DNA polymerase proofreading and processivity.pptx

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1. DNA polymerase proof reading and processivity

2. The term  proofreading  is used in genetics to refer to the error-correcting processes, first proposed by  John Hopfield  and Jacques Ninio , involved in  DNA replication ,  immune system  specificity, enzyme-substrate recognition among many other processes that require enhanced specificity.  The extent of proofreading in DNA replication determines the  mutation rate , and is different in different species. For example, loss of proofreading due to mutations in the  DNA polymerase epsilon  gene results in a hyper-mutated genotype with >100 mutations per Mbase of DNA in human colorectal cancers . The extent of proofreading in other molecular processes can depend on the  effective population size  of the species and the number of genes affected by the same proofreading mechanism.

3. DNA Polymerase Proofreading . A 3´→ 5´  proofreading   exonuclease domain is intrinsic to most  DNA polymerases . It allows the enzyme to check each nucleotide during  DNA  synthesis and excise mismatched nucleotides in the 3´ to 5´ direction . What is the main function of DNA polymerase? The  main function of DNA polymerase  is to make  DNA  from nucleotides, the building blocks of  DNA . There are several forms of  DNA polymerase  that play a role in  DNA  replication and they usually work in pairs to copy one molecule of double-stranded  DNA  into  two  new double stranded  DNA  molecules. Which DNA polymerase proofreads? In bacteria, all three DNA polymerases (I, II and III) have the ability to proofread, using 3' → 5'  exonuclease activity . When an incorrect base pair is recognized, DNA polymerase reverses its direction by one base pair of DNA and excises the mismatched base . In  eukaryotes  only the polymerases that deal with the elongation (delta and epsilon) have proofreading ability (3’ → 5’ exonuclease activity)

4. Proofreading and Repair DNA replication takes place only once each generation in each cell Errors in replication ( mutations ) occur spontaneously only once in every 10 9 to 10 10 base pairs Can be lethal to organisms Proofreading - the removal of incorrect nucleotides immediately after they are added to the growing DNA during replication Errors in hydrogen bonding lead to errors in a growing DNA chain once in every 10 4 to 10 5 base pairs

5. Proofreading Improves Replication Fidelity Cut-and-patch catalyzed by Pol I: cutting is removal of the RNA primer and patching is incorporation of the required deoxynucleotides Nick translation : Pol I removes RNA primer or DNA mistakes as it moves along the DNA and then fills in behind it with its polymerase activity Mismatch repair: enzymes recognize that two bases are incorrectly paired, the area of mismatch is removed, and the area replicated again Base excision repair: a damaged base is removed by DNA glycosylase leaving an AP site; the sugar and phosphate are removed along with several more bases, and then Pol I fills the gap

6. DNA Polymerase Repair

7. DNA polymerase is self-correcting. It makes only about one error in ever 10 7 nucleotide pairs replicated, but this is too many for survival of an organism. Error-correcting activity is called proofreading . Before adding the next nucleotide, DNA polymerase checks the one it just added. If there is a mispaired nucleotide, it is removed using the nuclease activity. DNA polymerase has both 5’ – 3’ polymerization activity and 3’ – 5’ nuclease activity DNA polymerase can not start a new strand of DNA, it only elongates an existing strand.

8. DNA polymerase can not polymerize 3’ to 5’. It can not add nucleotides to the 5’ end and there is no DNA polymerase that can. DNA polymerase can not start a new strand of DNA. It can only elongate an existing strand. Therefore primers are required These are RNA not DNA since they are only temporary. This is done by a primase . Primerase can not proofread and leaves many mistakes. Primers are removed by nucleases that recognize RNA/DNA helices. This leaves a gap filled in by a DNA repair polymerase . And DNA ligase joins the nucleotides at the nick.. One primer is required for the leading strand. Multiple primers are needed for the lagging strand . Okazaki fragments

