6. DNA POLYMERASE
3 important properties
Bacteria- 5 polymerases
Dna polymerase1
3 ' - 5 ' and 5 ' - 3 ' exonuclease activity
5 ' - 3 ' endonuclease activity
Klenow fragment
When DNA
polymerase can
replicate DNA why
other enzymes
required?
27. 4. REPLICATION
Primase associated with pol α—produces RNA primer
Adds deoxynucleotides to RNA and dissociates from
template
Pol δ adds deoxynucleotides on both
leading as well as lagging strand
29. DIFFERENCE
• PROKARYOTIC REPLICATION
1. Initiation point specific(ori)
2. DNA poly-1,2,3,4,5
3. No mitochondria
4. Replication with only 1
replication fork
5. Theta structure observed
6. Only unwinding takes place
7. RNA as primer
8. Okazaki fragments are large
• EUKARYOTIC REPLICATION
1. Many ori
2. Many types-α, β, γ, δ, ε
3. γ DNA polymerase found in
mitochondria
4. Many replication forks
5. Theta structure not observed
6. Histone seperation as well as
unwinding
7. RNA/DNA as primer
8. Okazaki fragments are small
30. Inhibitors of DNA replication
• That affect either template or priming ability of
the growing strand
1. Intercalating agents-
anthracyclins(daunorubicin,doxorubicin)
2. Chain breakage-bleomycin
3. Interstrand crosslinks-mitomycin,nitrogen
mustard
31. • Act directly on polymerase or other enzymes
1. Acyclovir
2. DNA gyrase inhibitor-nalidixic acid
3. Topoisomerase 1 inhibitor- quinolone
Dna –macromolecule-genetic info from 1 to other generation,bp rule mntd,new strand joijnd to old strand by h bonds,high fidelity-mtn genetic stability ,paradox
Parent dna -2 strands complementary to each other,both undergo simultaneous repli,
Prok-1,eukar-multiple,site mostly consist of short sequence of a-t,2 complementary strand sep at site of repli to form bubble,thetarepli
Dna-ds and antiparrallelREPLI IN 5- 3 DIRECTIONsimult, enzyme capable of polymerising in 3-5 doesn’t exist in any org,lead(frward)-cont towards replifork.,lagg(retrograde)-discont(against),sepr of 2 strands reults in y shaped repli fork
5-3 removed –klenow- in recombinant techno.dna poly-chain elongation,processivity,proofreadin
Nucleophilic attack by 3-oh grp of rnaprimeronα phosphate of the first enterindntp with release of pyrophosphatein order to drive the reaction to right,pp removed by pyrophosphataserelease of pp n its subsequent clevage provides energy that ddrivespolynerization
Nick translation-imp lab procedure for producing labelleddna-used as probes-diagnostic n forensic
Pol3 holoenzyme-cntns several diff subunits,retains sum activity even 1 or more subunit ismissin.b subunit-sliding clamp-protein is a dimer of c shaped monomer-pseudosymmetrical 6 ptdstar,+vely charged on inner surface,-ve charge on outer surfacegamma comp-clamp lloader.binds tightly to core
1Topoisomerase-nuclease+ligase(overcumsupercoils);2 topo(gyrase)-cuts both strandsn reseals. Ligase-catalyses formation of phosphodiesterbwdnasynth by dna pol3 n dna pol1,req energy
Helicases-zip opener,atpdependent,ssb-not enzyme-keep 2 strands separate;protect degradation by nucleases
Synth of newdna-abt 5- 50 ntrnareqd as primer(rna polymerase=primase),primase is imp as dna polymerase cant syn de novo,constantsynand supply of rna primer on lagging strand;leadin-1 primercomplex of helicase+primase=primosome
5-3 EXONUCLEASE FN,addsdeoxynt to 3 end of newly synthokazaki.in effect translates the nick at 5 end of rna to that occupied by 3 end
Dormant or non dividing- g0,g1 –active protein syn,g2-enlarment of cell cytoplasm.s phase-greater quantities of dna poly a,all enzymes increased.,nucleardna is replicated once n only once
Many ori aka autonomous replicatin sequence(ars);oricntns base baseseqcald as origin replication element(ore).origin recognition complex
Pol e-syn on lagg. pol a-analo to pol3-multisubunit enzyme.lacks 3- 5 exonuclease.a protein subunit in holoenzyme enables it to bind diadenosine tetra phosphate(small molecule that stimulatsrepli of mammalian cells n is growth signal)major n pcna=identical to b clamp
Shortenin-aplasticanemia,immortality-tumor cells,regulated by trf1(length of telomerase) n trf2(protects the ends).tankrase-alters activity of trf1