DNA Replication Chapter 25

DNA Polymerase (E. coli ex) Catalyzes synth of new DNA strand d(NMP)n + dNTP  d(NMP)n+1 + PPi 3’ –OH of newly synth’d strand attacks first phosphate of incoming dNTP Rxn thermodynamically favorable Why??

DNA Polymerase – cont’d Noncovalent stabilizing forces impt REMEMBER?? Base stacking  hydrophobic interactions Base pairing  multiple H-bonds between duplex strands As length of helix incr’d, # of these forces incr’d  incr’d stabilization

DNA Polymerase – cont’d Can only add nucleotides to pre- existing strand So problematic at beginning of repl’n Problem solved by synth of ….

DNA Polymerase – cont’d Primers (25-5) Synth’d by specialized enzymes Nucleic acid segments complementary to template Often RNA Have free 3’ –OH that can attack dNTP

DNA Polymerase – cont’d Once DNA polymerase begins synth of DNA chain, can dissociate OR can continue along template adding more nucleotides to growing chain Rate of synth DNA depends on ability of enz to continue w/out falling off Processivity

DNA Polymerase – Accuracy Enz must ACCURATELY add correct nucleotide to growing chain E. coli accuracy ~ 1 mistake per 109 – 1010 nucleotides added Geometry of enz active site matches geom. of correct base pairs (25-6) A=T, G=C fit Other pairings don’t fit

Fig.25-6

Accuracy – cont’d Enz has “back-up” proofreading ability Its conform’n allows recognition of improper pairing Has ability to cleave improperly paired bases (25-7) Called 3’  5’ exonuclease activity Enz won’t proceed to next base if previous base improper Then catalyzes add’n of proper base Increases accuracy of polymerization 102 – 103 X Note: cell has other enz’s/mech’s to find/repair mistakes (mutations) after new helix synth’d w/ repl’n

Fig.25-7

3 E. coli DNA Polymerases (Table 25-1) I -- impt to polymerase activity Slow (adds 16-20 nucleotides/sec) Has 2 proofreading functions Only 1 subunit II – impt to DNA repair Less polymerization activity Several subunits

3 DNA Polymerases -- cont’d III – principle repl’n enz Much faster than polymerase I (adds 250-1000 nucleotides/sec) Many subunits (Table 25-2) each w/ partic function Encircles DNA; slides along helix (25-10) One subunit “clamps” helix  better processivity

Fig.25-10

Replisome Many other enz’s/prot’s necessary for repl’n (Table 25-3) Complex together  replisome Helicases – sep strands Topoisomerases – relieve strain w/ sep’n Binding proteins – keep parent strands from reannealing Primases – synth primers Ligases – seal backbone What bonds hold nucleic acid backbone together?

Initiation – 1st Stage Repl’n E. coli unique site = ori C (25-11) 3 adjoining 13-nucleotide consensus seq’s Non-consensus “spacer” nucleotides 4 9-nucleotide consensus seq’s spaced apart Consensus seq’s contain nucleotides in partic seq common to many species

Initiation – cont’d At ori C (at 4 9- ‘tide seq area) (25-12) ~20 DnaA mol’s (proteins) bind Requires ATP  nucleosome- like structure

Initiation – cont’d  Unwinding of helix (at 3 13- ‘tide seq area) ~13 nucleotides participate in unwinding Requires ATP Requires HU (histone-like protein)

Initiation – cont’d Unwound helix is stabilized Requires DnaB, DnaC (proteins) These bind to open helix DnaB also acts as helicase Unwinds DNA helix by 1000’s of bp’s

Initiation – cont’d Result: Nucleotide bases now exposed for base pairing in semiconservative repl'n What does semiconservative mean? Yields 2 repl’n forks

Initiation – cont’d Other impt repl’n factors at repl’n forks (Table 25-4) SSB = Single Strand DNA Binding Protein Stabilizes sep’d DNA strands Prevents renaturation DNA gyrase -- a topoisomerase Relieves physical stress of unwinding Note: in E. coli, repl’n is regulated ONLY @ initiation

Elongation Second stage of repl’n Must synth both leading and lagging strands REMEMBER: 1 parent strand 3'  5; its daughter can be synth'd 5'  3' easily. What about the other parent strand (runs 5'  3')?? Follows init’n w/ successful unwinding  repl’n fork, stabilized by prot’s So have parent strands available as templates for base-pairing  2 daughter dbl helices

Elongation -- cont'd – Leading Strand Simpler, more direct (25-13) Primase (=DnaG) synthesizes primer 10-60 nucleotides NOT deoxynucleotides Short RNA segment Occurs @ fork opening Yields free 3’ –OH that will attack further dNTP’s

Leading Strand – cont’d DNA polymerase III now associates Catalyzes add’n of deoxy- nucleotides to 3’ –OH (25-5)

Leading Strand – cont’d Elongation of leading strand keeps up w/ unwinding of DNA @ repl’n fork Gyrase/helicase unwind more DNA  further repl’n fork SSB stabilizes single strand DNA til polymerase arrives Synth continues 5’  3’ along daughter strand