9. DNA Repair Rare beneficial DNA mutations (mistakes or damage) allow for evolution of organisms in the face of ever changing environments. However, in the short term, DNA mutations are almost always detrimental to an organism. Genetic stability is the result of the accuracy of DNA polymerase and the many mechanisms for proof-reading and DNA repair. When these processes fail, a permanent change in the DNA sequence occurs - mutation . Sickle-cell anemia Glutomic acid to valine at 6th position of 146 aa

10. Mutations in germ cells (reproductive cells) lead to genetic diseases like sickle-cell anemia. Somatic cells must also be protected from damage to the DNA. Gradual accumulation of mutations in the DNA of somatic cells can eventually lead to a lack of replication control and cancer - one cell grows uncontrollably. These mutations accumulate and cancer is more likely as we age.

11. DNA Mismatach Repair removes replication errors that escape from the replication machine. Corrects 99% of the replication errors. Accuracy is one in 10 7 base pairs. Mismatch repair proteins must excise (remove) the mismatched nucleotide from the new strand . How does the enzyme complex know which is which? The new strand may remain nicked for a short time, also old strands may have chemical modifications, such as methylation.

12. DNA mismatch repair proteins are thought to recognize (bind to) the distortion in the geometry of the DNA double helix resulting from an incorrect base pairing. They may recognize the correct (old) strand by occasional nicks (which would only be present for a short time after replication is complete.

13. Even though DNA is one of the most stable molecules, thermal collisions do sometimes cause changes - including depurination and deamination.

14. Ultraviolet radiation in sunlight damages DNA and promotes covalent linkage between two adjacent pyrimidine bases - forming thymine dimers. Many different repair enzymes exist to correct and repair the damage. When someone inherits a mutation in the gene responsible for producing one of the mismatch repair proteins, they may be predisposed to certain cancers.

15. If uncorrected, the result is a permanent mutation.

16. 1. There are a variety of enzymes that recognize and exise different types of DNA damage Step 2 and 3 are nearly the same for most types of DNA repair. Since the damaged strand usually has a distinct structure, different from normal DNA structure, it can be “recognized” by the DNA repair enzyme. And, because each strand is complementary to each other, there is a template to copy.

17. Single-celled organisms like yeast have more than 50 different proteins that function in DNA repair. Probably much more complex in humans. Xeroderma pigmentosum is a genetic disease in which the gene for one of these DNA repair enzymes is mutated. Severe skin lesions, including cancer, are the result of an accumulation of thymine dimers in cells exposed to sunlight.

18. DNA is extremely stable. Changes accumulate slowly in the course of evolution, as evidenced by the degree of homology between many genes even in unrelated organisms like fruit flies and humans.

19. DNA Polymerase processivity

20. In  molecular biology  and  biochemistry ,  processivity  is an  enzyme 's ability to  catalyze  "consecutive reactions without releasing its  substrate ". For example, processivity is the average number of  nucleotides  added by a  polymerase   enzyme , such as  DNA polymerase , per association event with the template strand. Because the binding of the polymerase to the template is the rate-limiting step in  DNA synthesis , the overall rate of  DNA  replication during  S phase  of the  cell cycle  is dependent on the processivity of the DNA polymerases performing the replication.  DNA clamp  proteins are integral components of the DNA replication machinery and serve to increase the processivity of their associated polymerases. Some polymerases add over 50,000 nucleotides to a growing DNA strand before dissociating from the template strand, giving a replication rate of up to 1,000 nucleotides per second.

