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Replication

Replication. Which is the most necessary process for life?. Is it translation ? Is it transcription ? Is it replication ?. Energy. DNA. RNA. Proteins. information flow. Information carryer replication. Outline. Overview Replication fork and involved enzymes

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Replication

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  1. Replication

  2. Which is the most necessary process for life? • Is it translation ? • Is it transcription ? • Is it replication ? Energy DNA RNA Proteins information flow Information carryer replication

  3. Outline • Overview • Replication fork and involved enzymes • Differences among eukaryotes and prokaryotes • DNA repair • Replication initiation • Replication termination

  4. What happens upon replication? 1. Double-stranded DNA unwinds 2.Two new strandsare formed by pairing complementary bases with theold strands

  5. OH OH OH OH OH OH OH O O O O O O O OH OH OH O P O P OH OH OH O 5' end of strand Chemistry P P CH2 CH2 Base Base O O P P CH2 CH2 Base Base O O H20 + P 3' P OH P + Synthesis reaction CH2 P Base O CH2 5' Base O OH 3' 3' end of strand OH

  6. What do you need for replication ? • 1) template - dsDNA • 2) Origin - some place in dsDNA, which is recognized by replication machinery • 3) polymerse & other replicating enzymes • 4) nucleotides

  7. Enzymatic activities of polymerases • 5’-3’ polymerase activity 5’- GTCACC-3’ 5’- GTCACCG-3’ 3’-TTCAGTGGCAA-5’ 3’-TTCAGTGGCAA-5’ NEVER 3’-5’ polymerase activity! +G 5’-3’ polymerase activity is present in all DNA and RNA polymerases

  8. Enzymatic activities of polymerases • 3’-5’ exonuclease (editing) activity 5’-AAGTCAC -3’ 5’-AAGTCAC-3’ 3’-TTCAGTGGCAA-5’ 3’-TTCAGTGGCAA-5’ -A A Normally, only one mismatched nucleotide is removed 3’-5’ exonuclease activity is present in most (but not all) DNA and RNA polymerases

  9. Enzymatic activities of polymerases • 5’-3’ exonuclease activity 5’-AA CACC-3’ 5’-AA CC-3’ 3’-TTCAGTGGCAA-5’ 3’-TTCAGTGGCAA-5’ -ACA A 5’-3’ exo activity requires a free 5’-end or a nick in dsDNA 5’-3’ exo activity can be combined with 5’-3’ polymerase activity. This results in a replacement of a part of strand. 5’-3’ exo activity is present only in some DNA polymerases, notably bacterial DNA polymerase I

  10. Replication enzymes (summary) • Polymerase III (E.coli) – adds nucleotides • Helicase – unwinds the DNA • Topoisomerase – releases tension on ds DNA • SSB – binds to ssDNA • Primase – makes RNA primer • Polymerase I (E.coli)– replaces RNA primers with DNA • Ligase – joins Okazaki fragments

  11. DNA REPLICATION (E.coli) Pol III synthesises leading strand 4 2 1 Helicase opens helix Primase synthesizes RNA primer 3 Topoisomerase nicks DNA to relieve tension from unwinding Pol I replaces RNA primer with DNA 5 6 7 SSB protein prevents ssDNA from base-pairing Pol III elongates primer; produces Okazaki fragment DNA ligase links two Okazaki fragments to form continuous strand

  12. DNA polymerases in E.coli • DNA pol I – excises RNA primer and fills the gap • DNA pol II – DNA repair • DNA pol III – main replicating enzyme • Recently discovered: • DNA pol IV – increase mutation rate upon starvation and stress conditions (“Mutate or die!”) • DNA pol V – “SOS” polymerase, active upon DNA- damaging conditions. Can bypass damaged DNA effectively at a cost of higher mutation rate (“Replicate or die”).