Fig.25-13

Elongation -- cont'd – Lagging Strand More complicated REMEMBER: still need 5’  3’ synth, AND still need to have antiparallel strands. Template strand here is 5’  3’ Can’t synth continuous daughter strand 5’  3’ Cell synth’s discontinuous DNA fragments (Okazaki fragments) that will be joined (25-13) Must have several primers AND coordinated fork movement

Fig.25-13

Lagging Strand – cont’d Lagging strand is looped next to leading strand (25-14) DNA polymerase III complex of subunits catalyzes nucleic acid elongation on both strands simultaneously Primosome = DnaB, DnaG (primase) held together w/ DNA polymerase III by other prot’s

Fig.25-14

Lagging Strand – cont’d One subunit complex of DNA polymerase III moves along lagging strand @ fork in 3’  5’ direction (along parent) Another subunit complex of polymerase III synth’s daughter strand along leading strand At intervals, primase attaches to DnaB (helicase) Here, primase catalyzes synth of primer (as on leading strand) Also (once primer synth’d), primase directs “clamp” subunit of polymerase III to this site This directs other polymerase III subunits to primer

Lagging Strand – cont’d Now polymerase III catalytic subunits add deoxynucleotides to primer  Okazaki fragment Book notes primosome moves 3’  5’ along daughter strand, but both primase & polymerase synthesize strands 5’  3’ along daughter

Fig.25-14

Lagging Strand – cont’d Okazaki fragments must be joined DNA polymerase I exonuclease cleaves RNA primer (25-15) DNA polymerase I simultaneously synth’s deoxynucleotide fragment 10-60 nucleotides Nicks between fragments

Lagging Strand – cont’d DNA ligase seals nicks between fragments (25-16) Catalyzes synth of phosphodiester bond NADH impt (coordination role?)

Fig.25-16

Termination Repl'n has occurred bidirectionally @ 2 forks concurrently E. coli genome is closed circular So 2 repl'n forks will meet

Termination – cont’d Ter = seq of ~ 20 nucleotides (25-17) Tus = prot's that bind Ter When replisome encounters Ter-Tus Replisome halted Repl'n halted Replisome complex dissociates

Termination – cont’d Result = 2 intertwined (catenated) circles Topoisomerase IV nicks chains One chain winds through other 2 Complete genomes sep'd

Eukaryotic DNA Replication Repl'n mechanism & replisome structures similar to prokaryotes, BUT: DNA more complex Not all is coding for peptides Chromatin packaging more complex REMEMBER: nucleosomes, 30 nm fibers, nuclear scaffold, etc. No single origination pt for repl'n Many forks develop  Simultaneous repl'ns bidirectionally Forks move more slowly than in E. coli But efficient because more forks

Eukaryotic DNA Replication Repl'n enzymes not yet fully understood DNA polymerase a In nucleus Has subunit w/ primase activity May be impt to lagging strand synth DNA polymerase d Assoc'd w/ a Impt to attaching enz to nucleic acid chain Has 3'  5' exonuclease ability (proofreading) DNA polymerase e Impt in repair

Eukaryotic DNA Replication Replisome proteins not yet fully understood Found prot's similar to SSB prot's of E. coli Termination seems to involve telomerases Telomeres = seq's @ ends of chromosomes

DNA Alterations Need unaltered, correct nucleotide seq to code for correct aa's  correct peptides  correct proteins Some changes acceptable Some "wobble" in genetic code Some DNA damage in mature cells can be fixed DNA repair mechanisms avail for TT dimers (ex) Have (more) other mature cells that can maintain homeostasis in organism BUT -- if mispaired bases during repl'n  mutation in daughter cell (and her subsequent daughters)

Definitions Lesion = unrepaired DNA damage Mammalian cell prod's > 104 lesions/day Mutation = permanent change in nucleotide seq Can be replicated during cell division Results if DNA polymerase proofreading fails May occur in unimpt region = Silent Mutation Doesn't effect health of organism

Definitions – cont’d Mutation -- cont’d May confer advantage to organism = Favorable Rare Impt in evolution May be catastrophic to organism health Correlations between mutations & carcinogenesis

DNA Repair Cell has biochem mech's to repair damage to DNA Though 104 lesions/day, mutations < 1/1,000 bp's If repair mech's defective  disease/dysfunction Ex: xeroderma pigmentosum UV light  DNA lesions No repair mech  Skin cancers Repair mech ex: base excision repair Takes advantage of complementarity of strands

Base Excision Repair N-Glycosylases Cleave N-glycosyl bonds What parts of nucleic acid are joined by N- glycosyl bonds? Several specific N-glycosylases Each recognizes a common DNA lesion Common -- bases altered by deamination events Yields apurinic or apyrimidinic (AP) site

Excision Repair – cont’d Uracil Glycosylase -- ex Deamination of cytosine  uracil (improper) Enz recognizes, cleaves ONLY U in DNA Not U in RNA Not T in DNA  AP site on DNA (25-22) Would this be apurinic or apyrimidinic? Leaves behind sugar-phosphate of original nucleotide

Excision Repair – cont’d Then other enz's (AP endonucleases) cleave several bases of mutated strand around AP site Then DNA polymerase I catalyzes polymerization of proper nucleotides at site Then DNA ligase seals nicks on sugar- phosphate backbone

Fig.25-22