Chen Yonggang Zhejiang University Schools of Medicine  Biochemistry
DNA Replication .
 
DNA Replication-Conservation of Information <ul><li>DNA replication must be carried out every time a cell divides </li></u...
DNA is replicated in a semi-conservative manner <ul><li>Messelson & Stahl showed, using  15 N-labelled DNA that the produc...
Each separated DNA strand is duplicated to give two new double helices.
DNA semiconservative replication <ul><li>Each DNA strand serves as a template for the synthesis of  a new strand, producin...
The process which appeared simple initially is complex <ul><li>Replication occurs just prior to cell division </li></ul><u...
To allow the replication to occur supercoiling must be relaxed <ul><li>A type I (single strand breaking) topoisomerase cle...
The first step in replication involves  oriC <ul><li>OriC  is a 245 bp region, the  origin of E coli replication </li></ul...
dnaA allows binding of two other important proteins <ul><li>dnaB  is a  DNA helicase  which carries out the ATP- driven un...
SSB, a single strand binding tetramer stabilizes the initiation complex McKee 18.7
DNA replication involves many enzymes <ul><li>An RNA primase binds to the SSB stabilized melted helix </li></ul><ul><li>Si...
At the replication fork two strands are managed differently <ul><li>The 5’ end of the primer(leading strand) is extended c...
The replication fork McKee 18.6
DNA Polymerase III holoenzyme contains 10 distinct types of subunits <ul><li>DNA Polymerase III is the primary replicase i...
The Pol III synthesizes DNA from dNTPs McKee  18.3
The Dimer moves in one direction and synthesizes 5’-3’  <ul><li>The leading strand is synthesized by addition of 5’dNTPs i...
DNA polymerase III forms phosphodiester bonds <ul><li>2’-deoxynucleoside 5’ triphosphates are the activated intermediates ...
Synthesis of a phosphodiester bond
DNA Polymerase III is at the center of Replication McKee 18.8
Top-down view of replication
While DNA polymerase III does the replicating, DNAP I cleans up <ul><li>Pol I(100kd) is a monomer of about 10% the size of...
The three activities are on one polypeptide <ul><li>The larger fragment(Klenow fragment) of 67kd contains the polymerase a...
As Pol III finishes, Pol I goes to work <ul><li>The 5’-3’ exonuclease removes the RNA primer </li></ul><ul><li>The polymer...
Supercoiling was taken out by dnaB, DNA gyrase replaces it <ul><li>Following synthesis of the strands, excision of RNA, re...
DNA ligase seals the Okazaki fragments and the completed double helical DNA <ul><li>This energy requiring enzyme uses NAD ...
Eucaryotic Replication is similar to that of procaryotes <ul><li>Both have initiation, elongation and termination phases a...
Eucaryotic replication is distinct from that of procaryotes <ul><li>There are 5 polymerases </li></ul><ul><li>The chromoso...
Initiation of replication occurs at multiple  ori <ul><li>A large complex of proteins assembles at an ori (Origin Recognit...
Binding of initiation factors to the lagging strand differs <ul><li>Pol    is the main eucaryotic replication polymerase ...
Finishing and sealing of the lagging strand is different <ul><li>Specific protein factors are important in finishing up re...
Telomeres are GC rich self-complementary sequences at chromosome ends <ul><li>Telomerase maintains the telomeres </li></ul...
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2,dna replication

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2,dna replication

  1. 1. Chen Yonggang Zhejiang University Schools of Medicine Biochemistry
  2. 2. DNA Replication .
  3. 4. DNA Replication-Conservation of Information <ul><li>DNA replication must be carried out every time a cell divides </li></ul><ul><li>Procaryotic growth involves cell division </li></ul><ul><li>Mitosis in eucaryotes involves cell division </li></ul><ul><li>DNA replication is template driven and synthesizes DNA in a semi-conservative manner </li></ul><ul><li>dNTP + DNA n DNA n+1 +PPi </li></ul>
  4. 5. DNA is replicated in a semi-conservative manner <ul><li>Messelson & Stahl showed, using 15 N-labelled DNA that the products of replication had intermediate density </li></ul>McKee 18.2
  5. 6. Each separated DNA strand is duplicated to give two new double helices.
