Replisome: The Complex of Proteins Involved in DNA Elongation, Study notes of Molecular biology

Download Replisome: The Complex of Proteins Involved in DNA Elongation and more Study notes Molecular biology in PDF only on Docsity! 1 Molecular Genetics PCB4522 Spring 2004 Lecture 2- Replication Dr. Eva Czarnecka-Verner Course web page: http://PCB4522.IFAS.UFL.EDU --Or go to Microbiology & Cell Science home page and look under course material. Chapter 13: DNA Replication (Chapter 15 in Gene VI &14 in VIII) Primosome: a protein complex that initiates synthesis of a DNA strand. Replisome: complex of proteins engaged in elongation of the newly synthesized DNA strand. Assembles at the replication fork. Identification of protein components involved in DNA synthesis 1. temperature sensitive mutants: conditional lethal mutants; replication at permissive conditions but fail to function at nonpermissive conditions (high temp.; 42°C). In E. coli identified loci- dna genes 2. dna genes: a. quick-stop mutants: immediate stop in replication; elongation enzymes defective & defects in precursors. b. slow-stop mutants: defective in reinitiation (smaller class). 2 Identification of protein components involved in DNA synthesis 3. in vitro complementation systems : combine extracts from mutant and wild-type strains. Can add back purified proteins to identify function of a specific dna gene product. Progress much slower in eukaryotes. DNA polymerases: enzymes that make DNA 1. Both bacteria and eukaryotes contain multiple DNA polymerases. 2. The ones that actually replicate the DNA are called “DNA replicases.” 3. All have the same type of synthetic activity: a) each can extend a DNA chain by adding nucleotides one at a time to a 3’ OH end b) the choice of dNTPs dictated by base pairing with the template strand DNA polymerases: enzymes that make DNA 5. Bacterial DNA replicases contain a large number of subunits (large protein assemblies). It is hard to say which proteins are actually subunits and which proteins are just loosely associated. 4. Some function as independent enzymes 5 Eukaryotic DNA polymerases: REPLICASE DNA polymerases δ (III) and ε (II)- 1) Heterodimeric 2) Need auxillary proteins: a) RF-C (replication factor C)- binds to RNA/DNA primer & stimulates assembly of pol- δ or pol- ε b) RF-C loads PCNA (processivity factor) onto DNA (PCNA binds to δ or ε & makes them stable on DNA 3) Intrinsic proofreading activities 4) Probably synthesize all cellular DNA DNA synthesis occurs by adding dNTPs to the 3’ end of the strand. 5’ 5’3’ 3’ OH 5’PPP 3’OH PP Choice of dNTPs: G-C ; A-T 5’ to 3’ 6. DNA synthesis has an extraordinary high fidelity: between 10-8 and 10-10 1 error per genome (4200 kb) per 1000 bacterial replications. Substitutions; frame shift 7. Proofreading function: all bacterial DNA polymerases have a 3’-5’ exonuclease activity. Operates in the reverse direction from synthesis. Processivity. 8. In proofreading the excised base is replaced by a different active site of the enzyme than the one used for the original synthesis. Expected error is:1 per 1000 bps replicated 6 Proofreading drastically reduces the errors made in replication Enzyme Synthetic domain Proofreading domain Error rate - proof. + proof. DNA pol I aa 200-600 N-terminal 10-5 5 x10-7 DNA pol III α subunit ε subunit 7 x10-6 5 x10-9 T4 DNA pol C-terminal N-terminal 5 x10-5 10-7 Rev. transcrip. none 10-5 1. easily cleaved into two fragments by proteinase: a. large fragment (Klenow fragment) contains polymerase and 3’-5’ exonuclease (proofreading domain). Used in vitro for synthesis reactions (DNA sequencing). E. coli DNA pol I (polA) N C Klenow fragment (68 kD) exonuclease 3’-5’ polymerase site small fragment (35 kD) C 5’-3’ proofreading exonuclease catalytic in vitro DNA synthesis using the Klenow fragment (DNA pol I) 5’ 5’3’ 32P Experimental Uses: a. Fill-in reaction to label recessed ends of DNA; EMSA, Northern, Southern b. DNA sequencing Unique ability to start replication at a nick in DNA * 7 Klenow fragment (DNA pol I) Catalytic domain of T7 DNA Polymerase Right hand structure= synthetic domain has 3 parts Thumb Fingers Palm Proofreading exo 3’-5’ dNTP Large cleft B DNA distorted A DNA 60o Inward rotation 8o Small fragment: has 5’ to 3’ exonuclease only. This activity allows pol I (intact) to be used for nick translation in vitro. DNA pol I (polA) N C Klenow fragment exonuclease 3’-5’ polymerase small fragment exonuclease 5’-3’ Proofreading domain 68 kDa 35 kDa Note: removes RNA primer & ~10 bp DNA 10 DNA synthesis is semidiscontinuous and primed by RNA The problem: DNA synthesis must always proceed from 5’ to 3’. As the replication fork moves, one of the template strands continuously exposes new “upstream” template. 3’ 5’ lagging strand leading strand 5’ 3’ 5’3’ leading strand: synthesis can proceed continuously in the 5’ to 3’ direction. lagging strand: synthesized in the reverse direction as a series of fragments which are later joined. Discontinuous synthesis. 3’ 5’ lagging strand leading strand 5’ 3’ 5’3’ Semidiscontinuous replication: The lagging strand fragments are known as “Okazaki fragments.” Usually 1,000 to 2,000 bases in length. 5’ lagging strand 3’ Semidiscontinuous replication: RNA primer (~11-12 bases) (RNA polymerase=dnaG primase) 5’-CTG-3” GAppp-5’ 1 2 11 The leading strand is also often isolated in fragments due to the misincorporation of UTP. Repair of the UTP leaves small gaps until they are filled in. (“pseudo-Okazaki fragments”) Semidiscontinuous replication: 3’ leading strand 5’ 3’ UTP gap Semidiscontinuous replication: Steps in lagging strand synthesis: a. synthesis of RNA primer. b. extension of Okazaki fragment (DNA). c. synthesis of next Okazaki fragment upstream of the last. d. removal of RNA primer (Who’s done it?) e. fill gap and seal (ligation) nick . DNA pol I (polA) starts synthesis at the nick between DNA and RNA and 5’-3’ exonuclease activity removes the RNA primer and replaces it with DNA. Semidiscontinuous replication: 3’ 5’ 3’ 3’ lagging strand 5’ 3’ nick DNA pol I nick translation RNA primer (1)(2) 12 DNA ligase: 1. Ligase-ATP complex formed. 2. The ATP changes to AMP as is releases pyrophosphate (PPi) and covalently attaches to the phosphate of the 5’ end of the DNA. 3. Original phosphate at the 5’ end covalently joins the OH of the 3’ end and AMP is released. NOTE: T4 phage ligase uses ATP. E. coli ligase uses NAD. 3’ 5’ 3’5’ -OH3’ O Ligation 3’ 5’ 3’ DNA ligase O-P-O O O -- -- 5’ P O O -- - -O 3’ -P-O O O -- -- -(NAD or ATP) T4 phage ligase uses ATP; E. coli ligase uses NAD. Ligase + AMP Ligase seals nicks in DNA Phosphodiester bond The end of lecture 2