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DNA Replication

DNA Replication . Lecture 7 20/02/2011. What Is DNA Replication. DNA Replication is the process in which the DNA within a cell makes an exact copy of itself. Why does DNA replicate? During which phase of the cell cycle does DNA replicate?. DNA Replication.

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DNA Replication

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  1. DNA Replication Lecture 7 20/02/2011

  2. What Is DNA Replication • DNA Replication is the process in which the DNA within a cell makes an exact copy of itself. • Why does DNA replicate? • During which phase of the cell cycle does DNA replicate?

  3. DNA Replication Replication occurs during Interphase Replication fork Replication fork Replication bubble Hydrogen bond DNA replication is the process where an entire double-stranded DNA is copied to produce a second, identical DNA double helix. Why does DNA need to copy? Simple: Cells divide for an organism to grow or reproduce, every new cell needs a copy of the DNA or instructions to know how to be a cell. DNA replicates right before a cell divides.

  4. DNA Replication • From the three-dimensional structure of DNA that Watson and Crick proposed in 1953, several important genetic implications were immediately apparent. • The complementary nature of the two nucleotide strands in a DNA molecule suggested that, during replication, each strand can serve as a template for the synthesis of a new strand. • The specificity of base pairing (adenine with thymine; guanine with cytosine) implied that only one sequence of bases can be specified by each template, and so two DNA molecules built on the pair of templates will be identical with the original. • This process is called semiconservative replication, because each of the original nucleotide strands remains intact (conserved), despite no longer being combined in the same molecule; the original DNA molecule is half (semi) conserved during replication.

  5. The Three Possible DNA Replication Models • Conservative- would leave the original strand intact and copy it. • Dispersive-would produce two DNA molecule with sections of both old and new along each strand. • Semiconservative –would produce DNA molecule with both one old strand and one new strand.

  6. Meselson and Stahl convincingly demonstrated that replication in E. coli is semiconservative.

  7. Requirements of Replication • Although the process of replication includes many components, they can be combined into three major groups: 1. a template consisting of single-stranded DNA, 2. raw materials (substrates) to be assembled into a new nucleotide strand, and 3. enzymes and other proteins that “read” the template and assemble the substrates into a DNA molecule.

  8. Bacterial DNA replication • Bacterial DNA ( chromosome) replication is stepwise process in which many cellular proteins participate. • Theta replication A common type of replication that takes place in circular DNA, such as that found in E. coli and other bacteria, is called theta replication. • Bacterial chromosomes contain a single origin of replication • Origin of replication is the place in which the DNA synthesis begins • The replication is bidirectional (in both direction) around the bacterial chromosome. • Two replication forks move in opposite directions outward from the origin • The two replication forks meet each other to complete the replication process.

  9. Steps of theta replication • In theta replication, double-stranded DNA begins to unwind at the replication origin, producing single-stranded nucleotide strands that then serve as templates on which new DNA can be synthesized. • The unwinding of the double helix generates a loop, termed a replication bubble. • The point of unwinding, where the two single nucleotide strands separate from the double-stranded DNA helix, is called a replication fork. • If there are two replication forks, one at each end of the replication bubble, the forks proceed outward in both directions in a process called bidirectional replication, simultaneously unwinding and replicating the DNA until they eventually meet. • If a single replication fork is present, it proceeds around the entire circle to produce two complete circular DNA molecules, each consisting of one old and one new nucleotide strand.

  10. Steps of replication in bacterial chromosome • The origin of replication (oriC) contain three type of DNA sequences 1- an AT-rich region 2- DnaA boxes sequences 3- GATC methylation sites

  11. Initiated by binding of DnaA protein to the origin of replication • four DnaA boxes sequences (recognition sites) where the initiation of DNA replication take place when 20 – 40 DnaA protein bind to this sequence forming complex proteins, result in the separation of AT base pairs. • Binding of two DNA helicase enzymes ( function is to separate the DNA strands within the oriC region). • The direction of the helicases traveling from 5’ to 3’. • Helicases break the hydrogen bond between the two strand. • Topoisomerase type II (DNA gyrase) is enzymes that travels ahead of helicases to removes the supercoils.

  12. DNA Replication DNA helicase • Helicase unwinds the double helix starting at a replication bubble. • The two strands separate as the hydrogen bonds between base pairs are broken. • Two replication forks form and the DNA is unwound in opposite directions.

  13. DNA Replication • Helicase has completed unwinding the DNA strand. • Single strand Binding Proteins (SSB) keep the two strands from re-annealing (coming back together).

