User talk:Hala Ouzon-Shubeita

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'''Methods'''
'''Methods'''
-
PFGE: is used to visualize the small difference between circular E.coli chromosome and linear E.Coli chromosome. PFGE uses a periodic change of field direction which makes different length DNA react to the field at different rates, thus enabling the separation of by size of large fragments of DNA.   
+
(Pulse-field gel electrophoresis(PFGE): is used to visualize the small difference between the very big circular E.coli chromosome and linear E.Coli chromosome. PFGE uses a periodic change of field direction which makes different length DNA react to the field at different rates, thus enabling the separation of by size of large fragments of DNA.   
-
In situ DNA footprinting is a method to investigate the sequence specificity of a DNA-binding protein. By using chemical or enzymatic activity, a DNA bound to a protein can be cleaved. DNA fragments can then be run on a gel to determine which bases are protected from attack when the protein is bound. [4]. Authors of this paper used chloroacetaldehyde(CAA), which is  a single strand-specific probe. CAA can react with unpaired and unprotected adenines and cytosine. In this experiment cells were treated with CAA then plasmids were extracted and the DNA region was analyzed by primer extension. CAA modified bases are revealed by their ability to terminate the extension of 32P end-labeled primer.  
+
In situ DNA footprinting: is a method to investigate the sequence specificity of a DNA-binding protein. By using chemical or enzymatic activity, a DNA bound to a protein can be cleaved. DNA fragments can then be run on a gel to determine which bases are protected from attack when the protein is bound. [4]. Authors of this paper used chloroacetaldehyde(CAA), which is  a single strand-specific probe. CAA can react with unpaired and unprotected adenines and cytosine[4]. In this experiment cells were treated with CAA then plasmids were extracted and the DNA region was analyzed by primer extension. CAA modified bases are revealed by their ability to terminate the extension of 32P end-labeled primer.  
To illustrate how the method works (see the figure), at the transcription bubble the DNA helix is unwound over 12-16 bp, with the downstream margin of the transcription bubble at the level of the catalytic center of RNAp. Most of the base residues on the non-transcribed strand are accessible to the single strand-specific probes and thus they are exposed to the solvent. The protection of 8-12 nucleotides of the template strand from single strand-specific chemical probes very often has been taken as an indication of the presence of an extended RNA:DNA hybrid within transcription elongation complexes. Modified bases were detected by primer extension and then by running the extension products on a 6% polyacrylamide gel. [4]
To illustrate how the method works (see the figure), at the transcription bubble the DNA helix is unwound over 12-16 bp, with the downstream margin of the transcription bubble at the level of the catalytic center of RNAp. Most of the base residues on the non-transcribed strand are accessible to the single strand-specific probes and thus they are exposed to the solvent. The protection of 8-12 nucleotides of the template strand from single strand-specific chemical probes very often has been taken as an indication of the presence of an extended RNA:DNA hybrid within transcription elongation complexes. Modified bases were detected by primer extension and then by running the extension products on a 6% polyacrylamide gel. [4]

Current revision

Hello, Hala Ouzon-Shubeita! This is a welcome message from OpenWetWare. By the way, we've announced you on the home page! You can leave messages to any OWW member by editing their User_talk pages like this one. And don't forget to personalize your User Page so that we can get to know you better! We've included some tips below to get you started.

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CH391L/S2013 Hala Ouzon Jan 30 2013

Purified human BRCA2 stimulates RAD51-mediated recombination [4]

Background:

DNA integrity is essential to life. However DNA is constantly subject to assaults from endogenous species, such as oxygen radicals, alkylating agents or replication errors. Or from exogenously generated agents like UV light and X-rays [1]. DNA damage could manifest in the form of single-strand breaks, double- strands breaks, insertions, deletions or the formation of bulky adducts. Figure 1 below shows some common DNA damaging agents and some repair mechanisms [1].

Figure 1. DNA damage, repair mechanisms and consequences [1].
Figure 1. DNA damage, repair mechanisms and consequences [1].

Chromosomes Double-strand breaks (DSB) in particular are troublesome because a single unrepaired DSB could lead to chromosomal instability or cell death [2].There are two ways in which organisms can repair DSBs; Non Homologous Ends Joining (NHEJ) and Homologous Recombination (HR). HR is a very important repair pathway in mitosis and in chromosomal exchange during meiosis [2]. Therefore HR is highly regulated. HR goes through three phases: presynapsis, synapsis and post-synapsis[2]. In the presynapsis phase as a response to DSBs several sensor proteins detect the break and send signals to mediator proteins that activate effectors that repair the damage. Figure 2 below is a simplified representation of this process [3].

Figure 2. Molecular mechanisms of the DNA damage response [3].
Figure 2. Molecular mechanisms of the DNA damage response [3].

