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Exploring the role of SeqA in chromosomal replication and damage

Presented by Juhi Arora

MSc (F)27.09.2016

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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

Gel shift assay

Filter binding assay

In situ Footprinting

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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.

https://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.

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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.

http://what-when-how.com/molecular-biology/filter-binding-assays-molecular-biology/

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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.

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Reaction Mechanism

Technique

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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.

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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.

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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.

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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.

http://www.bio-rad.com/en-in/applications-technologies/pulsed-field-gel-electrophoresis#related_content

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• 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.

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.

12To determine which stage is affected.

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Thank You!