  14. DNA primer • Single strand binding protein bond to both single strands for preventing the reforming of double- helix • The two single strands are kept in exposed condition to bond to single nucleotides • Then the synthesis of short single strand of RNA called RNA primer take place via DNA primasethrough linkage of ribonucleotiedes • length of primer are 10-12 nucleotides. • The function of these primer is to start replication of DNA. • Primers removed at the later stage of replication

  15. DNA Replication Leading Strand Primase RNA Primer Lagging Strand • Primase is an RNA polymerase that makes the RNA primer. • These primers “tell” the DNA polymerase where to start copying the DNA.

  16. DNA polymerases link nucleotides to synthesize the daughter strands • DNA polymerases are responsible for covalently attaching nucleotides together to make new daughter strands. • Five type of DNA polymerase • Two of them, DNA polymerase I and DNA polymerase III, carry out DNA synthesis associated with replication; the other three have specialized functions in DNA repair • DNA polymerase can elongate strand only from an RNA primer or pre-existing strand . • DNA polymerase can attach nucleotides only in 5’ to 3’ direction

  17. DNA polymerase • DNA polymerase III is a large multiprotein complex which is responsible for most of the DNA replication. • DNA polymerase III synthesizes nucleotide strands by adding new nucleotides to the 3’ end of growing DNA molecules. • Thisenzyme has two enzymatic activities. • Its 5’ 3’ polymerase activity allows it to add new nucleotides in the 5’ 3’ direction. • Its 3’ 5’ exonuclease activity allows it to remove nucleotides in the 3’ 5’ direction, enabling it to correct errors. • If a nucleotide having an incorrect base is inserted into the growing DNA molecule, DNA polymerase III uses its 3’ 5’ exonuclease activity to back up and remove the incorrect nucleotide. • It then resumes its 5’ 3’

  18. DNA polymerase III is a processive enzyme that use deoxynucleoside triphosphate • Energy for synthesis comes from the removal of the two phosphates of the in coming nucleotide • DNA polymerase catalyze the formation of covalent bond between the 3’ –OH group on the previous nucleotide and the inner most 5’ phosphate group on the incoming nucleotide and result in the release of pyrophosphate. • This enzymes is processive; it remain attach to the growing strand after it catalyze the covalent joining of the nucleotides this made the replication process fast.

  19. DNA Replication • Since DNA is antiparallel, synthesis occurs in opposite directions forming two strands • One strand in synthesized – • leading strand (5’3’) which synthesized continuously when one primer is made at the origin and then DNA polymerase III can attach nucleotides (DNTPs) in (5’3’) direction as it slides to the opening of replication fork. • lagging strand (3’5’) which synthesized discontinuously through short segments of DNA length in bacteria 1,000-2,000 nucleotides, each contains a short RNA primer at the 5’ end which made by primase the reminder of the fragment is strand of DNA ( called Okazaki fragments ) made by DNA polymerase III. • To complete the synthesis of Okazaki fragments within the lagging strand three additional events must occurs: • DNA polymerase I remove the RNA primers through its exonuclease activity (digestion). • DNA polymerase I synthesis of DNA in the area where primers have been removed (fill the gap) • DNA ligase catalyzes the covalent attachment of adjacent fragments of DNA • Because of this DNA synthesis is called Semidiscontinuous

  20. DNA Replication Leading Strand 5’ 3’ Direction of Replication DNA Polymerase 3’ 5’ Lagging Strand Direction of Replication • The DNA polymerase starts at the 3’ end of the RNA primer of the leading stand CONTINUOUSLY. • DNA is copied in 5’ to 3’ direction. • DNA polymerase copies the lagging strand DIS- continuously.

  21. DNA Replication • The dis-continuous pieces of DNA copied on the lagging strand are known as Okazaki fragments.

  22. DNA Replication Another DNA Polymerase removes the RNA primers and replaces them with DNA.

  23. DNA Replication ligase Finally the gaps in the sugar phosphate backbone are sealed by DNA ligase There are now 2 identical double helices of DNA.

  24. Despite their differences, all of E. coli’s DNA polymerases 1. synthesize any sequence specified by the template strand; 2. synthesize in the 5’ 3’ direction by adding nucleotides to a 3-OH group; 3. use dNTPs to synthesize new DNA; 4. require a primer to initiate synthesis; 5. catalyze the formation of a phosphodiester bond by joining the 5 phosphate group of the incoming nucleotide to the 3-OH group of the preceding nucleotide on the growing strand, cleaving off two phosphates in the process; 6. produce newly synthesized strands that are complementary and antiparallel to the template strands; and 7. are associated with a number of other proteins.

  25. Termination • In some DNA molecules, replication is terminated whenever two replication forks meet. • In others, specific termination sequences block further replication. • A termination protein, called Tus in E. coli, binds to these sequences. • Tus blocks the movement of helicase, thus stalling the replication fork and preventing further DNA replication.

