SeqA is known to bind and sequester hemi-methylated DNA. This is an important step in regulation of DNA replication. Its role in replication has been discussed. A possible role of SeqA has also been suggested in chromosomal damage based on a series of experimenents by researchers. Major techniques are outlined.

1. Exploring the role of SeqA in
chromosomal replication and
damage
Presented by
Juhi Arora
MSc (F)
27.09.2016
1

2. This study focuses on deciphering the in vivo role of SeqA and Dam methylase on oriC.
 How do they interact?
 Where do they interact?
Known so far:
 SeqA preferentially binds to hemi-methylated DNA. It sequesters oriC and leads to a 13
min delay in methylation after replication.
 SeqA null mutants show increased and asynchronous chromosome initiation
 SeqA was identified as a protein bound to hemimethylated bacteriophage P1 origin
2
Gel shift
assay
Filter binding
assay
In situ
Footprinting

3. Gel Shift Assay
• Also known as Electromobility shift assay (EMSA)
• When DNA is bound to protein, the complex migrates slower than free
linear DNA in non-denaturing conditions or agarose gel electrophoresis.
• That is, the rate of complex migration is shifted or retarded.
3https://www.thermofisher.com/in/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-
resource-library/pierce-protein-methods/gel-shift-assays-emsa.html
• Competitor blocks non
specific binding.
• Common competitors:
salmon sperm DNA,
poly (dI.dC)
• The oligonucleotides
maybe radiolabeled,
fluorescently labeled or
attached to DIG/hapten
for detection.

4. Filter Binding Assay
• Exploits differential binding of nucleic acids and proteins to nitrocellulose
filters.
• In a protein:DNA complex, protein interacts with nitrocellulose via
hydrophobic interactions, allowing the complex to bind to the filter.
• Free DNA passes through.
• DNA:protein complex must be stable. Reaction conditions (such as pH)
may vary depending on properties of the protein.
4
http://what-when-how.com/molecular-biology/filter-binding-assays-molecular-biology/

5. In situ footprinting using 1,10-phenanthroline-copper ion (OP-Cu)
• OP-Cu is an efficient chemical nuclease that generates single stranded
nicks in DNA.
• Cleavage with OP-Cu generates DNA footprints that help in studying the
interaction of proteins with specific DNA sequences.
• OP-Cu attacks the deoxyribose moeity thus breaking the phosphodiester
bond.
• Products of cleavage reaction include: Free base, DNA fragments
terminating in 5’ or 3’ends and 5-methylene-2-furanone (oxidation
product of deoxyribose)
• The 5’end of the oligonucleotide must be labeled (radioactive or
fluorescent).
• The region where the protein binds to DNA is not visible on the gel and
can be mapped by loading free DNA fragments.
5

6. 6
Reaction Mechanism
Technique

7. 7
Gel Shift Assay
shows preferential
binding of SeqA to
hemi-methylated
oligonucleotides.
Filter binding assays were also
performed with hemi-methylate,
fully methylated and unmethylated
DNA sequences. 1 unit is defined as
the activity that binds to a quarter
of the labeled probe. This assay also
shows preferential binding to HM
DNA.

8. 8
In situ Footprinting with OP-Cu
Gel shift assay reveals that SeqA
and Dam bind to the same sites.
Dam methylase is unable to
dissociate SeqA upto at least 4
minutes in vitro.

9. 9
SeqA is speculated to be involved in preventing chromosomal fragmentation. This
assumption is based on the observation that in one co-lethal mutant (recA-), seqA was
found to be inactivated.
 Does absence of SeqA lead to chromosomal damage? If so, how?
 What are some other factors affecting this?
 Which stage of the cell cycle is affected?
 Possible mechanism of action.
Known so far:
 SeqA is known to channel new DNA to the place of nucleoid condensation.
 SeqA mutants have increased no. of replication origins per cell yet normal ori/ter
ratios.
 Double-stranded breaks may form due to incompletely segregated nucleoids.
 SeqA mutants have increased supercoiling.

10. Pulse field gel electrophoresis (PFGE)
• Larger fragments (>20kb) of DNA tend to comigrate and appear as a single
bulky band on conventional gel electrophoresis.
• In PFGE, alternating electrical field is applied in different directions.
• DNA molecules elongate upon application of electric field and relax upon
removal of electric field.
• Rate of relaxation depends on size of DNA.
• When orientation of electric field is changed, DNA molecules must return
to their elongated form prior to re-orientation.
10
http://www.bio-rad.com/en-in/applications-technologies/pulsed-field-gel-electrophoresis#related_content

11. • A series of experiments had established that SeqA depends on RecA i.e.,
seqA, recA double mutants could barely grow.
• Various constructs were created wherein SeqA was either deleted or
interrupted. These were transduced into strains containing SOS proteins
with lacZ fusion. Thus, SOS response could be monitored and it was found
that seqA mutation led to SOS induction.
• Thus, seqA mutants exhibited chromosomal damage.
11
SeqA mutants were propagated
into recBC+ and recBC- strains.
RecBC was inactivated using Gam.
Chromosomal fragmentation in
SeqA mutants was observed more
in the absence of recBC.
This shows that while SeqA
mutants are prone to
chromosomal fragmentation, this
is reparable by recBC.
Thus, SeqA depends on
recombinational repair system.

12. 12To determine which stage is affected.

13. 13
Thank You! 