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DNA Replication 1-General Principles

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1 DNA Replication 1-General Principles
2-The enzymology of DNA polymerases A-General Properties: B-Bidirectionality and Priming problems C-Catalytic Properties of DNA polymerase I Polymerase 5’->3’ exonuclease 3’->5’ exonuclease and proofreading 3- Prokaryotic DNA Replication A - The prokaryotic Replisome B - Interference between Replication and Transcription • Not treated: Old experiments (Meselson & Stahl, Cairns) 4- Eukaryotic DNA Replication A-The eukaryotic Replisome B- Dealing with Chromatin C-Dealing with linear chromosomes: Telomeres and Telomerase

2 DNA Replication by DNA polymerases : Copying the genetic material to prepare for cell division
Parental DNA Replication is Semi-Conservative: 1 parental strand is transmitted into each daughter DNA molecule (Meselson & Stahl exp.) 2 Copies Replication is bidirectional from the Origin of Replication: (Cairns exp.) • 1 origin of replication in most eubacterial chromosomes • several origins of replications in archea, some eubacteria, and in eukaryotes

3 Fundamental properties of DNA Polymerases
Base P OH O- Base n 5’ HO Base n+1 : + 5’ 3’ primer template dNTPs 5’ 3’ New strand PPi Catalyze the polymerization of deoxyribonucleotides in the 5’->3’ direction: (dNMP)n+dNTP -> (dNMP)n+1 + PPi 2) Require a template (usually DNA) 3) Require a primer (DNA or RNA)

4 The bidirectionality problem
Synthesis of DNA on the lagging strand requires continuous synthesis of primers Synthesis of DNA is semi-discontinuous

5 Where do the primers used during DNA replication come from ?
The primers are made of RNA since RNA polymerases do not require primers. The existence of joint RNA-DNA molecules was demonstrated by alkaline hydrolysis of Okazaki fragments. DNA synthesis B B OH P B OH PPP PPP OH OH rNTPs a32P-dNTPs Alkaline hydrolysis B B B B B B B OH OH OH OH OH OH OH P P P P P P Ribonucleotide with a 3’P- : Diagnostic of a 5’RNA-3’DNA junction alkaline hydrolysis

6 DNA Polymerase I = the prototype DNA polymerase
- Discovered in the late ‘50s by Arthur Kornberg (1959 Nobel Prize in Medicine) - First DNA polymerase discovered - 3 Enzymatic activities associated to three distinct active sites on a single polypeptide chain The activities can be artificially separated by experimental treatment with the trypsin protease ( these fragments have no physiological relevance) 109 kD +NH3 COO- Limited Tryptic proteolysis 34 kD 75 kD = Klenow Fragment 5’ -> 3’ Exonuclease 5’ -> 3’ Polymerase 3’ -> 5’ Exonuclease

7 (T.Steitz) Two metal ion mechanism for DNA Polymerase I
- Me=divalent metal ion (usually Mg++) MeA=activates the 3’OH for attack on the a phosphate of the incoming dNTP (lowers pKa of 3’O) MeB=plays the dual role of stabili- zing the neg.charge that builds up on the leaving oxygen and chelating the  and  phosphates MeA and B stabilize both the structure and charge of the pentacovalent transition state

8 How does DNA Polymerase I select correct nucleotides from incorrect nucleotides ?
forming non Watson-Crick base pairs do not fit the active site and are ejected Suggests that H-bonding per se does not contribute to nt selection by DNA polymerases Nucleotides forming Watson-Crick base pairs fit the active site (BLUE SQUARE)

9 select the proper nucleotide: Testing the importance
How do DNA polymerases select the proper nucleotide: Testing the importance of H-bonding in base pairs for the fidelity of nucleotide incorporation Thymine Di-fluorotoluene Can H-bond with A Same size as Thymine but cannot H-bond with A 5’ 3’ X Template = 24 nt Labeled primer = 23 nt After extension = 24 nt A,C,G,T or F ?

10 Discrimination for deoxynucleotides vs ribonucleotides
by DNA polymerases Alignment of sequences of polymerases active site Use dNTP Use riboNTP Use dNTP Use riboNTP Catalytic Asp The aromatic/large side chain found in DNA polymerases close to the dNTP binding site provides a steric gate against riboNTPs Steric Clash with the Y416 residue if a 2’-OH is present Biochemistry, 41, 10256–10261

11 3’-> 5’ exonuclease activity of DNA Polymerase I
OH O- Base n (now last base) 5’ Base n+1 (Last Base of DNA) H : -O 3’-> 5’ exonuclease activity of DNA Polymerase I This reaction is not the reversal of the 5’->3’ polymerization: The attacking group is water rather than pyrophosphate (a hydrolysis rather than a pyrophosphorolysis). For this reason, the active sites of the polymerization and of the 3’->5’ exonuclease reactions must be different. This is essential for the biological role of the 3’->5’ exonucleolytic reaction, which is to edit newly polymerized sequences.

