_OBRIEN-5__DNA replication 39-48

_OBRIEN-5__DNA replication 39-48 - “CENTRAL DOGMA” DNA...

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Unformatted text preview: “CENTRAL DOGMA” DNA “MN RNA “w PROTEIN (0N3: POL WLICASE DNA MIL—A RNA _—9' PROTEIN REVERSE TRANSCRIPTASE N LI ’ 5 A. DNA: DOUBLE TEMPLATE MOLECULE 3 l. strands separate, each becoming template for assembly of a complementary strand RESULT: TWO IDENTICAL MOLECULES FROM ONE: ~ 2. Replication is “W” Meselson-stahl experiment confirmed basic hypothesis. Parental strands are conserved during subsequent rounds of replication. 3'7" 3. Base pairing rules completely dictate the sequence of the newly synthesized strand continuous discontinuous synthesis synthesis single stranded DNA template SI 3! ‘le L I “ L ” oli First enzyme isolated that catalyzes polymerization of deoxyribonucleotides. 1. Single polypeptide chain M; 109 KD; 1000 aa; 300-400/ce11 Has several different activities: a) Polymerase (5' 3'); ~ 10/sec b) 3' 5' exonuclease c) 5' 3' exonuclease ‘7‘! Growing strand: 9—»3' 3. Reaction requirements a DNA template b. 3 - OH primer; can be RNA or DNA c. dNTP’s and Mg2+ LIZ Nuclease activities of Pol I a. 3' -) 5' Exonuclease \ \rf‘fy‘, F‘F/‘P ' Attacks 3' end that is not C A T T G H-bonded to complementary \\ strand G T C mismatched ’ , t base pair Function: Proofreading Decreases copy rate error of polymerization by ~104 b. 5' -) 3' Exonuclease \ ,l/ \ “‘Pa‘rf‘rflr Function: C A T T G Primer removal .6 T A C ., ' \. f' \ \ 4 , .. ~ \er J (\Attacks 5' end that is H-bonded to complementary strand Removes successive nucleotides #3 5. Nick Translation Concerted 5' exonuclease and polymerase activities. Requires dNTP’s. (7 Kss nick has been “translated” six nucleotides on lower strand Functions: i_I_l vivo: primer removal in vitro: label any ds DNA 6. Other polymerases in E. Coli P0111 - error prone repair Pol III - major replication polymerase - lower abundance (1/40) than Pol I but polymerizes at higher rate (lOOO/sec) - 7 polypeptide subunits in holoenzyme ‘fo C. DNA LIGASES 1. Catalyze formation of phosphodiester bonds between adjacent DNA strands 2. Reaction _E. c li T4 phage and Animals 1 l Utilizes energy of NAD or ATP \ ' Mat/«w ~ CATTG EnzymezlwiNHf+ATPQ G T A A C H ,9 Enz-lys-N-O-P-O-Adenosine ’ I \ \ \ r a a $1wa a— rafl;;a [a ’ fl ’ ’ V ’ + Enz-lys-NH3 T A Nucleophilic attack of 3' OH on f activated P atom ' g 9 Mechanics of Replication 1. Initiation a. Starts from specific origin b. DNA synthesis may be uni-or bi directional origin :21: “uni” I A/\-§ unwinding *-\—————————' n it replication fork “bi” «- Al -) M \I/t replication forks Chain growth occurs on bpt_h parental strands at replication forks a. Composition of replication fork 1. Two locally unwound parental strands 2. Newly made Leading strand, continuous synthesis on the 3' -) 5' parental strand 3. Newly made Lagging strand, made on 5' -9 3' parental strand'in short fragments that become ligated later into one continuous strand #6 b. Replication fragments 5' 3' 1-2 kb Prok f 0.1-0.2" Euk RNA '\ DNA 10 - 60 nucleotides made by “primase” E. Enzymology of replication 1. P01 m, from RNA primer that is made during origin “activation” 2. Lagging strand (‘dna G’l a ’ : De novo synthesis of RNA primer DBP lagging strand 3' i9 % A 5' 3' 5' “If? -—r —7' RNA Primer b. Pol III Extends RNA primers into Okazaki fiagments 3' 5' 5' q— .7 .—7 v 0. P011 1) 3, l .. removes RNA primer, and 2) Bobmeraseactiyitx fills in gap with deoxynucleotides 3" i a, / d. Ligase joins DNA replication fragments F. Eukaryotic DNA replication 1. Pol erases Large, multisubunit structures 2. Multiple origins of replication Origins on same DNA molecule can be activated or deactivated as the developmental stage requires Example: Drosophila W—W supercoils 1. removed by TOPO ISOMERASES 2. Fusion of replication forks Replication faster if origins are activated closer to each other. 5‘8 ...
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