Introduction:

The focus of this study [4] is on the breast cancer susceptibility protein (BRCA2) and its goal in mediating the binding of the central protein in recombination (RAD51) to ssDNA, replacing replicating protein A (RPA) and stabilizing RAD51-ssDNA filament formation. Many factors affect the likelihood of developing breast cancer but individuals carrying mutation in BRCA1 or BRCA2 have 40-80% chance of developing breast cancer [ 5 ]. All the previous knowledge about this protein came from studying its fragments or exploring its orthologues. Because of the large size of BRCA2 it was very difficult to induce and purify a functional full length BRCA2.

Methods and Results: [4]

Dr. Kowalczykowski’s group was able to clone and purify the full length human BRCA2 (3,418 amino acid) . This was successfully done by adding two tandem repeats of the maltose binding protein tag (MBP) at the N terminus of the protein (increasing the size to 470KDa) and expressing the construct in human 293TD cells. Cloning was verified by Western blotting on the C-terminus region and the MBP tag. The two MBP tags did not affect the function of BRCA2 as tested by comparing cell survival between brca2 mutant cells and brca2 mutant cells transfected with 2XMBP-BRCA2.Furthermore to confirm that the purified BRCA2 protein was folded correctly and biochemically active, this group used pull-down assays to test its interactions with recombinant proteins that were previously shown to interact with BRAC2 fragments. BRAC2 interacts with the strand-exchange protein (RAD51) and its Meiotic counterpart DMC1. Despite a report that BRAC2 interacts with the ssDNA-binding factor, replication protein A (RPA), no significant interactions were detected.

Figure 3 shows the BRAC2 functional domains and some protein interactions [3].

Figure 3. BRCA2 functional domains [3].
Figure 3. BRCA2 functional domains [3].

As shown in figure 3 RAD51 binds the BRC repeats of BRCA2. Using known concentrations of BRCA2 and RAD51, this group was able to estimate that six RAD51 proteins bind to BRCA2 at once. Using mobility shift assays, it was shown that BRCA2 has preference binding ssDNA and tailed DNA over dsDNA and that this result does not change if BRCA2 was pre-incubated with RAD51. RAD51 has affinity for both dsDNA and ssDNA. Binding to dsDNA inhibits DNA strand exchange.

When incubated with ssDNA and dsDNA, RAD51 inhibits DNA strand exchange. However, this exchange is facilitated if RAD51 was pre-incubated with BRCA2 indicating that the latter either targets RAD51 to ssDNA or limits its binding to dsDNA or both. This effect was reduced if the mixture was supplemented with RPA, but the exchange was not fully suppressed. To confirm that BRCA2 limits binding of RAD51 to dsDNA, the authors incubated both proteins with ssDNA first then added dsDNA. The inhibitory effect of the excess RAD51 that binds the dsDNA was reduced in a BRCA2 concentration depended manner. BRCA2’s role in targeting RAD51 to ssDNA is accomplished by blocking the ATPase activity of RAD51. Addition of BRCA2 reduced the ATPase activity in a concentration dependent manner down to the levels measured in the absence of DNA. These findings confirm that BRCA2 plays two roles: it stabilizes RAD51 bound to the ssDNA by down-regulating its ATPase activity and limits its binding to dsDNA.

In vivo, there is competition between RAD51 binding to ssDNA and RPA binding. It was shown previously from studying fragments of BRCA2 that this protein also encourages RAD51 filament formation on RPA-coated ssDNA. To check whether full length BRCA2 also have similar effect this group used DNA strand exchange assay similar to that used to assist BRCA2’s role in mediating RAD51 binding to ssDNA but with ssDNA first complexed with RPA. And they found that increasing the concentration of BRCA2 increased DNA strand exchange as much as 20 fold and that its stimulatory effect was obvious even at 100-fold smaller concentration than RAD51.

Summary:

In summary, this work investigated the biochemical functions of full-length BRCA2. The authors showed that it enhances the functions of RAD51 in recombinatorial DNA repair of breaks. Surprisingly, they found that the protein can bind to ssDNA and tailed DNA of either polarity. This suggests that BRCA2 could also have a role in the repair of DNA gaps. The ability to purify full length BRCA2 should facilitate studying its important role in increasing the risk of cancer in individuals with mutations in the protein.


References:

1. Negritto, M.C., Repairing Double-Strand DNA Breaks. Nature Education, 2010. 3(9).

2. Krejci, L., et al., Homologous recombination and its regulation. Nucleic Acids Res, 2012. 40(13): p. 5795-818.

3. Roy, R., J. Chun, and S.N. Powell, BRCA1 and BRCA2: different roles in a common pathway of genome protection. Nat Rev Cancer, 2012. 12(1): p. 68-78.

4. Jensen, R.B., A. Carreira, and S.C. Kowalczykowski, Purified human BRCA2 stimulates RAD51-mediated recombination. Nature, 2010. 467(7316): p. 678-83.

5. James D.Fachenthal and Olufunmilayo I. Olopade, Breast cancer risk associated with BRCA1 and BRCA2 in diverse populations.Nature Reviews Cancer, 2007.7: p.937-948.