12 e P2 = P1 Not making mistakes during polymerization is
thermodynamically impossible 5’ 3’ A 5’ template P1 P2 5’ T 5’ C 3’ A 5’ 3’ A 5’ template template P2 = probability of incorporating one incorrect nucleotide e.g. A C mismatch P1 = probability of incorporating the right nucleotide A:T base pair P2 P1 = e (DGAxC DGA:T) / RT (derived from the Boltzman distribution) P2 DGAxC DGA:T = 3 kcal/mol. = 0.01 or 1% - at least P1 DNA polymerases are much more accurate than HOW ??

13 Editing of newly synthesized DNA by the 3’->5’ exonuclease activity
template Polymerization 5’ 3’ 5’ template 3’->5’ exo triggered by the mistake New strand 5’ 3’ 5’ 3’->5’ exo 5’ 3’ 5’ Polymerization 3’ 5’ 5’ Editing of mistakes require a switch between : polymerization mode and editing mode

14 Switch between Polymerizing and Editing Modes
in DNA Polymerase: Structural Basis for “Proofreading”

15 5’-> 3’ exonuclease activity of DNA Polymerase I
OH O- Base 1 Base 2 5’end -O H : (now base 1) New 5’end Biological significance: Allows the replacement of damaged or abnormal DNA sequences by “Nick translation” (important for DNA Repair Chapter) Also allows the removal of RNA sequences embedded in DNA (removal of replication primers). dNTPs PPi The nick has moved “bad” DNA nick New strand 5’ 3’ 5’ 3’ 3’ 5’ 3’ 5’

16 The replisome of E.coli 1) Helicases 6) DNA topoisomerase II
Unwind DNA at the replication fork in a reaction coupled to ATP Hydrolyis 2) Single-stranded DNA binding proteins (SSB) Bind and stabilize the DNA in a single stranded conformation after the melting by helicases 3) The Primosome Synthesizes RNA primers of the lagging strand Contains Primase 4) DNA Polymerase III :The replicase 6) DNA topoisomerase II Relaxes supercoiled DNA that forms ahead of the replication fork. Decatenates the final product 7) Rnase H Removes RNA primers 8) DNA Polymerase I Replaces RNA primers with DNA by nick translation 8) DNA Ligase Joins the Okazaki fragments

17 The Replisome of E.coli in action “old model”
Trends in Microbiology 15, (2007) DNA Polymerase III Core - 130 kD = Catalytic site for polymerization kD = 3’->5’ editing exonuclease q kD = structural role ? b Subunit = Sliding Clamp A homodimer of 2 X 41 kD ATP-dependent processivity factor = b clamp Pol.III Core is poorly processive by itself Complex = Clamp loader t Subunit The Replisome of E.coli in action “old model” 4 polypeptides ATP-dependent conformational changes Facilitates the loading of the b clamp onto DNA Dimerization factor Holds two Pol. III cores together

18 Sliding b clamps provide processivity to DNA polymerase III
3’ b- Clamps Newly Replicated Strand 5’ homodimer of 2 X 41 kD wrapped around dsDNA Template Strand 3’

19 The replisome of E.coli in action: Cycles of molecular events during Lagging Strand synthesis (1)
Step1: The primase synthesizes a new RNA primer upstream in the lagging strand; the two polymerase replicate DNA Step2: A sliding clamp is assembled around the new RNA primer; primase dissociated Trends in Microbiology 15, (2007)

20 The replisome of E.coli in action: Cycles of molecular events during Lagging Strand synthesis (2)
Step2: A sliding clamp is assembled around the new RNA primer; primase dissociated Step3: the lagging strand polymerase detaches and associates with the newly deposited sliding clamp Trends in Microbiology 15, (2007)

21 The replisome of E.coli in action: Cycles of molecular events during Lagging Strand synthesis (3)
Step3: the lagging strand polymerase detaches and associates with the newly deposited sliding clamp Step4: the lagging strand polymerase start to synthesize the next Okazaki fragment; primase will reinitiate synthesis of the next RNA primer  back to Step1 Trends in Microbiology 15, (2007)

22 The Clamp loader use cycles of ATP hydrolysis to open and load
the sliding clamps around the primed DNA Binding of the Clamp loader to b-clamps; Opening of the b-clamps; Loading the b-clamps on a primer-template duplex Recycling of the clamp loader Kelch et al. - Science 23 December 2011: Vol. 334 no pp

23 Newsflash – Recent works shows that There are 3 core DNA polymerases
associated with most replisomes in vivo Nature Structural & Molecular Biology Volume: 19, Pages: 113–116 (2012) What is the advantage of 3 Polymerase replisomes vs 2 Polymerases ? Science. 2010 328(5977): 498–501

