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DNA replication Three models of DNA replication. 1 Most DNA replication is bidirectional Starts at the origin -defined sequence of base pairs Each region served by one DNA origin is called a replicon Some linear DNA viruses Certain plasmids Most common for eukaryotes and prokaryotes 2 Bidirectional DNA replication Bidirectional DNA replication in eukaryotes 3 Such studies have revealed clusters of active replicons Replicating mammalian cells were exposed first to high then to low concentration of H3Thymidine – DENA will be heavily labeled near the origin and lightly later. Most DNA replication is bidirectional Prokaryotic chromosomes have a single origin of replication with two replication forks Much larger eukaryotic chromosomes have many origins of replication Each region served by one DNA origin is called replicon. First evidence of bidirectional fork growth came from fiber autoradiography of labeled DNA molecules from mammalian cultured cells. Such studies revealed clusters of active replicons, each of which contain 2 growing forks moving away from a central origin. 4 Number of growing forks and their rate of movement In E. coli cells it takes 42 minutes to replicate the single circular chromosome that has 4 639 221 bp and is about 4.1mm in length. Since the chromosome is duplicated from one origin by two growing forks, we can calculate that the rate of the fork movement is about 1000bp/second/fork. Number of growing forks and their rate of movement The rate of fork movement in human cells, based on fiberlabeling experiments, is only about 100bp/second/fork. The entire human genome of 3 x 109 bp replicates in about 8 hours, suggesting that human genome might have about 1000 forks. However, fiber autoradiography and electron microscopy indicate that growing forks are spaced closer than 3 x 106 apart. A most likely estimate is that human genome contains 10 000100 000 replicons, each of which is actively replicating for only part of the 8 hours required for replication of the entire genome. 5 Replication bubbles DNA Replication Origin Replication origin = site on the DNA double helix where replication is initiated. •site where the double helix first opens ---> replication bubble. •consist of specific nucleotide sequences recognized by initiator proteins. oA-T rich (easier to separate) o100 bp (base pairs) in length 6 Number of replication origins •Prokaryotes o1 replication origin per chromosome oreplication rate = 500 nucleotides per sec. •Eukaryotes omultiple replication sites on each chromosome. oreplication rate = 50 nucleotides per sec. In eukaryotes replication origins are activated in clusters of 20 to 80 adjacent origins = replication units. The pattern of replication is controlled, temporally and spatially. DNA Replication Origin of E.coli E. coli replication origin oriC is an ≈240bp DNA segment present at the start site for the replication of the E. coli chromosomal DNA. Plasmids or any other circular DNAs containing oriC are capable of independent and controlled replication in E. coli cells. 7 DNA Replication Origin of E.coli 1 13 17 5’ GATCTNTT TATTT 29 GATCTNTT TATTT CTAGANAAATAAA 3’ CTAGANAAATAAA 58 32 44 GATCTNTT TATTT CTAGANAAATAAA 66 TGTGGATAA ACACCTATT 166 174 TTATACACA AATATGTGT 201 209 240 TTTGGATAA AAACCTATT 248 TTATCCACA AATAGGTGT 3’ 5’ Consensus sequence of the minimal bacterial replication origin based on analyses of genomes from six bacterial species 13 bp repetitive sequences are rich in A and T. The 9bp sequences exist in both orientations. These sequences are referred as 13-mers ans 9-mers. DNA Replication Origin of E.coli Cell wall Plasma membrane origins The origins of the replicated chromosomes have independent points of attachment to the membrane and thus move further apart as new membrane and cell wall forms midway along the length of the cell. 8 Yeast autonomously replicating sequences Each yeast chromosome has multiple origins of replication: about 400 origins exist on 17 chromosomes of S. cerevisiae. Each yeast origin, called autonomously replicating sequence (ARS), confers on a plasmid the ability to replicate in yeast and is a required element for YACs. Detailed mutational analysis of one ≈180 bp ARS called ARS1 revealed only one element, a 15-bp segment, designated element A, stretching from position 114 to 128. Three other short segments – elements B1, B2 and B3 – increase the efficiency of ARS functioning. Comparison of the sequences required for functioning of many different DNA segments that act as ARSs led to recognition of an 11-bp consensus sequence: (5’) A/T-T-T-T-A-T-A/G-T-T-T-A/T (3’) Element A in ARS1 is identical in 10 out of 11 positions of the consensus sequence, and element B2 – in 9 of 11. DNA footprinting revealed that 6 different proteins called the ORC (origin recognition complex) binds specifically to the element A in ARS1 in an ATP-dependant manner. This complex also binds to other ARSs. The ORC remains bound to an ARS throughout the cell cycle and during replication becomes associated with other proteins –this triggers DNA synthesis. Yeast mutants defective in any of the proteins of ORC are defective in DNA replication. 