Lecture 7 Notes: MITx: 7.28.

1x Molecular Biology: DNA Replication and Repair

GOALS:
1.Understand the genetic screening process used to identify DNA replication mutants.
2.Learn different assays to probe DNA-protein binding interactions and DNA unwinding events.

OBJECTIVES:
After completing the materials for this week, you should be able to...
1. Explain the necessity of conditional alleles and give examples.
2.List the steps for generating a pool of temperature-sensitive DNA replication mutants and evaluate
which of the pool are DNA replication initiation mutants.
3.Define specific activity of a protein and summarize the process for maximizing the specific activity
during purification.
4.Given a desired resolution and DNA of interest, design the best assays for DNA unwinding or DNAprotein interaction.
5.Interpret data from DNA unwinding or DNA-protein interaction experiments.
6.Analyze protein structures to infer functional information.

Segment 1:
Genetic screen for DNA replication proteins
Need conditional alleles (alleles that are dead at one condition and alive in another), typically
temperature-sensitive (e.g. active @ 30C vs inactive @ 42C)
1. Protocol:

2. Mutagenize E. coli cells with ethylmethyl sulfate (EMS)

3. Incubate mutagenized cells at 42C for 1.5x cell doubling time (30-45 min). Waiting 1.5 x
doubling time ensures that mutants are fully penetrated, that is they have fully turned off
replication prior to adding tritiated thymidine which will kill any cell that incorporates even a
little bit of it.
4. Add 3H-thymidine (tritiated thymidine a DTTP precursor) to growth media
5. Incubate mutagenized cells + 3H-thymidine for an additional 1.5x cell doubling time (30-45
min) at 42C. The 3H-thymidine will be incorporated into replicating cells and will kill them
but it does not kill cells that are not replicating since the radiation it produces does not penetrate
cell walls.
6. Wash away 3H-thymidine ; resuspend cells in growth media with excess unlabeled thymidine

7. Freeze cells at -80C; leave for one month. This will allow tritium radiation from any 3Hthymidine incorporated into DNA in replicating cells to kill the cell.
8. Plate cells and grow at 30C. Cells not killed by radiation from incorporated 3H-thymidine will
grow.

Segment 2:
In E. coli different mutations stop replication at different points in the cycle resulting in either fast or
slow stops.

Mutations in the proteins that load only once at the beginning of the replication cycle (i.e. dnaA, dnaC)
usually cause slow stops whereas mutations in proteins that are actively involved in replication (dnaB,
dnaE and dnaG) generally cause fast stops.
Segment 3:
How to use mutants?
1. Sort by fast vs slow stop
2. Clone genes sequence genes [over]express gene/protein
3. Epitope tag protein (attaching genetically a short sequence encoding peptide that you have
antibodies (B) to)
1. Immunoprecipitate your protein + any associated proteins
4. Biochemical complementation
1. Take advantage of mutant extract lacking one activity
2. Mame extracts from wild type and temperature sensitive (42C) mutant strains
3. Test the extract for replication activity
1. Wild type Active
2. Mutant Defective (inactive)
4. Add fractions derived from wild type extracts to the mutant extract and assay for replication

Segment 4:
How do you know when a replication protein extract is pure?
Purify until specific activity no longer increases
Specific activity: Number of units of activity (typically arbitrarily defined) per mg total protein
As you purify you remove proteins that are not involved in the specific activity from those that are
involved
Maximize specific activity of protein
Segment 5:
The E. coli origin has these two sequences, the 13-mer and the 9-mer and the 9-mers are what are
recognized by the initiator protein.

The initiator protein binds the 9-mer sequence using a double stranded DNA binding domain located at
its C-terminus. It only binds the 9-mer sequence when already bound to ATP.
The binding of the initiator protein drives the unwinding of the DNA at the 13-mer region (AT-rich,
easier to unwind) of the origin via a ssDNA binding domain that is separate from its dsDNA binding
domain.

Process is much more efficient with negatively supercoiled DNA

dnaC is an inhibitor of dnaB
The dnaC/dnaB complex interacts with the bound dnaA and opens the dnaB ring and places it around
the ssDNA. The dnaB ring is placed in opposite orientations around the opposite strands of DNA.

Synthesis of an RNA primer separates dnaC from dnaB

dnaC disintegrates into monomers when it separates from dnaB and dnaB becomes active

In the absence of dnaC, dnaB begins to unwind DNA

Sliding clamp loaders recognize and bind the leading strand RNA primers and load sliding clamps on
the leading strand primer template junctions oriented to synthesize DNA in the opposite direction of the
helicase

DNA polymerase then extends the primer by adding nucleotides at the 3' end

Primase rebinds and adds a primer to the lagging strand

The primase disengages and the sliding clamp loader loads a sliding clamp and a DNA polymerase on
the PTJ created by the new primer on the lagging strand

The DNA polymerase extends the primer on the lagging strand to create dsDNA and the sliding clamp
loader engages the third polymerase to continue extending the lagging strand

dnaA is the only sequence specific binding protein required for the replication process. All other
binding is either protein-protein (i.e. between dnaA and dnaC) or non-sequence specific protein-DNA
interaction (i.e. helicase to DNA). Most proteins involved recognize structures (i.e. PTJ) rather than
specific DNA sequences.