  13. The subunits of E. coli DNA polymerase III 5’ to 3’ polymerizing activity 3’ to 5’ exonuclease activity a and e assembly (scaffold) Assembly of holoenzyme on DNA Sliding clamp = processivity factor Clamp-loading (“g”) complex g complex g complex g complex,binds to SSB g complex 2x a 2x e 2x q 2x t 2x b g d d’ c y Function Subunit Core Enzyme dimer Holoenzyme

  14. Sliding clamp around the DNA Clamp ensures processivity of nucleotide addition Clamp is loaded only once on the leading strand Clamp is re-loaded on the lagging strand upon synthesis of new Okazaki fragment

  15. Structure of clamp • pseudo-6-fold symmetry • prokaryotes – dimer • eukaryotes – trimer • Domains within the monomer have very similar structure but no detectable sequence similarity

  16. Subunits of pol III in E.coli Pol III =a+e+q Why a dimer?

  17. DNA looping during replication

  18. How about eukaryotes? • General mechanism of replication similar to prokaryotes with some minor differences

  19. DNA REPLICATION (Eukaryotes) RPA protein prevents ssDNA from base-pairing Pol d synthesises leading strand 5 2 1 Helicase opens helix Primase synthesizes RNA primer 3 4 Topoisomerase nicks DNA to relieve tension from unwinding RNase H excises RNA primer Pol a extends the RNA primer a little bit 5 6 7 Pol d replaces Pol a; produces Okazaki fragment DNA ligase links two Okazaki fragments to form continuous strand

  20. Main differences among eukaryotic and prokaryotic replication forks • In eukaryotes RNA primer is first extended by Pol a, then by Pol d. In prokaryotes extension is done solely by Pol III • In eukaryotes, RNA primer is excised by RNase H and then gap filled by Pol d. In prokaryotes Pol I is able to both excise RNA and fill in DNA • Okazaki fragments in eukaryotes are about 200 nt long, while in bacteria 2000 nt (yes, not the other way around)

  21. Eukaryotic DNA polymerases

  22. Reasons for differences in replication among prokaryotes and eukaryotes • 1. Eukaryotic chromosomes are typically much longer than prokaryotic • 2. Eukaryotic chromosomes are linear, not circular

  23. Multiple origins in chromosomes Bacteria Eukaryotes

  24. Rate of DNA synthesis and the need for multiple origins Origins Genome Fork speed Repl. time Comment 30 kb/min 40 min 1 4.6 Mbp E. coli Repl. would last 80hr if only 1 ori 14 Mbp (1 cm) 3 kb/min 20 min 330 Yeast 1 l culture = 4.1010 cells --> 400 000 km DNA synthesized (Earth-Moon distance) Repl. would last 1 year if only 1 ori 3 Gbp (2 m) 3 kb/min 7 h >10 000 ? Human 2.1013 km DNA synthesized (2 light-years) during life time (1016 cell divisions)

  25. Linear DNA needs special treatment: Telomeres and telomerases • Telomeres: short, repetitive sequences in the ends of eukaryotic chromosomes • Telomerase: polymerase, making those sequences • What are they good for?

  26. Telomerase in action Telomerase contain internal RNA, wich acts as a template After one round of nucleotide addition, telomerase translocates to the next ttttgggg repeat

  27. T-loops TRF1 and 2 – telomere binding proteins Formation of T-loops controls the lenght of telomeres

  28. Is telomerase always active? • Active in children and germ cells of adults • Inactive in somatic cells of adults • So, chromosomes actually get shorter – this is why we get old and die... • For the same reason, cultivated primary animal cells do not divide infinitely • Activation of telomerase in adult mice increase their life span • Telomerase is active in most tumours

  29. DNA damage • 1. Base damage: deamination, depurination, alkylation... • 2. Thymine dimerisation • DNA damage can lead to: - 1. prevention of base pairing - 2. incorrect base paring • Those types of DNA damage are NOT caused by DNA polymerase errors

