Initiation Initiation of DNA replication means primer synthesis Different organisms use different mechanisms to make primers Different phages infect E. coli using quite different primer synthesis strategies Coliphages were convenient tools to probe DNA replication as they are so simple they must rely primarily on host proteins to replicate their DNAs
21-3 Priming in E. coli Primosome refers to collection of proteins needed to make primers for a given replicating DNA Primer synthesis in E. coli requires a primosome composed: –DNA helicase –DnaB –Primase, DnaG Primosome assembly at the origin of replication, oriC uses multi-step sequence
21-4 Priming at oriC Source: Adapted from DNA Replication, 2/e, (plate 15) by Arthur Kornberg and Tania Baker.
21-5 Origin of Replication in E. coli Primosome assembly at oriC occurs as follows: –DnaA binds to oriC at sites called dnaA boxes and cooperates with RNA polymerase and HU protein in melting a DNA region adjacent to leftmost dnaA box –DnaB binds to the open complex and facilitates binding of primase to complete the primosome –Primosome remains with replisome, repeatedly primes Okazaki fragment synthesis on lagging strand –DnaB has a helicase activity that unwinds DNA as the replisome progresses
21-6 Priming in Eukaryotes Eukaryotic replication is more complex than bacterial replication Complicating factors –Bigger size of eukaryotic genomes –Slower movement of replicating forks –Each chromosome must have multiple origins Started study with a simple monkey virus Later consider yeast
21-7 Origin of Replication in SV40 The SV40 origin of replication is adjacent to the viral transcription control region Initiation of replication depends on the viral large T antigen binding to: –Region within the 64-bp ori core –Two adjacent sites Exercises a helicase activity that opens up a replication bubble within the ori core Priming is carried out by a primase associated with host DNA polymerase
21-8 Origin of Replication in Yeast Yeast origins of replication are contained within autonomously replicating sequences (ARSs) These are composed of 4 important regions: –Region A is 15 bp long and contains an 11-bp consensus sequence highly conserved in ARSs –B1 and B2 –B3 may allow for an important DNA bend within ARS1
Elongation Once a primer is in place, real DNA synthesis can begin An elegant method of coordinating the synthesis of lagging and leading strands keep the pol III holoenzyme engaged with the template Replication can be highly processive and so very rapid
21-10 Speed of Replication The pol III holoenzyme synthesizes DNA at the rate of about 730 nt/sec in vitro The rate in vivo is almost 1000 nt/sec This enzyme is highly processive both in vitro and in vivo
21-11 The Pol III Holoenzyme and Processivity of Replication Pol III core alone is a very poor polymerase, after assembling 10 nt it falls off the template Takes about 1 minute to reassociate with the template and nascent DNA strand Something is missing from the core enzyme –The agent that confers processivity on holoenzyme allows it to remain engaged with the template –Processivity agent is a “sliding clamp”, the - subunit of the holoenzyme
21-12 The Role of the -Subunit Core plus the -subunit can replicate DNA processively at about 1,000 nt/sec –Dimer formed by -subunit is ring-shaped –Ring fits around DNA template –Interacts with -subunit of the core to tether the whole polymerase and template together Holoenzyme stays on its template with the - clamp Eukaryotic processivity factor, PCNA forms a trimer, also forms a ring that encircles DNA and holds DNA polymerase on the template
21-13 The Clamp Loader The -subunit needs help from the complex to load onto the DNA template –This complex acts catalytically in forming this processive complex –Does not remain associated with the complex during processive replication Clamp loading is an ATP-dependent process –Energy from ATP changes conformation of the loader so that -subunit binds to one of the -subunits of the clamp –This binding opens the clamp and allows it to encircle DNA
21-14 The Clamp and Loader Source: Adapted from Ellison, V. and B. Stillman, Opening of the clamp: An intimate view of an ATP-driven biological machine. Cell 106(2001), p. 657, f. 3. Source: Adapted from Henderson, D.R. and T.J. Kelly, DNA polymerase III: Running rings around the fork. Cell 84:6, 1996.
21-15 Lagging Strand Synthesis The pol III holoenzyme is double-headed There are 2 core polymerases attached through 2 -subunits to a complex –One core is responsible for continuous synthesis of the leading strand –Other core performs discontinuous synthesis of the lagging strand –The complex serves as a clamp loader to load the clamp onto a primed DNA template –After loading, clamp loses affinity for complex instead associating with core polymerase
21-16 Simultaneous Strand Synthesis The complex and clamp help core polymerase with processive synthesis of an Okazaki fragment When fragment completed, clamp loses affinity for core Associate clamp with complex which acts to unload clamp Now clamp recycles
21-17 Lagging Strand Replication Source: Adapted from Henderson, D.R. and T.J. Kelly, DNA polymerase III: Running rings around the fork. Cell 84:7, 1996.
Termination Termination of replication is straightforward for phage that produce long, linear concatemers Concatemer grows until genome-sized piece is snipped off and packaged into phage head Bacterial replication – 2 replication forks approach each other at the terminus region –Contains 22-bp terminator sites that bind specific proteins (terminus utilization substance, TUS) –Replicating forks enter terminus region and pause –Leaves 2 daughter duplexes entangled –Must separate or no cell division
21-19 Decatenation: Disentangling Daughter DNAs At the end of replication, circular bacterial chromosomes form catenanes that decatenated in a two step process –First, remaining unreplicated double-helical turns linking the two strands are melted –Repair synthesis fills in the gaps –Left with a right-handed parallel torus catenane with an even number nodes that is decatenated by topoisomerase IV Linear eukaryotic chromosomes also require decatenation during DNA replication
21-20 Termination in Eukarytoes Unlike bacteria, eukaryotes have a problem filling the gaps left when RNA primers are removed at the end of DNA replication If primer on each strand is removed, there is no way to fill in the gaps –DNA cannot be extended 3’ 5’ direction –No 3’-end is upstream –If no resolution, DNA strands would get shorter with each replication
21-21 Telomere Maintenance At the ends of eukaryotic chromosomes are special structures called telomeres One strand of telomeres is composed of tandem repeats of short, G-rich regions whose sequence varies from one species to another –G-rich telomere strand is made by enzyme telomerase –Telomerase contains a short RNA serving as template for telomere synthesis C-rich telomere strand is synthesized by ordinary RNA-primed DNA synthesis –This process is like lagging strand DNA replication This mechanism ensures that chromosome ends can be rebuilt and do not suffer shortening with each round of replication
21-22 Telomere Formation
21-23 Telomere Structure All eukaryotes protect their telomeres from nucleases and ds break repair enzymes Ciliates have TEBP to bind and protect the 3’-single-strand telomeric overhang Budding yeast has Cdc13p which recruits Stn1p and Ten1p that all bind ss telomeric DNA Mammals and fission yeast also have a protein similar to TEBP binding specifically to ss telomeric DNA
21-24 Mammalian Telomere Structure Mammalian telomeres form looped structures that protect ss telomeric DNA Proteins TRF1 and TRF2 appear to help telomeric DNA in mammalian cells form a t loop in which ss 3’-end of telomere invades the ds telomeric DNA upstream TRF1 may help bend the DNA into shape for strand invasion TRF2 binds at point of strand invasion, may stabilize the displacement loop