  6. 7. DNA semiconservative replication <ul><li>Each DNA strand serves as a template for the synthesis of a new strand, producing two new DNA molecules , each with one new strand and one old strand, this is semiconservative replication. </li></ul>
  7. 8. The process which appeared simple initially is complex <ul><li>Replication occurs just prior to cell division </li></ul><ul><li>The E coli chromosome is a single circular DNA double helix associated with proteins in a nucleoid </li></ul><ul><li>The E coli chromosome is negatively supercoiled and thus is quite compact and inaccessible </li></ul>McKee 17.16
  8. 9. To allow the replication to occur supercoiling must be relaxed <ul><li>A type I (single strand breaking) topoisomerase cleaves and relaxes the negative supercoiling ahead of the replication complex </li></ul><ul><li>The topoisomerase has a central hole through which the double helix passes. An intermediate is an enzyme-linked 3’OH </li></ul><ul><li>dnaA is displaced providing access for the next component needed for replication </li></ul>
  9. 10. The first step in replication involves oriC <ul><li>OriC is a 245 bp region, the origin of E coli replication </li></ul><ul><li>OriC contains 3 tandem repeats of a 13 bp sequence beginning in GATC and rich in AT bp </li></ul><ul><li>These repeats are weakly H-bonded and serve to provide 4 binding sites for a protein, dnaA , a start signal for replication </li></ul><ul><li>Replication proceeds in two directions - bidirectional </li></ul>
  10. 11. dnaA allows binding of two other important proteins <ul><li>dnaB is a DNA helicase which carries out the ATP- driven unwinding of the DNA double helix </li></ul><ul><li>dnaC is an important accessory protein which binds and is soon released </li></ul><ul><li>Together the three proteins utilize ATP to bend and separate the two strands of the bacterial chromosome </li></ul>
  11. 12. SSB, a single strand binding tetramer stabilizes the initiation complex McKee 18.7
  12. 13. DNA replication involves many enzymes <ul><li>An RNA primase binds to the SSB stabilized melted helix </li></ul><ul><li>Since DNA polymerases require a primer and only extend that primer, the RNA primase (dnaG) in association with other proteins (primasome) synthesizes a 5-7 nucleotide primer using information from the template strand </li></ul>
  13. 14. At the replication fork two strands are managed differently <ul><li>The 5’ end of the primer(leading strand) is extended continuously by DNA polymerase III in a 5’-3’ direction by dNTPs </li></ul><ul><li>The primer on the lagging strand is also extended 5’-3’ by DNA polymerase III, in a discontinuous manner </li></ul><ul><li>Thus primer is made once on the leading strand and every 1000 nucleotides on the lagging strand </li></ul>
  14. 15. The replication fork McKee 18.6
  15. 16. DNA Polymerase III holoenzyme contains 10 distinct types of subunits <ul><li>DNA Polymerase III is the primary replicase in E. coli </li></ul><ul><li>It has polymerase and 3’-5’ exonuclease activities </li></ul><ul><li>It functions as a dimer </li></ul><ul><li>It has great fidelity, only 1 error in 10 10 bp </li></ul><ul><li>It is highly processive, sticking to the DNA for the entire trip through the chromosome </li></ul><ul><li>It has a rapid biosynthetic rate, synthesizing 1000 nt/sec </li></ul>
  16. 17. The Pol III synthesizes DNA from dNTPs McKee 18.3
  17. 18. The Dimer moves in one direction and synthesizes 5’-3’ <ul><li>The leading strand is synthesized by addition of 5’dNTPs in response to the template </li></ul><ul><li>The looped lagging strand is synthesized in Okazaki fragments using 5’dNTPs </li></ul><ul><li>The lagging strand must be pieced together using DNA Polymerase I </li></ul>
  18. 19. DNA polymerase III forms phosphodiester bonds <ul><li>2’-deoxynucleoside 5’ triphosphates are the activated intermediates needed for synthesis </li></ul><ul><li>Information from the parental strand provides the information for 5’-3’ synthesis (parental strand is read 3’-5’) </li></ul><ul><li>Thus each parental strand serves as the template for synthesis of a complementary strand </li></ul>
  19. 20. Synthesis of a phosphodiester bond
  20. 21. DNA Polymerase III is at the center of Replication McKee 18.8
  21. 22. Top-down view of replication
  22. 23. While DNA polymerase III does the replicating, DNAP I cleans up <ul><li>Pol I(100kd) is a monomer of about 10% the size of Pol III(900kd) </li></ul><ul><li>It has three activities </li></ul><ul><ul><li>It is a DNA polymerase </li></ul></ul><ul><ul><li>It is a 3’-5’ exonuclease </li></ul></ul><ul><ul><li>It is a 5’-3’ exonuclease </li></ul></ul><ul><li>It is a processing and proofreading enzyme </li></ul>
