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Pharm. 304 Biochemistry Fall 2014 Dr. Brad Chazotte 213 Maddox Hall Web Site:

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1 Pharm. 304 Biochemistry Fall 2014 Dr. Brad Chazotte 213 Maddox Hall Web Site: Original material only ©2000-14 B. Chazotte DNA REPLICATION & REPAIR Student Edition 9/27/13

2 GOALS Examine the mechanism of DNA replication. Learn what steps are involved in replicating DNA. Examine which enzymes/proteins are involved in DNA replication. Understand how and why DNA is repaired Examine the similarities and differences between prokaryotic and eukaryotic DNA replication. Examine repair mechanisms that insure replication fidelity.


4 Voet, Voet & Pratt 2013 Fig 25.1 DNA Replication is Semiconservative The Meselson & Stahl Experiment

5 Voet, Voet & Pratt 2013 Fig 25.2 DNA Replication by DNA Polymerase DNA is replicated using a single strand as the template. The template strand is read in the 3’ → 5’ direction The synthesis of the new (replicated) complementary strand by DNA polymerase occurs only in the 5’ → 3’ The new strand forms a double helix with the template strand that is antiparallel. 3’ OH group of sugar 5’ PO 4 group on sugar Incoming dNTP Voet, Voet & Pratt 2013 Fig 3.3

6 Voet, Voet & Pratt 2013 Fig 25.3; & 2008 Fig. 25.4 Replication of the E. Coli Chromosome “θ structure” DNA synthesis occurs at a replication fork in the replication eye ( θ structure). There may be one fork (unidirectional replication) or two forks (bidirectional replication. Found that θ replication in bacteria is almost always bidirectional.

7 Voet, Voet & Pratt 2013 Fig 25.4 DNA Replication is Semidiscontinuous DNA synthesis is ALWAYS 5’ →3’ direction. Leading strand synthesis is ALWAYS 5’→3’ as well and is able to be CONTINUOUS. Lagging strand synthesis is STILL 5’→3’ direction but must be DISCONTINUOUS. Lagging strand synthesis proceeds with 1K to 2K size fragments called Okazaki fragments that are subsequently linked together by another enzyme, DNA ligase.

8 DNA polymerase requires a free 3’OH on the sugar moiety. RNA primers synthesized complementary to template strand provide a free 3’OH group for the DNA polymerase. In E. Coli PRIMASE synthesizes the RNA primers 1-60 nucleotides long (primer length is species dependent). RNA primer is replaced by DNA later. The leading strand needs one primer the lagging strand needs multiple primers (for each Okazaki fragment). Voet, Voet & Pratt 2013 Fig 24.5 DNA Replication Requires an RNA Primer

9 Major Steps of Prokaryote Replication Initiation Find the designated replication initiation site Separate the strands Unwind the DNA Prevent the single strands from reannealing Synthesis (Elongation) Replisome (has 2 polymerases) moves along the template strands and synthesizes DNA. DNA Polymerase III is E. Coli’s replicase. Check fidelity of replication (proofreading) Joining of Okazaki fragments on the lagging strand (DNA ligase) after removing RNA primer Termination Replication terminus has a number of terminator sites

10 Lehninger (Nelson & Cox) 2005 Fig 25.11 Sequences in E. Coli Replication Origin “oriC Sequences in E. Coli Replication Origin “oriC ” Enzymes need to “know” where to start replication process. Want a start site that has familiar features “consensus” that a protein and/or enzyme can recognize plus certain chemical/physical properties. Note that the sequence on the left is AT-rich.

11 Initiation of Replication In E. Coli replication begins at a region know as oriC – highly conserved sequence in gram(-) bacteria. Multiple copies of protein DnaA bind to oriC. Cause ~45 bp AT-rich sequence (13 bp repeats) to separate into single strands. (“melting”). Hu histone-like protein binds and stimulates initiation Helicase enzyme DnaB recruited by DnaA to oriC. One DnaB binds to each melted strand to further separate the strands. (Helicases act to unwind DNA by traveling along one strand of the double helix.) Single Strand binding protein (SSB) electrostatically binds to DNA behind the advancing helicase to prevent reannealing of the strands. DNA Gyrase (a topoisomerase II) relieves the stress on DNA molecule due to helicase driven unwinding Lehninger (Nelson & Cox) 2005 Fig 25.12

