Molecular Biology Lec 3 PDF

 Continued… DNAis a directional molecule The complementary strands run in opposite directions ✓One strand runs 3’-5’ ✓The other strand runs 5’ to 3’ ( the end of the 5’ has...

 Continued… DNAis a directional molecule The complementary strands run in opposite directions ✓One strand runs 3’-5’ ✓The other strand runs 5’ to 3’ ( the end of the 5’ has the phosphates attached, while the 3’ end has a hydroxyl exposed) DNAhelix has two external grooves: ✓major groove (a deep & wide ) and ✓minor groove (a shallow & narrow ) Both of these grooves are large enough to allow protein molecules to come in contact with the bases. Polymorphism of DNAHelix DNAwas thought to have the same monotonous structure (for about 20yrs): ✓exactly 36º of helical twist between its adjacent base pairs ✓10 nucleotide pairs per helical turn and ✓uniform helix geometry  The polymorphism of DNAhelix can be classified into A-, B-, & Z-DNA(forms) based on: ✓number of residues (monomers) per turn (“n”) ✓ the spacing of residues along the helical axis (“h”). CONTINUED… D N A p o 1y n u c Ie o t:iide sll"' and CH 3 ', 0 C 4 - ' H / - o - - - P = 0 c?' - c \ N H b " T N - / c _ c ,.. c # I :!:c s · c e - NI - - -..... ·.:.. A · I s \ ,.. H ,C 0 / H / C -. ; N 0 0 = I o- P- l 0 \ HC = N, 'N - H c - c / -a - c \ N... A / 'i:L L - C / C H3 =c" \ - N \ T CH H "\ · c -.? r/ \ \ E.oC-c/ H C C \ N ·· N...._c/ tOe \ H H  Heterocatalytic function Autocatalytic functions ✓Directs the synthesis of other ✓Directs the synthesis of DNA molecules, RNAand protein itself Why does DNAself-replicate or autocatalytic? Because of: ✓ the specificity of base pairing, ✓ the sequence of base along one chain automatically determines (directs) the base sequence along the other. The mechanism: Strand separation, unwinding , followed by copying of each strand. Each separated strand acts as a template for the synthesis of a new complementary strand. DNAREPLICATION Models for DNAreplication 1) Semiconservative model Daughter DNA molecules contain one parental strand and one newly-replicated strand. 2) Conservative model Parent strands transfer information to an intermediate & the intermediate gets copied. The parent helix is conserved, the daughter helix is completely new 3) Dispersive model Parent helix is broken into fragments, dispersed, copied then assembled into two new helices. New and old DNA are completely dispersed.  ENZYMES OF DNAREPLICATION Three main enzymes act on DNAin both prokaryotic and eukaryotic cells are: ✓Nucleases ✓Polymerase ✓Ligase І. Nuclease enzymes ✓hydrolyze or break down a polynucleotide chain into its component nucleotides ✓will attack either the 3´ or the 5´ end of this linkage. ✓digest phosphodiester backbone There are two types nuclease enzymes:- exonuclease and endonuclease. a). Exonuclease ✓begin its attack from a free end of a polynucleotide ✓It begins at a free 3’-OH end or at a free 5'-Phosphate end (5'→3’) ✓It travels along the strand & digest the entire polymer ( liberates NMP) B). Endonuclease ✓digest phosphodiester bond within the interior of a polynucleotide chain ✓Single strand-cut the chain into two pieces, double helix, it forms nick  II) POLYMERASE / REPLICASE ENZYMES Catalyze the synthesis of one polynucleotide chain that is a copy of another. In vitro DNA polymerization Biologists understood the DNAreplication in living cells(in vivo) tried in vitro polymerization using the ff 3 molecules in addition to DNApolymerase: ✓deoxynucleotide triphosphates ( dATP, dCTP, dGTPand (d) TTP), ✓Primer: single-stranded DNA(ssDNA)/ RNA polynucleotide chains with free 3'-OH ends ❖Primers cannot initiate the de novo (the synthesis of complex molecules from simple molecules) synthesis of a new strand ✓DNAtemplate – strand that directs polymerization The DNApolymerase observe the rule of complementary base pairing. If a free 3'-OH on a primer strand lies opposite a thymine on a template strand: ✓a polymerase enzyme will add only an adenine group to the primer ✓Formation phosphodiester bond with a free 3'-OH ✓a molecule of pyrophosphate (P~P) is simultaneously released. The