  23. 24. The three activities are on one polypeptide <ul><li>The larger fragment(Klenow fragment) of 67kd contains the polymerase and the 3’-5’ exonuclease </li></ul><ul><li>The smaller, 36kd contains the 5’-3’ exonuclease activity </li></ul>
  24. 25. As Pol III finishes, Pol I goes to work <ul><li>The 5’-3’ exonuclease removes the RNA primer </li></ul><ul><li>The polymerase synthesizes DNA to fill the gap </li></ul><ul><li>Errors in Pol III synthesis are removed by 3’-5’ exonuclease </li></ul>
  25. 26. Supercoiling was taken out by dnaB, DNA gyrase replaces it <ul><li>Following synthesis of the strands, excision of RNA, replacement by DNA using Pol I, the supercoiling can be reinstated </li></ul><ul><li>DNA gyrase, an ATP- linked, energy requiring enzyme introduces negative supercoils to restore the original twist in the leading strand </li></ul>
  26. 27. DNA ligase seals the Okazaki fragments and the completed double helical DNA <ul><li>This energy requiring enzyme uses NAD as the activated AMP donor in this reaction </li></ul><ul><li>Pyrophosphate cleavage drives the reaction to completion </li></ul><ul><li>Termination occurs at a ter region and is mediated by a binding protein TBP </li></ul><ul><li>A type II(double stranded) topoisomerase is probably involved in helix dissociation </li></ul>
  27. 28. Eucaryotic Replication is similar to that of procaryotes <ul><li>Both have initiation, elongation and termination phases and are bidirectional </li></ul><ul><li>Both involve multiple DNA polymerases </li></ul><ul><li>Both involve multiple copies of the primary replicase which replicates strands differently </li></ul><ul><li>Replication rate is slower, but replication is rapid due to multiple replicons </li></ul><ul><li>Both require topoisomerases to unwind and rewind the DNA </li></ul><ul><li>Both require ligases </li></ul>
  28. 29. Eucaryotic replication is distinct from that of procaryotes <ul><li>There are 5 polymerases </li></ul><ul><li>The chromosomes are linear </li></ul><ul><li>There are multiple ori , and replication units </li></ul><ul><li>Replication only occurs during the S phase of the cell cycle </li></ul><ul><li>Telomeres restrict the number of times a replicon can be expressed </li></ul>
  29. 30. Initiation of replication occurs at multiple ori <ul><li>A large complex of proteins assembles at an ori (Origin Recognition Complex-ORC) </li></ul><ul><li>Details not for testing </li></ul><ul><ul><li>A complex with helicase activity must bind and be activated </li></ul></ul><ul><ul><li>Replication Protein A (RPA ) binds and separates the strands(like SSB in E.coli) </li></ul></ul><ul><ul><li>RFC(replication factor C– a clamp loading factor) and PCNA(proliferating cell nuclear antigen) allows binding of Pol  to both the leading and lagging strand </li></ul></ul>
  30. 31. Binding of initiation factors to the lagging strand differs <ul><li>Pol  is the main eucaryotic replication polymerase (Details not for testing) </li></ul><ul><ul><li>Replication protein A(RPA) binds to the single strands </li></ul></ul><ul><ul><li>Pol a and a primase complex binds to the lagging strand </li></ul></ul><ul><ul><li>An RNA primer and 15-30 dNTPs are synthesized </li></ul></ul><ul><li>Pol  binds and replicates one nucleosomes worth of Okazaki fragment </li></ul>
  31. 32. Finishing and sealing of the lagging strand is different <ul><li>Specific protein factors are important in finishing up replication (details not for testing) </li></ul><ul><ul><li>DNA polymerase  remove the primers and DNA polymerase  excise errors </li></ul></ul><ul><ul><li>Topoisomerases induce supercoiling </li></ul></ul><ul><ul><li>DNA ligase seals the breaks </li></ul></ul><ul><ul><li>Chromosomes segregate </li></ul></ul><ul><li>Replication bubbles merge </li></ul><ul><li>Telomeres determine the end of replication </li></ul>
  32. 33. Telomeres are GC rich self-complementary sequences at chromosome ends <ul><li>Telomerase maintains the telomeres </li></ul><ul><li>Telomeres are repeat structures with a terminal loop </li></ul><ul><li>At each replication the telomeres are modified using an integral RNA template </li></ul><ul><li>Loss of telomeres limits replication </li></ul><ul><li>Cancer cells lose control of their telomeres </li></ul>

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