12 DNA Synthesis (Elongation) Involves two related, yet distinct, operations: Lagging and Leading strand syntheses. Accomplished via the replisome, a single multiprotein particle, that includes TWO polymerase III enzymes Problem? - DNA polymerase synthesis is 5’→3’ and strands are antiparallel. How can you synthesize the lagging strand? Berg, Tymoczko & Stryer 2012 Fig 28.9 Basic Concept

13 PRIMING for DNA Synthesis Primosome – a protein assembly that creates a RNA primer complementary to the template strand so DNA polymerase can synthesize DNA. In E. Coli the primosome includes the DnaB helicase and DnaG an RNA-synthesizing primase. Primosome moves in 5’→3’ direction along the LAGGING strand, i.e. towards the replication fork, while displacing SSB. The primosome must initiate EACH Okazaki fragment on the lagging strand. (The leading strand needs only one primer.)

14 Lehninger (Nelson & Cox) 2005 Fig 25.13 Okazaki Fragment Syntheses the Lagging Strand DNA Topoisomerase relieves strain while unwinding DNA. Type II cuts both strands and then reseals. Helicase separates dsDNA. Binds at initiator site and uses energy to separate strands SSB keep single strands separate Primase synthesizes RNA primer DNA polymerase III synthesizes DNA Has to add to a free 3’ OH group

15 Voet, Voet & Pratt 2006 Fig 24.10 E. Coli DNA Polymerase I Klenow Fragment with helical dsDNA Polymeri- zation adds to 3’OH Voet, Voet & Pratt 2013 Fig 25.9

16 Voet, Voet & Pratt 2013 Table 25.1 Comparison of E. Coli DNA Polymerases Replaces RNA primers Main DNA Synthesis

17 Voet, Voet & Pratt 2008 Fig 24.16b β-Clamp of E. Coli DNA Polymerase III around a DNA Molecule and Processivity Processivity: the number of consecutive residues an enzyme can synthesize. The β-clamp of the polymerase III holoenzyme keeps the enzyme synthesizing for >5,000 residues versus 12 in its absence for the Polymerase III core enzyme. DNA molecule

18 Lehninger (Nelson & Cox) 2005 Fig 25.14a Leading & Lagging Strand DNA Synthesis I helicase Topoisomerase II not shown

19 Lehninger (Nelson & Cox) 2005 Fig 25.14b Leading & Lagging Strand DNA Synthesis II

20 Lehninger (Nelson & Cox) 2005 Fig 25.14c Leading & Lagging Strand DNA Synthesis III

21 Lehninger (Nelson & Cox) 2005 Fig 25.14d Leading & Lagging Strand DNA Synthesis IV

22 Lehninger (Nelson & Cox) 2005 Fig 25.14e Leading & Lagging Strand DNA Synthesis V

23 Berg, Tymoczko & Stryer 2002 Fig 27.32 Simplified Schematic Overview of the Overall DNA Replication Process The helicase unwinds the two parental strands SSB proteins prevent the parental strands from rejoining The primase puts on an RNA primer; multiple primers on the lagging strand on the right. DNA polymerase IIIs on the replicase complex synthesize the leading and lagging strands DNA polymerase I on the lagging strand removes the RNA primer and replaces it with DNA DNA ligase seals the nick left by the DNA polymerase I on the lagging strand.

24 Voet, Voet & Pratt 2013 Fig 25.19 E. Coli Chromosome with Termination Sites Replication terminus - A 350 kb region flanked by 7 nearly identical nonpalindromic terminator sites ~25 bp each. TUS protein specifically binds to a Ter site and prevents strand separation by Dna B helicase Termination sites act as one way valves – insures bidirectional replication forks will meet in replication terminus Counter clockwise clockwise

25 Accurate Replication is Important Cells maintain a balance of dNTP’s Polymerase itself uses a two stage process. dNTP complementary binding followed by polymerization 3’→5’ polymerase (I & III) exonuclease activity detects and removes occasional errors. Other cellular repair mechanisms for errors in new DNA or later damage to DNA exist. Using an RNA primer that is later replaced with DNA reduces errors that can arise when the amount of bases for cooperative base-pairing is low.


27 Voet, Voet & Pratt 2008 Table 25.2 Eukaryotic DNA Polymerases - Properties Polymerases and names are different in eukaryotes. PCNA proliferating cell nuclear antigen – a sliding clamp protein

28 Eukaryotic Initiation & Elongation Multiple origins in eukaryotic chromosomes, but each replicon is replicated only once during a cell cycle Eukaryotic DNA is packaged in nucleosomes (more complex chromosome structure) – histones disassemble immediately ahead of replication fork then reassociate with daughter duplexes STEPS: Helicase needed to prepare DNA for replication Separated strands are coated with protein to prevent reassociation replication protein A (viz. SSB). Along with accessory proteins DNA Pol α/Primase start new strand. Pol δ replace Pol α and extends the strand until another replication fork is met (no termination sequence viz. Ter).