hydrolyzes of pyrophosphate into 2 phosphates release energy that drives polymerization. prlmor 5 tomplato 3 HO OH 5' 3' 5' Prokaryotic DNApolymerases Three d/t types of DNA polymerase in prokaryotes (E. coli) a). DNApolymerase I ✓was 1st isolated in 1960 byArthur Kornberg ✓suggested to be involved in DNAreplication ✓now considered to be a DNArepair rather than a replication enzyme ✓mainly involved in removing RNA primers from Okazaki fragments ✓5’ → 3’exonuclease activity & polymerization. b). DNApolymerase IІ - DNArepair enzyme. c). DNApolymerase IІІ - plays an essential role in DNAreplication ✓ a multimeric enzyme or holoenzyme having 10 subunits (alpha (α), beta (β), epsilon (ε), theta (θ), tau (τ), gamma (γ), delta (δ), delta dash (δ´ ), chi (χ), and psi (ψ) ✓The core enzyme comprises three subunits α, β and θ) ✓The seven subunits increase processivity (rapidity & efficiency) Eukaryotic DNApolymerases There are 5 types of eukaryotic DNApolymerases: ✓DNApolymerase α (alpha) - used as primase (formation of primer). ✓DNApolymerase β (beta) - concerned with DNArepair. ✓DNApolymerase γ (gamma) - mitochondrial polymerase and replication of DNAin mitochondria. ✓DNApolymerase δ (delta) –polymerization of leading strand and okazaki fragments ✓DNApolymerase ε ( epsilon) - DNArepair (III) DNA ligase Ligate (bind) a nick of DNA which is created by endonucleases enzyme / removal of primer. catalyzing phosphodiester bond formation between free 3´ -OH and free 5´ -P groups ligase enzyme from E.coli requires a cofactor oxidized nicotinamide adenine dinucleotide (NAD+)  Roles of RNAPrimers in DNA Replication DNApolymerase cannot initiate synthesis of DNAwithout the availability of a primer RNAstrand. primer RNAstrand - short RNAoligonucleotide segments synthesized by primase enzyme. RNAprimer is synthesized by copying a particular base sequence from one DNAstrand. Primer is about 10- 20 nucleotides.  DNAreplication in prokaryotes and eukaryotes is started in specific units called replicons (origin of replication). Prokaryotic cells (E. coli) has only one origin of replication – single genome Eukaryotes have many origin of replication (replicons). Eg. Yeast has 500 OriC. It is A- Trich regions. The termination of DNAreplication requires the product of tus gene is called ter binding protein (TBP) which recognizes termination sites. Table: prokaryotes and eukaryotes replicons REPLICONS PROTEINS INVOLVEDINOPENINGOFDNAHELIX The three types of proteins need to open and to expose DNAtemplate for the DNApolymerase: 1). DNA helicases ✓are ATP- dependent unwinding enzymes which promote separation of the two parental strands ✓establish replication forks that will progressively move away from the origin of replication. ✓DNAhelicases utilize ATPto move rapidly along a DNAsingle strand 2). Single strand DNA-bindingprotein ( SSBP) ✓it is also called Helix-destabilizing strand protein. ✓prevent from rewinding of the single DNAstrands/ duplexes ✓bind to exposed DNAstrands without covering the bases ✓It also prevents nuclease enzymes from attacking single strands. ✓coat and straighten out the regions of single-strand DNA 3). Topoisomerases (DNA gyrases) relax the supercoil by attaching to the transiently supercoil duplex → nicking one of the strands and rotating it through the unbroken strand → the nick is then resealed  D N A helicase b"inds  DNAtopoisomerase can be viewed as a ‘‘reversible nuclease’’ b/c: It holds energy released when phosphodiester bond is broken to seal the nick. Type of topoisomerase Topoisomerase I - a single-strand breaks or nick. Topoisomerase II – the double-strand break in the helix. Replisome and Primosome Replisome ✓The association of DNA polymerases and helicases at origin of replication. Why? ✓Ans:- increases the speed of helicase 10- fold, prevents helicase from “running away” from replication fork. ✓Called replication fork enzymes. ✓Important for the synthesis of leading and lagging strands in a coordinated fashion. CONTINUED… Primosome ✓the primase is linked directly to the DNA helicase at replication fork to form a unit on the lagging strand. ✓Activate primase 