9 SV 40 origin of replication A 65-bp region in the SV40 chromosome is sufficient to promote DNA replication both in animal cells and in vitro. Researchers have used mammalian proteins and plasmids carrying the SV40 origin to study the molecular mechanisms of DNA replication. Common features of replication origins Although the specific nucleotide sequences of replication origins from E.coli, yeast, and SV40 are very different, they share several properties: Replication origins are unique DNA segments that contain multiple short repeated sequences. These short repeat units are recognized by multimeric originbinding proteins. These proteins play a key role in assembling DNA polymerases and other replication enzymes on the sites where replication begins. Origin regions usually contain an AT-rich stretch. Origin-binding proteins control initiation of DNA replication by directing the assembly of replication machinery to specific sites on the chromosome. 10 General features of chromosomal replication 1. The general features of chromosomal replication seem to apply with little modification to all types of cells. 2. DNA replication is semiconservative. 3. Once replication has started it continues until the entire genome has been duplicated. 4. It starts at origin. An origin “fires” ones and only ones during the cell cycle. 5. Replication is bidirectional. 6. At the place of the replication start (origin) helix unwinds and creates two replicational forks. The DNA replication machinery DNA polymerases are unable to melt duplex DNA (I.e. break certain hydrogen bonds) in order to separate strands that are to be copied All DNA polymerases so far discovered can only elongate a preexisting DNA or RNA strand, the primer; they can not initiate chains. The two strands in the DNA duplex are opposite (5’→3 and 3’ →5’) in chemical polarity, but all DNA polymerases catalyze nucleotide addition at the 3’hydroxyl end of a growing chain – only 5’→3 direction. 11 DnaA protein initiates replication in E.coli Genetic studies suggested that initiation of replication at oriC in E.coli is dependent upon protein coded by dnaA gene. DnaA protein binds with oriC. Although DnaA can bind to duplex E.coli origin DNA in the relaxed-circle form, it can initiate replication only when the DNA is negatively supercoiled. Supercoiling is controlled by enzymes called topoisomerases. Binding of DnaA to oriC 9-mers facilitates melting of duplex DNA, which occurs at oriC 13-mers. This process requires ATP and yields so called open complex. 12 DnaA protein initiates replication in E.coli 13 DnaB is a helicase that melts duplex DNA Helicases constitute a class of enzymes that can move along a DNA duplex utilizing the energy of ATP hydrolysis to separate the strands. SSB protein - binds ssDNA Helicases exhibit directionality with respect to unwinding reaction. DnaB moves along the single strand of DNA to which it binds in the direction of it’s free 3’ end – it unwinds DNA 5’→3’ direction. E. coli primase catalyzes formation of RNA primers for for DNA synthesis 14 E. coli primase catalyzes formation of RNA primers for for DNA synthesis The primers used during DNA replication in eukaryotes and prokaryotes are short RNA molecules whose synthesis is catalyzed by the RNA polymerase primase. Primase is usually recruited to a segment of single-stranded DNA by first binding to DnaB hexamer already attached at that site. The term primosome is now generally used to denote a complex between primase and helicase, sometimes with other proteins. In initiation of E. coli DNA replication, a primosome is formed by binding of primases to DnaB in prepriming complex. After bound primases synthesize short primer RNAs complementary to both strands of duplex DNA , they dissociate from the single stranded template. E. coli primase catalyzes formation of RNA primers for for DNA synthesis 15 Replication, Okazaki fragments 16 Ligation reaction: Polymerases DNA polymerases are important enzymes involved in DNA replication. Three polymerases have been purified from E.coli. In addition to important role in filling the gaps between Okazaki fragments, DNA polymerase I is the most important enzyme for gap filling during DNA repair. DNA polymerase II functions in the inducible SOS response; this polymerase fills the gap and appears to facilitate DNA synthesis directed by damaged templates. DNA polymerase III catalyzes chain elongation at the growing fork of E. coli. 17 DNA polymerase I 1957 – Arthur Kornberg isolated an enzyme (DNA polymerase I) from E. coli that was able to direct DNA synthesis in vitro. Major requirements for in vitro DNA synthesis were: 1. All four deoxyribonucleoside triphosphates (dATP, dCTP, dGTP, dTTP = dNTP). 