29 Voet, Voet & Pratt 2013 Fig 25.23 Eukaryotic Primer Removal Two enzymes involved in primer removal: RNase H1 Flap endonuclease-1 (FEN-1)

30 Voet, Voet & Pratt 2013 Fig 25.25 Avoiding a Bad End: Telomeres and Telomerase Problem: DNA polymerase cannot synthesize the extreme 5’ end of lagging strand. Could result in shorter chromosome each replication cycle Solution: Special enzyme, telomerase, to synthesize DNA at end of chromosome, the region called a telomere. Telomeric DNA – 1000+ tandem repeats of short G-rich sequence on 3’ ending strand of each chromosome end.

31 Voet, Voet & Pratt 2013 Fig 25.26 Synthesizing Telomeric DNA Telomerase – a ribonuclear protein -Telomerase contains an RNA sequence complementary to the repeating DNA sequence (i.e. its own template) -Repeatedly translocates to the new 3’ end of the DNA – adds multiple telomeric sequences. -Normal cellular “machinery” for lagging strand synthesizes the complementary DNA strand to the telomeric sequence leaving a 3’ overhang on the G-rich strand

32 Extension of Chromosome Ends by Telomerase and Polymerase Weaver 2005 Fig 21.34 a) Telomerase hybridizes with G-rich 3’ end of telomere strand. b) Adds TTG (complmentary to AAC on telomerase) to 3’ end of daughter strand c) Then add GGGTTG sequence using RNA template to 3’ end. {Steps a – c can repeat many times} d) When 3’ strand much longer a RNA primer is synthesized by primase complementary to G-rich strand e) DNA pol. Uses primer to fill remaining gap in C-rich strand f) Primer removed leaving 12-16 nt G- rich strand 3’ overhang

33 NEED FOR DNA REPAIR Undesirable changes to DNA if not fixed can become part of the permanent genome. Mutation – a heritable alteration of the genetic information. Notable when they occur in germ-line cells of multicellular organism Changes result from: Point mutations  Transition: a purine (or a pyrimidine) replaced by another  Transversion: a purine replaced by a pyrimidine or vice versa Insertion/Deletion mutations: one or more nucleotide pairs are inserted or deleted.

34 Types of Repair Direct Reversal There are several enzymes that recognize and repair several types of DNA damage – DNA photolyases for pyrimidine dimers. Alkyltransferases for base methylation. Base Excision Repair (BER) – remove bases that cannot be directly repaired, e.g. by DNA glycosylases. Nucleotide Excision Repair (NER) – correct pyrimidine dimers and other DNA lesions where bases are displaced from their normal position or have bulk substituents. Mismatch Repair (MMR) - correct replication mispairing and insertions or deletions up to 4 nucleotides. Bad MMR system then higher incidence of cancer!

35 Lehninger (Nelson & Cox) 2005 Fig 25.23 DNA Repair by Base-Excision Repair Pathway A glycosylase cleaves the glycosidic bond of corresponding type of altered nucleotide removing the base An AP endonuclease removes the apurinic or apyrimidinic site. DNA Pol I – synthesizes replacement DNA DNA ligase seals nick Voet, Voet, &Pratt 2013 Fig 25.33

36 Voet, Voet & Pratt 2013 Fig 25.35 Nucleotide Excision Repair of Pyrimidine Dimers Repair prompted by distortion of the helix structure. Pathway similar in all organisms. In E. Coli. ATP-dependent process cleaves 11nt sequence. Cleaved nt displaced by UvrD (Helicase II) Pol I and DNA ligase repair

37 Voet, Voet & Pratt 2013 Fig 25.36 Mismatch Repair (MMR) - E. Coli MutS binds to mismatched pair or unpaired base. MutS dimer then binds MutL. MutS 2 MutL 2 complex translocates along DNA in both directions to form DNA loop. Parent strand distinguishable due to existing methylation that occurs later to new daughter strand. MutH recruited and nicks strand. UvrD helicase separates strands. DNA polymerase III replaces daughter strand sequence.

38 Voet, Voet & Pratt 2013 Box 25.4 DNA Methylation

39 End of Lectures

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