1000-fold (not tightly associated) ✓It moves with the fork, synthesizing RNAprimers as it moves  Replication complex in eukaryotes. The lagging strand is shown looped around the replication complex to demonstrate that all DNAsynthesis is in the 5’ to 3’ direction. Single-strand binding proteins (not shown) are bound to the unpaired, single-stranded DNA. Other proteins also participate in this complex  MECHANISM OF DNAREPLICATION IN PROKARYOTES In E. coli, the process of DNAreplication involves the following three main steps: ❖ Initiation of DNAreplication ❖Elongation of DNAchain ❖Termination of DNAreplication 1). Initiation of DNA replication Initiation comprises three steps: ❖recognition of the origin of replication (Ori C) ❖The specific sites at which DNAunwinding and initiation of replication occur are called origins of replication ✓A-Trich region ✓DnaAprotein (initiator protein) , ATPdependent protein, opening promoter the only protein that identify DNAsequence ❖DnaAprotein separate, “ melt” opening of DNAduplex to generate a region of single stranded DNA. ❖capture of DnaB (helicase): ✓unwinding of the DNAin the presence of ATP, SSB protein and DNA gyrase (topoisomerase) ✓ also acts as the activator of primase.  Continued… ✓SSB binding occurs on single stranded regions of the single strand DNA. ✓DNAcovered by single-strand binding proteins is rigid, without bends or kinks. ✓The junction between the newly separated template strands and the un- replicated duplex DNAis known as the replication fork.  During elongation , the following events occur: ✓Formation of a replication fork by opening the DNAduplex- DnaB (helicase). ✓The DNAstrand having helicase becomes the lagging strand, Primosomes- synthesizes multiple primers for lagging strand ✓single RNAprimer for the leading strand ✓The DNA poll III has to work on the lagging strand but it travels in opposite direction to which DnaB helicase is bound ✓Dna-B helicase, primase and DNApoll III work together in strand elongation 2.ELONGATION OF DNACHAIN NB. Synthesis (elongation) of lagging and leading strands takes place by somewhat different; it is far more complex for lagging strand than for the leading strand.  Discontinuous synthesis on lagging strand On the laggingstrand, Primase is taken up from solution and is activated by helicase (Dna-B)to synthesize a RNAprimer (10-20nucleotides long). DNApoll III on the laggingrecognizes RNAprimers on strand and synthesis okazaki fragments. On completion of the okazaki fragments, the RNAprimers are excised by DNA polymeraseI, which then fills the resulting gaps with DNA- gap left The enzyme DNAligase forms the phosphodiester bond that links the free 3´ end of the primer replacement of the 5´ end of the okazaki fragment. Continuous synthesis on leading strand The leading strand is primed once on each of the parental strands DNA poll III causes elongation of the leading strand Finally DNApol I and ligase enzymes give final touch to the leading strand as in case of the lagging strand.  Polymerase I l l a) Initiation· RNA Lagging strand , I /1 SSB(single-strand DNAbinding proteins) RNAprimer for 2nd Okazaki fragment made by DNAprimase primer madeby 5 DNAprimase DNAhelicase 3' starts replication 5' of lagging strand (synthesis of 1st Polymerase I l l Okazaki fragment) DNAsynthesized by DNApolymerase I l l Polymerase III dissociates Discontinuous synthesis on this strand 5 s· a b)Further untwisting , RNAprimer for 3rd Okazaki fragment and elongation of 3' new DNAstrands· 1st Okazaki 5' 2nd Okazaki fragment ---- Continued untwisting and fork movement fragment elongated  Polymerase I I I dissociates c) Process continues; 2nd Okazaki fragment finished 3rd being 3' 5' synthesized · DNA primase beginning 4th fragment d) Primer removed by 3' DNA polymerase I 5' 5' RNA primer being replaced with DNA by polymerase I e) Joining of adjacent 3' DNA fragments by 5' DNA ligase 5' 3' 3.Termination of DNAReplication DNA replication terminates when another replication fork or the telomere is reached. Termination is mediated by DNA binding proteins called Replication Terminator Proteins (TUS).