DNA REPLICATION: Eukaryotes and Phages Paul D. Brown, PhD firstname.lastname@example.org BC21C: Molecular Biology I Replication in eukaryotes Mammalian DNA polymerases Eukaryotic Replication Machinery • Eukaryotic machinery is generally similar to that of E. coli. • Can get virus SV40 to replicate in vivo with 8 different purified components from mammalian cells. (SV40 virus, causes cancer, is model for eukaryotic replication) • Replication occurs bidirectionally with RNA primers. It is semidiscontinuous. • But two distinct DNA polymerases work at the growing fork, a and either d or e. The eukaryotic replication machinery is generally similar to that of E. coli Figure 12-12 1. The SV40 encoded protein T-antigen (T-ag) binds to the SV40 origin, melting it with its helicase activity; requires ATP. Local melting occurs then the hexameric form of T- ag binds, which has helicase activity. 2. It binds around both leading strands (NB there are two). 3. Replication protein A (RPA), a host cell SSB binds to the ssDNA. One molecule of DNA pol a, tightly bound to primase, binds to each template strand. Primases form the RNA templates, these are elongated for a short stretch by Pol a. These form the origins of the two leading strands. The activity of Pol a is stimulated by replication factor C (RFC). 4. Binding of PCNA (proliferating cell nuclear antigen) at the primer template 3’ termini displaces Pol a, this interrupts leading strand synthesis. 5. Pol d associates with PCNA, which increases the processivity of the enzyme and it makes the rest of the leading strand. PCNA is analogous to the b clamp of E. coli pol III. PCNA is a trimer, not a dimer. As T-ag helicase progresses along from the origin, primase-pol a complex associates with the melted template, downstream from the leading strand primers. Synthesis of the lagging strand is carried out by combination of primase, Pol a and RFC. Eukaryotic DNA polymerases: PRIMASE DNA Pol a(I):primase complex- bifunctional Heterotetrameric phosphoprotein 48 kDa PRIMASE- initiates DNA synthesis (makes complementary RNA primer) 58 kDa protein- tethers primase to 180 kDa subunit 180 kDa polymerase A subunit- extends RNA primer by making a short DNAi (3-4 bases) (only in Drosophila has proofreading activity 70 kDa subunit- no known catalytic function (may recruit pola:primase to the replication fork) Eukaryotic DNA Replication: 1. DNA pol a (I) + 2 primase subunits: initiate synthesis of lagging and leading strands. RNA- DNAi (3-4 b) primer. 2. DNA pol d (III) : elongates both lagging and leading strands. 3. PCNA: (proliferating cell nuclear antigen) acts as processivity factor for leading strand (like b clamp); elongation. 4. Replication factors C (RF-C; clamp loading, ATPase) and RF-A (single strand binding). 5. Topoisomerases I & II: maintains DNA winding. Similar functions at bacterial and mammalian replication forks: Function E. coli HeLa/SV40 •helicase DnaB T antigen •loading helicase DnaC T antigen • single strand SSB RF-A • priming DnaG Pol a(I)/2primases • sliding clamp b PCNA • clamp loading gd RF-C • catalysis Pol III core Pol d(III) • holoenzyme t ??? dimerization • RNA removal Pol I MF 1 exonuclease • ligation ligase ligase 1 Minimal oriC DnaA binding Region of melting L M R 1 2 3 4 13-mers 9-mers 245 bp GATCTNTTNTTTT TTATNCANA Note: GATC is Dam methylation site E. coli has a covalently closed circular chromosome 4 DNA polymerase complexes (2 per replication fork) at oriC Proceed in opposite directions and meet at opposite side. Circular daughter chromosomes are untangled Eukaryotic chromosomes are linear DNA primase can not start at an end Ends are called telomeres Repetitive sequence GGGGTTA Telomerase required to complete the ends DNA-RNA dependent DNA polymerase Rolling circle replication • Used by bacteriophage and Xenopus laevis. • This is applicable to circular DNA • Nick opens in ds DNA, 3’ end is extended, displacing the original parent strand. Rolling circle replication is commonly used by bacteriophages. Monomers of the genome are cleaved off and packaged. Phage fX174 has a ssDNA genome known as plus strand. When it replicates, the – strand is made to make the ds duplex. This replicated by the rolling circle mechanism. • When the duplex is made, it is covalently closed and it becomes supercoiled. The phage coded A protein nicks the + strand at the origin. The A protein remains attached to the 5’ end while the 3’ end is extended. The DNA can only be nicked when it is supercoiled. • The A protein binds to a ssDNA decamer at the site of this nick. An enzyme (relaxase) binds to supercoiled DNA, cleaves it and binds to the released 5’ end. The 3’ OH end of the nick is extended into a new chain. The chain is elongated around until it reaches the start point again. • The A protein stays with the rolling circle as well as to the 5’ end of the displaced tail as it passes, so it is able to recognise the origin again as it passes. When it does, the A protein nicks it, attaching to the new 5’ end. The A protein is involved in the circularisation process. 174 phage as a simple model for replication: Rolling “Lagging circle strand” replication synthesis (+) (+) + strand Replicative (+) form (RF): ds plasmid + strand packaged to form virion Rolling circle replication is a model for leading strand synthesis. 174 phage replication provides a model for DNA replication RF (randomly nicked) (+) SSB = = Rep (helicase) (+) (-) + (+) (-) Background for Rolling circle replication: Note: no DNA polymerase just to separate single strands Two kinds of activities are needed to convert double- stranded RF DNA to single-stranded DNA without synthesis of new DNA. a. Helicase: separates the strands using ATP to provide the energy. b. single-strand binding protein. (SSB). 174 as a simple model for replication DNA pol III elongates Rolling circle 3’-end of the nick. replication: SSB Gene A (+) (+) Rep (+) protein + (+) (-) (-) (-) (-) Proteins needed for rolling circle replication: a. “gene A” protein to nick at origin (pas). Covalently linked to 5’end of the displaced strand. b. SSB protein to keep DNA single-stranded. Binding is highly cooperative. c. Rep protein provides helicase function. d. DNA pol III holoenzyme How would a dsDNA phage replicate?
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