21. DNA binding interactions Polymerases interact with the  phosphate  backbone and the minor groove of the DNA, so their interactions do not depend on the specific nucleotide sequence. The binding is largely mediated by  electrostatic  interactions between the DNA and the "thumb" and "palm" domains of the metaphorically hand-shaped DNA polymerase molecule. When the polymerase advances along the DNA sequence after adding a nucleotide, the interactions with the minor groove dissociate but those with the phosphate backbone remain more stable, allowing rapid re-binding to the minor groove at the next nucleotide . Interactions with the DNA are also facilitated by  DNA clamp  proteins, which are multimeric proteins that completely encircle the DNA, with which they associate at  replication forks . Their central pore is sufficiently large to admit the DNA strands and some surrounding water molecules, which allows the clamp to slide along the DNA without dissociating from it and without loosening the  protein-protein interactions  that maintain the toroid shape. When associated with a DNA clamp, DNA polymerase is dramatically more processive ; without the clamp most polymerases have a processivity of only about 100 nucleotides. The interactions between the polymerase and the clamp are more persistent than those between the polymerase and the DNA. Thus, when the polymerase dissociates from the DNA, it is still bound to the clamp and can rapidly reassociate with the DNA. An example of such a DNA clamp is PCNA (proliferating cell nuclear antigen) found in  S. cervesiae .

22. Polymerase processivities Multiple DNA polymerases have specialized roles in the DNA replication process. In  E. coli , which replicates its entire  genome  from a single replication fork, the polymerase  DNA Pol III  is the enzyme primarily responsible for DNA replication and forms a replication complex with extremely high processivity. The related  DNA Pol I  has  exonuclease  activity and serves to degrade the  RNA primers  used to initiate DNA synthesis. Pol I then synthesizes the short DNA fragments that were formerly hybridized to the RNA fragment. Thus Pol I is much less processive than Pol III because its primary function in DNA replication is to create many short DNA regions rather than a few very long regions. In  eukaryotes , which have a much higher diversity of DNA polymerases, the low-processivity initiating enzyme is called  Pol α , and the high-processivity extension enzymes are  Pol δ  and  Pol ε . Both  prokaryotes  and eukaryotes must "trade" bound polymerases to make the transition from initiation to elongation. This process is called polymerase switching. [3] [4]

23. DNA polymerase’s rapid catalysis is due to its processive nature.  Processivity  is a characteristic of enzymes that function on polymeric substrates. In the case of DNA polymerase, the degree of processivity refers to the average number of nucleotides added each time the enzyme binds a template. The average DNA polymerase requires about one second locating and binding a primer/template junction. Once it is bound, a nonprocessive DNA polymerase adds  nucleotide s at a rate of one nucleotide per second. Processive DNA polymerases, however, add multiple nucleotides per second, drastically increasing the rate of DNA synthesis. The degree of processivity is directly proportional to the rate of DNA synthesis. The rate of DNA synthesis in a living cell was first determined as the rate of phage T4 DNA elongation in phage infected  E. coli . During the period of exponential DNA increase at 37 °C, the rate was 749 nucleotides per second . DNA polymerase’s ability to slide along the DNA template allows increased processivity. There is a dramatic increase in processivity at the  replication fork . This increase is facilitated by the DNA polymerase’s association with proteins known as the sliding  DNA clamp . The clamps are multiple protein subunits associated in the shape of a ring. Using the  hydrolysis  of ATP, a class of proteins known as the  sliding clamp loading proteins  open up the ring structure of the sliding DNA clamps allowing binding to and release from the DNA strand.  Protein-protein interaction  with the clamp prevents DNA polymerase from diffusing from the DNA template, thereby ensuring that the enzyme binds the same primer/template junction and continues replication. DNA polymerase changes conformation, increasing affinity to the clamp when associated with it and decreasing affinity when it completes the replication of a stretch of DNA to allow release from the clamp.

24. DNA polymerase delta  is an  enzyme  complex found in  eukaryotes  that is involved in  DNA replication  and  repair . The DNA polymerase delta complex consists of 4 subunits:  POLD1 ,  POLD2 ,  POLD3 , and  POLD4 . [1]  DNA Pol δ is an enzyme used for both leading and lagging strand synthesis. [2]   [3] It exhibits increased processivity when interacting with the proliferating cell nuclear antigen ( PCNA ). As well, the multisubunit protein  replication factor C , through its role as the clamp loader for PCNA (which involves catalysing the loading of PCNA on to DNA) is important for DNA Pol δ function.