2. Template DNA DNA Polymerases II and III 1969 – Peter DeLucia and John Cairns discovered a mutant strain of E. coli that was deficient in polymerase I activity. Observation: the mutant strain duplicated its DNA and reproduced itself but cells are highly deficient in DNA repair (UVsensitive). Conclusions: 1. At least one more enzyme is able to replicate E. coli DNA. 2. DNA polymerase I may serve a secondary (at least for replication) function which is associated with DNA fidelity. Two other unique DNA polymerases have been isolated 18 Properties of Three Bacterial DNA Polymerases Initiation of chain synthesis 5’-3’ polymerization 3’-5’ exonuclease activity 5’-3’ exonuclease activity Molecules of polymerase/cell Synthesis from Intact DNA Primed single strands Primed single strands plus SSB Protein In vitro chain elongation rate Mutation lethal? I + + + 400 II + + ? III + + 15 + - - + 600 + ? - + 30000 + DNA Polymerase III Holoenzyme The DNA polymerase III holoenzyme is a very large (>600 kDa), highly complexed protein composed of 10 different polypeptides. The so called core polymerase is composed of 3 subunits. The α subunit contains active site for nucleoride addition, and the ε subunit is a 3’-5’ exonuclease that removes incorrectly added (mispaired) nucleotides at the end of growing chain. The function of θ is still unknown. The central role of the remaining subunits is to convert the Polymerase III from distributive enzyme which falls the template after forming short stretches of 10-50 nucleotides to processive enzyme which can form stretches of up to 5 x 105 nucleotides before being released from the template. 19 DNA Polymerase III Holoenzyme The key to the processive activity of polymerase III is β subunit that forms a donut-shaped dimer around the DNA duplex and then associates with and holds the catalytic core polymerase near the 3’ terminus of growing strand. Once associated with DNA , the β subunit functions like a “clamp” which can slide freely along the DNA as the associated core polymerase moves. In this way active sites of core polymerase remain near the growing fork and the processivity of the enzyme is maximized. DNA Polymerase III Holoenzyme Out of the six remaining subunits 5 (γ,δ, δ1,χ and ψ) form socalled γ complex that mediates two essential tasks: 1) Loading of β subunit clamp onto the duplex DNA-primer substrate in a reaction that requires hydrolysis of ATP; 2) unloading of β subunit clamp after a strand of DNA has been completed. Loading and unloading of the β subunit clamp require opening of the clamp ring, but exactly how the γ complex does it is still unknown. The final τ subunit acts to dimerize two core polymerases and is essential to coordinate the synthesis of leading and lagging strands. 20 Subunits of DNA Polymerase III Holoenzyme Subunit Function α 5’-3’ ε polymerization 3’θ 5’ exonuclease γ ?? Loads enzyme on δ template (Serves δ’ as clamp loader) χ ψ β τ Groupings “Core” enzyme: Elongates polynucleotide chain and proofreads γ complex Sliding clamp structure (Processivity Factor) Holds together the two core polymerases at the replication fork Subunits of DNA Polymerase III Holoenzyme 21 Leading and lagging strands are synthesized concurrently Synthesis of leading and lagging strands 22 Cycling of polIII complex Replication fork in E. coli 23 Replication in eukaryotics is very similar to that in prokaryotic cells. Because eukaryotic cells have more DNA they also have more origins of replication. A mammalian cell for example has about 1 x 109 basepairs of DNA. There is an origin of replication about every 30,000 basepairs of DNA, though the structure of these sites is not clearly understood. The DNA synthesis is also much slower than in prokaryotic cells because of the chromatin proteins, synthesis is about 100 nucleotides per second. Mammalian DNA polymerases Much less is known about mammalian proteins involved in DNA replication. It had been thought that polymerase α synthesizes the lagging strand because of its low processivity. Polymerase δ is much more processive than α, as it is associated with the PCNA clamp. PCNA is enriched in proliferating cells and enhances the processivity of pol δ about 40 times. Pol β is not processive at all – it can do just 1 nucleotide – fits to its repair enzyme role. Pol γ if found only in mitochondria. 