Transcript Structure of a DNA polymerase

免疫学信息网
http://www.immuneweb.com
Looping the
lagging
strand to
make both
polymerases
move in the
same
direction
免疫学信息网
http://www.immuneweb.com
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.
免疫学信息网
http://www.immuneweb.com
免疫学信息网
http://www.immuneweb.com
免疫学信息网
http://www.immuneweb.com
免疫学信息网
http://www.immuneweb.com
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. coli
that carried out DNA
synthesis in the polA
strains.
免疫学信息网
http://www.immuneweb.com
免疫学信息网
http://www.immuneweb.com
免疫学信息网
http://www.immuneweb.com
Which polymerase is processive?
免疫学信息网
http://www.immuneweb.com
POLIII, b subunit
免疫学信息网
http://www.immuneweb.com
PCNA
Clamp loaders hydrolyze ATP to load clamp
How does one prove
that the clamp ring
is opened during
loading?
免疫学信息网
http://www.immuneweb.com
免疫学信息网
http://www.immuneweb.com
免疫学信息网
http://www.immuneweb.com
Structure of a DNA polymerase (gp43 from phage RB69)
Side view:
Polymerase active site
Top view with
template-primer:
Polymerase site
And
proofreading site
免疫学信息网
http://www.immuneweb.com
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.
免疫学信息网
http://www.immuneweb.com
* 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.
免疫学信息网
http://www.immuneweb.com
Topoisomerase action can be divided into three steps:
nicking (1), strand passage (2); resealing (3).
免疫学信息网
http://www.immuneweb.com
Cycle of topoisomerase activity inferred from structure
1
2
4
3
How would you test that the subunits have免疫学信息网
to open at the lower end to release the T segment?
http://www.immuneweb.com