Lecture Notes 12-13
Biological Physics, Spring 2006
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ydrolysis
MARCH 7th, TUESDAY
4. How can one measure FRET in a single protein having two cysteines? By random labeling and choosing the molecules
having one donor and one acceptor by looking at single molecules.
5. What are leading and lagging strands? What’s Okazaki fragment? Why does the replication stop? What proteins are
involved in replication? The key players in DNA replication include helicase, DNA polymerase, primase and SSB. DNA
polymerase can make new strand only in the 5’-3’ direction. No problem in making the leading strand but the lagging strand
needs to be made in the reverse direction of fork progression.
6. what is the diffusion limited reaction? How can one calculate the theoretical values for diffusion limited reaction?
7. What is ATPγS and why is it a useful research tool? It is an
example of so called ‘non-hydrolyzable’ ATP analogue. An ideal
analogue would bind to the ATPase with the same binding and
dissociation rate as the ATP itself but would have zero rate for
hydrolysis. That is, it will be close enough in structure to ATP but
not too close. In reality, there is always finite hydrolysis of these
non-hydrolyzable analogues and they should really be called
‘slowly-hydrolyzing’ ATP analogue. They are very useful if one
wants to study the effect of ATP binding on varous properties of
the enzyme such as structure, binding affinity, assembly state
(monomer, dimer, oligomer,…). A well known fact is that kinesin
binds extremely tightly to microtubules in the presence of
AMPPNP, another slowly hydrolyzing ATP analogue, and people
use this fact in order to purify kinesin from bovine brain as large
and heavy microtubules are easy to separate from other proteins.
Another use of slowly hydrolyzing ATP analogues (ATPγS,
AMPPNP, ..) is to test if a certain reaction requires ATP h
or just ATP binding.
8. What’s recombination? There are two types of DNA recombination, homologous (or genetic) recombination vs. site-
specific recombination. We will discuss only the homologous recombina tion here. If there are two homologous (meaning
nearly identical or very close sequences) DNA, celluar proteins can catalyze a reaction where the two DNA molecules join to
form an intermediate called Holliday junction (or DNA four-way junction), followed by branch migration and finally
resolved back to two dsDNA molecules. The net result could be sequence exchange between the two DNA on a local or
global level depending on how the junction is resolved. A traditional view of homologous recombination has been that this is
very useful in generating genetic diversity for evolution to choose from. The modern view is that its main function is DNA
repair during replication (3R’s, repair, replication, and recombination are all highly coupled!). Note that in bacteria with just
one copy of chromosome, homologous DNA would be routinely encountered only during DNA replication as there are two
daughter DNA molecules being made. If there is a DNA damage causing problems during replication, the Holliday junction
intermediate can form using the two daughter DNA molecules (they are highly homologous of course), and in still poorly
understood manner damage is repaired and replication can begin. Out of 12 helicases in E. coli, at least of them are
implicated in recombination (UvrD, Rep, RecQ, RecBCD, RecG, RuvB, and DnaB).
9. What is RecA filament? How is it used in DNA recombination? Where does the name come from? In genetics, it is
common to denote the gene itself (that is the DNA portion encoding the protein) is written in lowercase (such as recA, rep,
uvrD,…) while their protein products’ names start in uppercase (RecA, Rep, UvrD,…). In genetics, mutants are identified
and named according to the consequences of genetic mutations. For example, recA, recB, recC, … were first identified as
mutants having defects in DNA recombination, and named alphabeticaly. Some genes are later found to be identical to each
other and those redundant names then disappear from literature. Obviously RecA is the first gene identified and is the most
important protein for homologous recombination in bacteria.
