Single-strand binding proteins

DNA replication, part 2

Table 20.1

Figure 20.19

Stimulation of DNA synthesis by T4 phage SSB,

gp32

amB17(gene23)

tsP7 (gene 32), amB17(gene23)

amE727 (gene 49), tsP7 (gene 32),

Effect of the SSB gene 32

Topoisomerases

Rubber band model of

supercoiling in DNA

Nicking one strand - relaxation of supercoiling

Problem of Strain due to Unwinding of DNA by Helicase is solved by the Swivel Concept

DNA gyrase is a a DNA topisomerase

relaxed

supercoiled

•Topoisomerase II forms a covalent DNA-enzyme intermediate with its Tyr residues

Cyrstal structure of yeast DNA topoisomerase

Model in segment passing in topoisomerase II

DNA polymerases

Proofreading in DNA synthesis

DNA polI

Crystal structure of the Klenow fragment

Purification of DNA polymerases by DEAE sephacel

polA1 strain

Wild type with & w/o N-

ethylmaleimide (blue line)

Separation of polIII core and alpha subunit

Exonuclease activity of the epsilon subunit

Perfectly paired DNA substrate

Imperfectly paired DNA substrate

Termination of DNA replication

Replication Leaves Catenated Daughter Chromosomes

Two-step Model for Decatenation in Prokaryotes

Cozarelli and Colleagues: Topo IV is Responsible for Decatenation in Bacteria

•Studied decatenation of intertwined pBR322 plasmids

•Used ts mutations S. typhimurium; Mutations were in gryA (encodes gyrase), parC and parE (encode two subunitsof topo IV) and parF (unrelated gene linked to parC and E)

•Found mutations only in parC and parE (and not gyrA) prevented decatenation. Therefore, topo IV acts in decatenation.In eukaryotes topo II is the probably the decatenating enzyme.

The problem with replication ofLinear DNA

Telomerase activity on four repeats of TTGGGG

Tetrahymena

Telomeres in the protozooan Tetrahymena

Telomere binding proteins in ciliates and

yeast