Helices 23 Taq DNA Polymerase Exonuclease What process in DNA replication requires a 5’  3’ exonuclease process? Need to remove RNA primers from lagging strand!

Forms of DNA Helices 24 DNA Ligase Needed to seal the phosphodiester backbone following removal of the RNA primer by Pol I Energy Dependent reaction Hydrolysis of ATP or NADH

Forms of DNA Helices 25 DNA Ligase pdbID 2OWO

Forms of DNA Helices 26 DNA Ligase AMP covalently bound to active site pdbID 2OWO AMP phospoamide intermediate with Lysine shown biochemically

Forms of DNA Helices 27 DNA Ligase Reaction AMP phospoamide intermediate forms with Lys

Forms of DNA Helices 28 DNA Ligase Covalent Lys-AMP intermediate displaced by 5’ Phosphate of nicked DNA

Forms of DNA Helices 29 DNA Ligase 3’ OH attacks 5’ Phosphate displacing the AMP

Forms of DNA Helices 30 DNA Helicase Helicase functions as a hexamer Each subunit has a  fold Forms a clamp around the DNA that slides and unwinds dsDNA Unwinds dsDNA by ‘active rolling’ mechanism

Forms of DNA Helices 31 DNA Primase Primase must be able to oppose the direction of translocation Forms a non-covalent bond with DnaB (helicase) Synthesizes primers ~11 nt in vivo but ~60 in vitro Basic groove nonspecifically binds to ssDNA and directs it to the active site

Forms of DNA Helices 32 Single Stranded DNA Binding Protein SSB binding to DNA prevents: reannealing Stem loop structures (self complimentary sequences) Nuclease degradation Very basic proteins Bind to ssDNA with no sequence specificity Binds as a tetramer Can bind in a number of different conformations

Forms of DNA Helices 33 Replication Termination There are multiple termination elements in E. coli’s genome. Each are similar sequences long and promote binding of TUS proteins Consensus Sequence: ANNTAGTATGTTGTAACTA Clockwise moving replication forks pass through TerE, TerD and TerA but stop at TerC CCW moving replication forks pass through TerG, TerF, TerB and TerC but stop at TerA This verifies that each replication fork will finish at same place in genome Makes specific contacts with DnaB – termination is dependent on this interaction