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 activity2) Needed a primer annealed to the template.

Wait a minute!

John Cairns mutated the gene for DNA polymerase, polA, and the bacteria grew just fine!

Either the polymerase hypothesis was all wrong,…… or there were other DNA polymerases in E. colithat carried out DNA synthesis in the polA strains.

0.2M

0.4M

100

200

20 30 40

Fractions

polA- (Cairns)

I

III

II

100

200

600

3H Thymidine incorporation (pmol)

0.2M

0.4M

Phosphate (M)

polA+ (wild type)

+NEM

IIIII

I

Subunit

kDa Gene Subass embly

α 130 dnaE | 5 -' 3' polymeraseε 27.5 dnaQ

(mutD)| POL I I I CORE 5 -' 3'

exonucleaseθ 10 | 3 -' 5'

exonuclease

τ 71 dnaX

γ 47.5 dnaX |δ 35 | AT P dependentδ' 33 | γ COMPLEX clamploaderχ 15 |ψ 12 |

β 40.6 dnaN β CLAMP processivityfactor

DN A POLYMER ASE III

Subunit

Gene Bacterial Function Eukaryotic

α dnaE | 5'-3'po lyme rase

DNA POL δ

ε dnaQ(mutD)

| POL IIICORE

3'-5'exonuc leas e

DNA POL δ

θ | 5'-3'exonuc leas e

Fen1

τ dnaX

γ dnaX |δ | ATPδ' | γ

COMPLEXde pend entclamploade r

RF-C

χ |ψ |

β dnaN β CLAMP proc ess ivityfactor

PCNA

CONSERVATION FROM PROKARYOTES TOEUKARYOTES

P POL dNTP

Challenge with vast excess of cold primer-template

Gel electrophoresis of products

Challenge - + - +

POL-X POL-Y

Which polymerase is processive?

POLIII, β subunit PCNA

ATP

ATP

3‘OH

ATP

ADP+ PPi

Clamp

Clamp-loader

Clamp loaders hydrolyze ATP to load clamp

How does one provethat the clamp ringis opened duringloading?

Structure of a DNA polymerase (gp43 from phage RB69)

Side view:Polymerase active site

Top view withtemplate-primer:Polymerase siteAndproofreading site

* 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 relax DNA by changing the DNA linking number

* 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 gatesegment is covalentlylinked to the OH oftyrosine in the activesite of topo.

1

3

2

4

Cycle of topoisomerase activity inferred from structure

How would you test that the subunits have to open at the lower end to release the T segment?