 I'm Umair Phan, as you can see, and I'm from the Institute of Bioinformatics and Biotechnology at the University of Pune, near Bombay. And I'm working in the lab of Michael Cox with Pharming Ego, who is a graduate from there. And the title of my work is The Role of SSB in Homologous Recombination Catalyzed by Rekha in Dinakoka, St. Louis. I'll start by introducing the organism I'm working with. This is the worst of bacteria that has been listed in the history of the world record. It is capable of standing at a acute dose of some sort of radius of idealizing radiation without almost no loss of vitality. And to give a perspective on this, to kill an average human being's 5 to 10 grains of idealizing radiation, you know, so you can imagine how tough this is. So why is DRAT so radio-resistant? So there are a lot of characteristics which have attributed to this radio resistance of this organism. If they say that it is extremely good at DNA reconstruction, why is it so? They say that it has highly condensed chromosomes so that after a double-stranded break that occurs in the response to idealizing radiation, the strands of DNA do not diffuse out so that they can be joined together. And it has a high manganese to ion ratio in the cell. What manganese does is it sequesters out the hydroxyl radicals that are produced in the response to the idealizing radiation which causes the DNA damage. It has 4 to 10 copies of the genome depending on the stage of the cell cycle. This has been, I would say, to decrease the chances that a particular gene will be obliterated during a DNA damage. And most importantly for us that what we work on and what is most fascinating is that it has really efficient DNA repair system that's compared to other prototypical DNA repair systems in other bacteria. So here are the DNA repair pathways which have been reported in DRAT that is the homologous recombination. The extendance in this is even standardly the single-strand annealing. These two occur early on in the response to radiation and that is the non-homologous end-joining which has been recently reported. What it does is it joins non-homologous ends and this homologous recombination pathway which is the most important pathway for DRAT resistance which I'll be talking about over here. The RK protein plays a central role in the homologous recombination pathway which I have shown over here. It is important for the SOS response which happens after cell damage to activate a lot of things in response to the DNA damage. It acts as a recombinant during homologous recombination repair pathway. It is 80% dependent DNA protein over here. This is how homologous recombination by RK happens. One step, RK forms a nuclear protein on the single-stranded DNA and it recognizes the double-stranded homologous DNA. There is a synapsis which happens and then in the post-synapsis there are the inhibitors which are formed and which leads to the formation of a NIC double-stranded new product and dissociated single-strand. Over here in recombination, as you can see, RK binds to the single-strand overhangs. It invades homologous regions on the double-stranded region of other chromosomes and polymerases, helicases and nucleases are recruited to repair this damage. One thing that became quite apparent early on in DRAT research was that RK is extremely important for radiation resistance. As you can see, there is a stain with a mutation in RK. There are 74 degrees in viability in response to DNA-davaging agents which is extremely important for the radiation resistance of DRAT. Here I will introduce another protein which I have been working on which is the single-stranded DNA binding protein. It is a single-stranded DNA binding protein. It is really efficient at mounting on secondary structures in single-stranded DNA. People should think it is an inert sequence. SSVs are slightly inert and they really don't associate with other proteins and they just are involved in binding to single-stranded DNA. But work in Jim Keck's lab in Wisconsin has shown that SSVs are not quite that inert. They have been linked to various aspects of genome maintenance. They have been shown to interact physically with the overall area of cellular genome maintenance proteins. What we are interested in is that SSVs have been reported to facilitate standard change by RK. You know how it is written in SSV. It is slightly different from the prototypical E. coli SSV. It has two oligolimprote binding poles instead of the four in E. coli SSV. It forms a dimer whereas the E. coli SSV forms tetramer. It has been implicated in chromosome repair and its importance was simulating the DNA standard change by RK. The aim of the project was to determine the role of SSV in recombination reaction by RK and to study the role of phospholation in the regulation of SSV. Talking about the role of SSV in recombination reaction by RK first. So over here, this is the method I have used. This is the in vitro three-strand exchange assay. This is used to simulate the recombinase activity of RK in vitro. As you can see, we indicate the single-stranded DNA with RK. RK forms the equipment and then we introduce the double-stranded DNA. There is a synapsis formation and then the products are formed as the recombination progresses. Here the beauty of this assay is that we can monitor the recombination reaction as it is happening by taking time points. So as you can see, there is a single-stranded DNA and if recombination has started, the synapsis happens, the intermediates are formed and at around 30 minutes the product is starting to form. The previous work in the lab has shown that SSV is extremely important for initiation of standard exchange. As you can see over here, the same standard reaction with DRRK and DRSV we show that in the presence of DRSV, we can see the product formation, but in the absence of it, there is not even the initiation of standard exchange. So DRSV is extremely important for catalyzing the DRRK SSV immediately. So we wanted to know what is the role of DRSV in the overall recombination. Two models for this. One that DRSV removes secondary structures so that on the single-stranded DNA so that it can form nucleophilaments on it. And the second model was that DRSV sequesters the display single-stranded DNA to drive the reaction forward. So as you can see, these are the secondary structures in the single-stranded DNA. SSV makes sure it cleans out the secondary structures so that the RK can come