24 Probable roles of eukaryotic polymerases Polymerase α - priming replication on both strands Polymerase δ - elongation of both strands Polymerase β - DNA repair Polymerase ε - DNA repair Polymerase γ - replication of mitochodrial DNA Properties of mammalian DNA polymerases Mammalian polymerases 5’-3’ polymerization 3’-5’ exonuclease proofreading activity Synthesis from RNA primer DNA primer Associated DNA primase Sensitive to aphidicolin (inhibitor of cell DNA synthesis) Cell location Nuclei Mitochondria α + - β + - γ + + δ + + ε + + + + + + - + - + + + + + + - + - + + - + - 25 SV40 DNA can replicate in mammalian cells. Replication is initiated by binding of a virus-encoded protein called T-antigen to the SV 40 origin of replication. This multifunctional complex binding melts DNA through its helicase activity. Opening of the duplex at the SV40 origin also requires ATP and replication protein A (RPA), a host cell single stranded binding protein, with a function similar to that of SSB of E.coli. One molecule of polimerase α (Pol α) tightly associates with primase, then binds to each unwound template strand. Eukaryotic replication machinery is generally similar to that of E. coli The primases form RNA primers, which are elongated for a short stretch by Pol α, forming first leading strands, which grow from the origins in to different directions. The activity of Pol α is stimulated by replication factor C (RFC). 26 PCNA (proliferating cells nuclear antigen) then binds to the primer-template 3’ termini, displacing Pol α from both leading strand templates and thus interrupting leading strand synthesis. Next Pol δ binds to PCNA at the 3’ ends of the growing strands. The association of Pol δ with PCNA increases the processivity of the polymerase so that it can continue the synthesis of the leading strand without interruption. PCNA and DNA PCNA is a trimer that forms a “ clamp” around duplex DNA. 27 Eukaryotic replication machinery is generally similar to that of E. coli Thus the function of PCNA is highly analogous to that of the β subunit clamp of the E.coli polymerase III, as both proteins form rings around DNA. But, the amino acid sequences of them are different and β subunit clamp is a dimer and PCNA is a trimer. As melting of the duplex DNA, catalyzed by a hexameric form of Tag progresses further away from the origin, the primase-Pol α complex associates with melted template downstream from leading-strand primers. Synthesis of the lagging strand is then carried out by combined action primase and Pol α, along with RFC, Pol δ and PCNA while leading strand synthesis on the other side of the origin also proceeds. Finally, in eukaryotes as in E. coli topoisomerases play an important role in relieving torsional stress induced by growing fork movement and separating the strands. 28 A model for eukaryotic chromosome replication •Unwinding at origin of replication •DNA pol α-prim initiates DNA synthesis •PCNA, RF-C, pol δ, ε bind •Polymerase switch on lagging strand Termination of DNA Replication Several steps are involved in the termination of DNA replication: 1) Removal of RNA primers by DNA polymerase •When DNA polymerase encounters an RNA primer in its path, its proofreading mechanism recognizes that it is not DNA-DNA duplex and: oremoves the RNA primer, one ribonucleotide at a time (exonuclease activity) oinserts a deoxyribonucleotide, complementary to the base of the template strand. orepeats the process until all of the RNA is removed and replaced with DNA double strand. •This process occurs: oon the lagging strand, at the beginning of Okazaki fragments. oon the leading strand at the replication origin. 29 2) Closing the DNA-DNA gaps Problem: Removal of RNA primers by DNA polymerase leaves gaps between the 5'-P end of one nucleotide and the 3'-OH end of another. Therefore: There is no high energy phosphate bond to supply energy to close the gap with a phosphodiester bond. Solution: DNA ligase Unlike bacterial chromosomes, that are circular, eukaryotic chromosomes are linear and carry specialized ends called telomeres. Termination in prokaryotes Replication has a beginning and an end. In bacterial replication two forks approach each other in the terminus region – which contains 22-bp terminator sites that bind specific proteins. In E. coli the terminator sites are called TerA-TerF (E,D,A,C,B,F). They are the binding sites for the Tus proteins (terminus utilization substance). Sequences must be disentangled. 30 Termination in eukaryotes Telomeres – ends of eukaryotic chromosomes are composed of GC-rich sequences. The GC-rich strand of a telomere is added at the very 3’ end of DNA strands, in a semiconservative replication, by an enzyme telomerase. The exact repeat of telomere is species specific. In vertebrates, including humans, it is TTAGGG/AATCCC Telomerase add many repeated sequences at the ends of chromosome. Telomerase contains short RNA that serves as a template for telomere synthesis. Priming can then occur within these telomeres to make a C rich strand Telomeres and telomerase. 31 Forming of telomeres Mechanism of action of telomerase 32 Literature: Weaver, Molecular Biology, 2005. Brown, Genomes, 1999 Lodish et al. Molecular Cell Biology 2001 and 2006. Lewin, Genes VII. 33