  30. Deamination [O] R-NH2 R=O

  31. Thymine dimers Produced by UV light Results in no base-pairing with the complementary strand

  32. Repair of damaged bases

  33. Repair of G-T and G-U base pairs • The most usual mutation is deamination of cytosine or methylcytosine • As a result, uracil or thymine is produced, which both base-pair to adenine • Special repair mechanism has been developed for this mutation

  34. Excision of thymine dimers

  35. How do those repair enzymes know, which strand to repair? • Upon introduction of mutation in one strand, a mismatch is produced: • The template strand has to be distinquished from the newly made strand • In prokaryotes template strand has been previously labelled by methylation

  36. ....... ....... ....... ....... N-6-methyldeoxyadenosine Dam methylation deoxyadenosine

  37. Dam methylation Dam methylation in E.coli : A’s in GATC sequences get methylated CH3 C CACGATC ATT GTGCTAG TAA CH3 Error CACGATCCATT GTGCTAGGTAA T Replication Correct CH3 CACGATCCATT GTGCTAGGTAA CH3 Replication machinery recognizes the methylated strand and corrects the other strand. This is valid for prokaryotes, mechanism for eukaryotes has not been established yet

  38. Repair of dsDNA breaks • Under certain mutagenic conditions, break of dsDNA can occur • If this happens during late S or G2 phase, the sister chromatid is around which can be used as a reference • Otherwise – error prone ligation is a option (can be dangerous!)

  39. DNA damage bypass • Necessary, if a replication fork reaches damaged region of DNA • Two main types of bypass exist: • 1. Bypass by recombination • 2. Translesion

  40. Bypass by recombination • Damage (blue circle) hopefully occurs only in one parental strand • Newly made DNA strand temporarily base-pairs with the other newly made strand

  41. Translesion • Damage (lesion) bypass without information of other parental strand • Can be mutagenic or unmutagenic • In humans, polymerase eta is responsible for translession past thymine dimers • Individuals, lacking eta pol, use alternative, thymine dimer translesion pathway by pol zeta • zeta pathway is more mutagenic than eta • As a result, risk of cancer development under UV exposure is significantly increased

  42. Error rates during replication • DNA pol without proofreading: 1:105 • DNA pol with proofreading: 1:107 • Most errors will be corrected by repair enzymes. This leaves error rate of 1:1010 • Since human genome is 3.2x109 base pairs long, about one mutation is made upon each genome replication

  43. Question • Errors in replication can lead to cancer, genetic diseases, etc • Why Mother Nature has not eliminated DNA replication errors completely ? • Or at least, why the error rate has not been decreased still more ?

  44. When to replicate? • DNA replicates only during S phase and only once • This implies some sort of switch... • Cyclins take care of that Go

  45. What are those cyclins anyway? • Cyclins are proteins, which give a signal that it is time to proceed to the next cell cycle phase • Cyclins bind to and activate cyclin dependent kinases (CDKs) • CDKs phosphorylate and thereby activate various regulatory proteins

  46. Origin Recognition Complex ORC DNA origin Origin Recognition Complex (ORC, six subunits) binds specifically to origin DNA sites on the chromosome. ORC isbound to origin DNA regardless of whether replication is occurringor not.

  47. CDC6 and Cdt1 proteins CDC6 ORC Cdt1 DNA origin CDC6 and Cdt1 proteins are expressed only during S-phase and they bind to ORC

  48. MCM2-7 helicase CDC6 ORC Cdt1 MCM2-7 DNA origin CDC6 and Cdt1 bring the MCM2-7 helicase to the origin The whole complex still needs activation

  49. Phosphorylation of initiation complex Cycline dependent kinases phosphorylate the complex P P CDC6 ORC Cdt1 MCM2-7 P P P DNA origin Now the complex can activate replication

  50. Initiation P P CDC6 Geminin ORC Cdt1 P P MCM2-7 DNA P origin MCM2-7 moves along the DNA and opens the double helix. Other replication proteins can come into action now To prevent further initiation rounds, Geminin protein binds to CDC6 and CDT1, blocking binding of another MCM2-7

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