in, form an equilibrium on it, and proceed with the standard exchange. And for this, the external set of the design was we are going to use five polygons and we are going to use substrates with and without and with secondary structures so that the secondary structures can see how the reaction progresses in the absence and in the presence of SSVs. The second model is that SSV sequesters the display single-stranded to drive the reaction forward. As you can see, we added the RL standard DNA, there is SSV formation, and we introduced the SSV and it would bind to the stand which is being displaced and pull it out and literally pulling the reaction forward. To test this, we are going to replace the SSV which we used in the reactions with other agents such as nucleases or other DNA binding proteins to make it the activity of pulling the displaced stand, so the results. So as we all know, a lot of roadblocks we do along the way, we work on that science. So what we are doing was we are using untied polygons at first and what we realized that was these small polygons we could resolve on the polygons gel so we have started using spam type polygons to monitor the recombination reaction and we have seen quite a lot of mobility shift due to SSV binding so we have been treating the samples with protein S8 and SSVs. There is a result over here where we did a standard reaction with substrates which have no secondary structures, so over here this is the road standard DNA and as you can see recombination is occurring as a type of SSV and in the absence of SSV also there is the reaction happening. So we can say that probably SSV is required to remove the secondary structures so that the recombination reaction can proceed and another interesting observation on this result was that after the formation of the product, you can see a lot of intermediary higher heavier intermediary being formed and you wanted to know what these are and what would be the structure of these so that it would shed a light on the activity of SSV and recombination. So obviously you can see that the role of the SSV might be to remove the secondary structures to facilitate the recombination mutual treatment formation and as you can see I have shown you that there are some interesting intermediary that have been formed, you want to see what these would look like so you can speculate these kind of structures that have been formed and this would shed a light on how recombination reaction is performing or we might end up realizing that SSV and recombination are still born to it. So in the future we would be using electron microscopy to determine the structure of these intermediaries electron microscopy and resonance analysis will show whether the SSV and recombination are still attached to these substrates and we need to perform the other experiments which I showed earlier to establish a definitive model for SSV function. So here I will briefly describe the second part of the project that we are doing over here that is to study the role of phospholation in the regulation of SSV. Post-structural codification is extremely well known in heat carriers but it has only recently reported in low-carriers reports have shown that single-studied DNA-vining proteins get phosphorylated and paper last year showed that they have discovered a protein kinase that is active and which responds to DNA damage. So we wanted to study if SSV really gets phosphorylated, the D-Ride SSV and then perform best-in-dot and master catalyst to identify the phosphorylation in SSV and to study if there is a change in the phosphorylation states of these proteins in response to radiation. So the workflow, the cloning the SSV and the recombination to D-Ride these are the over-extractors and then transfer this cloning to D-Ride over-extract these proteins to verify the amount of different external conditions that is radiation without radiation and then use anti-tyrosine phosphate as the body is on a western cloud to detect phosphorylation and then to perform mass-prick analysis to see whether where the site of phosphorylation in these proteins is. Till now we have been able to clone the SSV and recade protein into the over-extractors and transfer which is constructed to D-Ride and we will try to, we will try to over-extract these proteins whether D-Ride has purified them and see if they are getting phosphorylated I still have 10 days here, unlike others and I hope to get something from it. I would like to acknowledge Dr. Michael Cox for he gets this one and the rational I will work with Khandin Ego, me and all the other COXLAB members my fellow Parama fellows you used to have sponsored medicines for giving the opportunity to come here and the Purao program and IUSS, TEF and DBT India for funding my project. So what do you think those intermediate sites look like? That is what I am speculating for that. So you think these are the only structures that you would see? You have more species than just two so these are the different parts those are the same, those are the different structures that have been formed So these are the two species that I have seen which I think there might be other intermediate sites that I am speculating And what do you think those structures might look like? The other ones that you can see if you just go back to your job space here So the two crossovers that you just showed which one do you think those are in this series of bands? We are not sure yet what they are but we think the higher one would be the other one the second one and the lower ones would be two single strands at a time the smaller 50-way space attached to the bigger 100-way space and a lot of in-between intermediate and complex So I am surprised that without your SSB still forming these complexes so how do you think that happens? So what is interesting here that in the presence of SSB these complexes are formed Here we can see the product being formed as similar to what in the presence of SSBs but these are not formed yet So probably SSB is stabilizing these structures Stabilizing the intermediate structures Why would they do that? Do you think that happens in a cell? Or the thing is that the RK has to join a lot of molecules together in the center of the 100-way space So it might be using this strategy that it associates with two or three molecular regions to form the request or it could be for associative performing the molecular search probably How does it tighten for all the switch and all that?