  26. The fidelity of DNA • replication Overall, replication results in an error rate of less than one mistake per billion nucleotides. How is this incredible accuracy achieved? • DNA polymerases are very particular in pairing nucleotides with their complements on the template strand. • Errors in nucleotide selection by DNA polymerase arise only about once per 100,000 nucleotides.

  27. Proofreading • Most of the errors that do arise in nucleotide selection are corrected in a second process called proofreading. • When a DNA polymerase inserts an incorrect nucleotide into the growing strand, the 3-OH group of the mispaired nucleotide is not correctly positioned for accepting the next nucleotide. • The incorrect positioning stalls the polymerization reaction, and the 3:5 exonuclease activity of DNA polymerase removes the incorrectly paired nucleotide. • DNA polymerase then inserts the correct nucleotide. • Together, proofreading and nucleotide selection result in an error rate of only one in 10 million nucleotides.

  28. Mismatch repair • A third process, called mismatch repair, corrects errors after replication is complete. • Any incorrectly paired nucleotides remaining after replication produce a deformity in the secondary structure of the DNA; the deformity is recognized by enzymes that excise an incorrectly paired nucleotide and use the original nucleotide strand as a template to replace the incorrect nucleotide. • Mismatch repair requires the ability to distinguish between the old and the new strands of DNA, because the enzymes need some way of determining which of the two incorrectly paired bases to remove. • In E. coli, methyl groups (CH3) are added to particular nucleotide sequences, but only after replication. • Thus, methylation lags behind replication: so, immediately after DNA synthesis, only the old DNA strand is methylated. • Therefore it can be distinguished from the newly synthesized strand, and mismatch repair takes place preferentially on the unmethylated nucleotide strand.

  29. Linear eukaryotic replication • The large linear chromosomes in eukaryotic cells contain far too much DNA to be replicated speedily from a single origin. • Eukaryotic replication proceeds at a rate ranging from 500 to 5000 nucleotides per minute at each replication fork (considerably slower than bacterial replication). • Even at 5000 nucleotides per minute at each fork, DNA synthesis starting from a single origin would require 7 days to replicate a typical human chromosome consisting of 100 million base pairs of DNA. • The replication of eukaryotic chromosomes actually occurs in a matter of minutes or hours, not days. This rate is possible because replication takes place simultaneously from thousands of origins.

  30. Typical eukaryotic replicons are from 20,000 to 300,000 base pairs in length. • At each replication origin, the DNA unwinds and produces a replication bubble. • Replication takes place on both strands at each end of the bubble, with the two replication forks spreading outward. • Eventually, replication forks of adjacent replicons run into each other, and the replicons fuse to form long stretches of newly synthesized DNA. • Replication and fusion of all the replicons leads to two identical DNA molecules.

  31. Eukaryotic replication • Eukaryotic cells utilize thousands of origins, and so the entire genome can be replicated in a timely manner. • DNA replication is divided into two distinct steps. • In the first step, the origins are licensed, meaning that they are approved for replication. This step is early in the cell cycle when a replication licensing factor attaches to an origin. • In the second step, initiator proteins cause the separation of DNA strands and the initiation of replication at each licensed origin. • The key is that initiator proteins function only at licensed origins. • As the replication forks move away from the origin, the licensing factor is removed, leaving the origin in an unlicensed state, where replication cannot be initiated again until the license is renewed. • To ensure that replication takes place only once each cell cycle. • the entire genome must be precisely replicated once and only once in each cell cycle so that no genes are left unreplicated and no genes are replicated more than once.

  32. Unwinding • Several helicases that separate double-stranded DNA have been isolated from eukaryotic cells, as have singlestrand- binding proteins and topoisomerases (which have a function equivalent to the DNA gyrase in bacterial cells). • These enzymes and proteins are assumed to function in unwinding eukaryotic DNA in much the same way as unwinding in bacterial cells.

  33. Eukaryotic DNA polymerases • A significant difference in the processes of bacterial and eukaryotic replication is in the number and functions of DNA polymerases. Eukaryotic cells contain a number of different DNA polymerases that function in replication, recombination, and DNA repair

  34. Nucleosome assembly • Eukaryotic DNA is complexed to histone proteins in nucleosome structures that contribute to the stability and packing of the DNA molecule. • Synthesis of new histone occur during the S phase of the cell cycle. • Following the replication each daughter strand contains a random mixture of original histone octamers and newly assembled histone octamers.

  35. The end of eukaryotic chromosomes are replicated by telomerase • Telomerase enzymes attach to the telomere at the end of the chromosomes. • Telomerase recognize telomeric sequence at the end of the chromosomes and synthesize additional repeats of telomeric sequences. • Why we need telomerase? • Because the DNA polymerase unable to replicate the 3’ end of DNA strands. • DNA polymerase synthesize from 5’ to 3’ • Need an RNA primer to complete synthesis

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