24 Why is a triPolymerase replisome ?
 TriPol.III diPol.III • TriPol.III replisome is more processive • TriPol.III replisome leaves less gaps to be filled by Pol.I -> DNA Replication is overall more efficient Nature Structural & Molecular Biology Volume: 19, Pages: 113–116 (2012)

25 Problem of Coordinated Nucleic Acids Synthesis in vivo:
What happens when DNA Polymerases and RNA polymerases collide ? (speed of DNA polymerase >> speed of RNA polymerase ) Nature 456, (2008)

26 Collision between DNA Polymerases and RNA polymerases result in polymerases dissociation and in the use of the RNA synthesized by RNA Polymerase as a primer for replication Nature 456, (2008)

27 Eukaryotic DNA Replication: it’s similar to bacterial DNA replication….
but it’s different ! • Machinery is overall similar to that use for bacterial DNA replication (names are different…) • Idiosyncraties of eukaryotic DNA replication are linked to the size and organization of eukaryotic genomes: • large size of eukaryotic chromosomes and limited time for DNA synthesis requires multiple origins of replication • Replication machinery needs to deal with nucleosome packaging of eukaryotic DNA • Problem of linear chromosomes

28 Architecture of the Eukaryotic Replisome
is similar to that of the bacterial Replisome Pol e - Replicates leading strand Pol d - Replicates lagging strand PCNA: (proliferating cells Nuclear antigen): = trimeric sliding clamp MCM = heterohexameric helicase FEN1 = nuclease that removes RNA primers Pol -primase Complex containing both primase and DNA polymerase a activities Mol.Cell 30, (2008) Replication Protein A (RPA) = SSB

29 The eukaryotic cell cycle
Eukaryotic DNA Replication: The limited time for DNA replication (6-8 hours) combined to the increased size of the genomes (>107 base pairs) explain the requirement for multiple replication origins

30 Histone chaperones Eukaryotic Replication needs to deal with the
nucleosome packaging of eukaryotic DNA • Needs to Remove Histones upstream from the replication fork such that replication is not impeded • Needs to reassemble histones/nucleosomes on newly replicated DNA to maintain chromatin structure and epigenetic marks Histone chaperones Example of Asf1, an H3/H4 histone chaperone that interacts with MCM MCM Replication Asf1 remove H3/H4 histones upstream from MCM and reloads them after replication has proceeded H3/H4 Asf1 can also add “new” H3/H4 histones since there is only ½ of the histones needed on the parental DNA Science 21 December 2007: Vol no. 5858, pp

31 Eukaryotic DNA Replication: Problem of maintaining the ends of linear chromosomes is linked to the degradation of RNA primers last primers on each 5’-end are removed but the gaps cannot be filled because of the lack of 3’-OH group Each cycle of replication would result in progressive chromosome shortening

32 Preservation of Telomeres by the telomerase
Telomerase (TERT): 1 RNA subunit (template) several proteins: 1 “reverse transcriptase” After extension of the upper strand by telomerase, the replication machinery can now use this strand to make a new RNA primer using primase, then a new okazaki fragment and fill in the lower strand to “elongate” this strand.

33 © 2010 European Molecular Biology Organization
The problem of Processivity in Telomerase : Telomerase is poorly processive in vitro - this cause issues because of the repeated nature of telomeric repeats The Pot1-Ttp1 complex provides processivity to telomerase in vivo Pot1-Ttp1 form a protein complex that binds to ss telomeric DNA Pot1-Ttp1 Pot1-Ttp1 Complex increase processivity by: decreasing telomerase dissociation from tel. DNA increase effciency of the translocation rate The EMBO Journal 21 January 2010/emboj © 2010 European Molecular Biology Organization

34 Connections between telomerase and aging
Humans Somatic tissues lack telomerase, possibly contributing to the normal physiological symptoms of aging. Shortening of telomeres may lead to senescence in cultured human cells Stem cells, and cancer cells all contain telomerase activity possibly explaining their ability to divide indefinitely. Germ cells and early embryos also contain telomerase. Mice Telomerase protein or RNA mutant mice are fine for a few generations, perhaps because of the extraordinarily long telomeres in laboratory mice After a few generations, the telomerase-mutant mice exhibit reduced fertility, signs of premature aging, and shortened life-span Rudolph et al (1999) Longevity, stress response, and cancer in aging telomerase-deficient mice.Cell 96, The picture is of a 3rd generation telomerase RNA mutant mouse. Gonzalez-Suarez E, Geserick C, Flores JM, Blasco MA. (2005) Antagonistic effects of telomerase on cancer and aging in K5-mTert transgenic mice.Oncogene Jan 31; Prematurely grey, balding

35 Accelerated telomere shortening in response to life stress
PNAS 101 (49), (2004) “Women with the highest levels of perceived stress have telomeres shorter on average by the equivalent of at least one decade of additional aging compared to low stress women”.

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