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Looping the lagging strand to make both polymerases move in the same direction The discovery of DNA polymerase. Arthur Kornberg and Bob Lehman pursued an enzyme in bacterial extracts that would elongate a chain of deoxyribonucleic acid just like glycogen synthase elongates a chain of glycogen. The enzymatic activity was unusual: 1) Needed a template which dictates what nucleotide was added: substrate was directing enzymatic activity 2) Needed a primer annealed to the template. I Wait a minute! Either the polymerase hypothesis was all wrong,…… or there were other DNA polymerases in E. coli that carried out DNA synthesis in the polA strains. 200 polA + (wild type) II 0.4M III 0.2M 100 +NEM 200 polA- (Cairns) II 0.4M III 0.2M 100 I 20 30 Fractions 40 Phosphate (M) John Cairns mutated the gene for DNA polymerase, polA, and the bacteria grew just fine! 3H Thymidine incorporation (pmol) 600 Sub kDa unit Gene 130 27.5 10 dnaE | dnaQ | POL III CORE (mutD) | 71 dnaX ' 47.5 35 33 15 12 dnaX 40.6 dnaN Subassembly | | | COMPLEX | | CLAMP DNA POL YMERASE III 5'-3' polymerase 5'-3' exonuclease 3'-5' exonuclease ATP dependent clamploader processivity factor Sub Gene unit Bacterial Function Eukaryotic dnaE | DNA POL dnaQ (mutD) | POL III CORE | 5'-3' polymerase 3'-5' exonuclease 5'-3' exonuclease dnaX ' dnaX dnaN | | | COMPLEX | | CLAMP ATP dependent clamploader processivity factor DNA POL Fen1 RF-C PCNA CONSERVATION FROM PROKARYOTES TO EUKARYOTES Which polymerase is processive? P POL dNT P Challenge with vast excess of cold primer-template Gel electrophoresis of products - + Challenge + POL-X POL-Y POLIII, subunit PCNA Clamp loaders hydrolyze ATP to load clamp Clamp-loader ATP ATP Clamp How does one prove that the clamp ring is opened during loading? ATP ADP + PP i 3‘OH Structure of a DNA polymerase (gp43 from phage RB69) Side view: Polymerase active site Top view with template-primer: Polymerase site And proofreading site Topoisomerases relax DNA by changing the DNA linking number * Topoisomerases II change the linking number in steps of 2 by passing both strands of double-stranded DNA through a break. * Eukaryotic topoisomerases isolated to date only relax supercoiled DNA, while prokaryotic topoisomerases (gyrases) can, given ATP, add supercoils. * TopoII releases catenated daughter molecules at the end of replication. Inhibitors like etoposide are used in chemotherapy. * Topoisomerases I change the linking number in steps of 1. They pass a single DNA strand through a nick.Topoisomerase I is a protein of the metaphase chromosome scaffold. * In interphase, topoisomerase is bound to the nuclear matrix. * The DNA replication machinery also appears bound to the matrix. * Inhibitor (camptothecin) also used in chemotherapy. Topoisomerase action can be divided into three steps: nicking (1), strand passage (2); resealing (3). 5‘ end of DNA in gate segment is covalently linked to the OH of tyrosine in the active site of topo. Cycle of topoisomerase activity inferred from structure 1 2 4 3 How would you test that the subunits have to open at the lower end to release the T segment?