CH391L/S2013 Hala Ouzon Feb 13 2013

Linking RNA Polymerase Backtracking to Genome Instability in E. coli [1]


Background

Unlike eukaryotes where translation and transcription take place in different cellular compartments, in bacteria, like E.Coli, the two events are coupled in time and space. During transcription RNA polymerase (RNAp) forms a very stable complex with DNA which is called the elongation complex (EC). This complex is the target of transcriptional regulation in both prokaryotes and eukaryotes. The rate of replication is higher than the rate of transcription which frequently causes collisions between the replisome and RNAp leading to DNA damage. Furthermore EC might slide in the reverse direction along DNA and RNA in an attempt to fix an incorrectly incorporated base which cause the EC to pause and increase the chance of DNA damage. Using in situ chloroacetaldehyde (CAA) DNA footprinting and pulse-field gel electrophoreses (PFGE) , this group shows that codirectional collision between the replisonme and EC (paused or backtracked) damaged DNA. And using these same techniques they showed that this damage is actually double strand breaks (DSB) located at the position of the arrested EC.


Methods

(Pulse-field gel electrophoresis(PFGE): is used to visualize the small difference between the very big circular E.coli chromosome and linear E.Coli chromosome. PFGE uses a periodic change of field direction which makes different length DNA react to the field at different rates, thus enabling the separation of by size of large fragments of DNA.

In situ DNA footprinting: is a method to investigate the sequence specificity of a DNA-binding protein. By using chemical or enzymatic activity, a DNA bound to a protein can be cleaved. DNA fragments can then be run on a gel to determine which bases are protected from attack when the protein is bound. [4]. Authors of this paper used chloroacetaldehyde(CAA), which is a single strand-specific probe. CAA can react with unpaired and unprotected adenines and cytosine[4]. In this experiment cells were treated with CAA then plasmids were extracted and the DNA region was analyzed by primer extension. CAA modified bases are revealed by their ability to terminate the extension of 32P end-labeled primer. To illustrate how the method works (see the figure), at the transcription bubble the DNA helix is unwound over 12-16 bp, with the downstream margin of the transcription bubble at the level of the catalytic center of RNAp. Most of the base residues on the non-transcribed strand are accessible to the single strand-specific probes and thus they are exposed to the solvent. The protection of 8-12 nucleotides of the template strand from single strand-specific chemical probes very often has been taken as an indication of the presence of an extended RNA:DNA hybrid within transcription elongation complexes. Modified bases were detected by primer extension and then by running the extension products on a 6% polyacrylamide gel. [4]

Figure 1. DNA footprinting [4].
Figure 1. DNA footprinting [4].


Glossary of terms

Cl857 is a temperature sensitive repressor allows for activation of pL promoter at 42°C.[1]

λ N codes for an antitermination protein (stimulates elongation), binds to nutL site on RNA

HK022 Nun factors: inhibits elongation and therefore arrests EC, binds to nutL site on RNA [5]

mfd: transcription-repair coupling factor

RBS: Expression of foreign genes in Escherichia coli requires the combination of prokaryotic transcription and translation elements with a coding region for the foreign gene. Commonly, this results in only modest expression of the foreign gene product. Therefore ribosome-binding site RBS is needed to enhance the translation of foreign genes. [2]

Rho: a prokaryotic protein involved in termination of transcription.

HU: replication inhibitor.

BCM: antibiotic bicyclomycin, specifically targets and inhibits Rho.

Gre A and GreB elongation factors.

Cm: Translation elongation inhibitor chloramphenicol. (it slows down translation inducing pauses of ECs)


Results

Two plasmids were used, one to monitor codirectional collisions and the other to monitor head-on collisions. Both had the phage λ cl857 pL-nutL-RBSN-UTR cassette with CoIE1 origin of replication. In pCODIR plasmid the phage λ was oriented codirectionally with the origin of replication, whereas the pHDON plasmid had phage λ oriented head-on with origin of replication.

Simulated ECs arrest was done by inducing the pL promoter in wild type and Mfd deficient (∆mfd) E.Coli cells, in the presence and absence of HK022 nun+ prophage. The effect of Nun arrested ECs on the chromosome stability was then asessed by CCA footprinting. Both DNA strands showed the same pattern of DNA breaks regardless of promoter orientation. Mfd relieved codirectional collision DNA damage but did not for the head-on collision DNA damage. If cells were treated with replication inhibitor (HU) or if the promoter was not induced no DNA breaks was detected in either plasmid. These results showed that both collisions between Nun-induced ECs and replisome induced DSB and Mfn actually cannot release Nun-arrested ECs in head-on collision. These findings are contradict a previous study that stated otherwise.

To test whether spontaneously arrested ECs also interfere with replication and cause DSB, they manipulated the rate of RNAp backtracking using the GreA and GreB, enzymes used in cells to minimize backtracking. These enzymes reactivate ECs by by stimulating the formation of a new 3’OH terminus. To demonstrate the role of GreA and GreB as anti-backtracking machinery and to see the anti-backtracking effect on genome stability, this group noticed that when deleting greA and greB a big cluster of DSB around 30 nucleotides downstream of RBS in pCODIR is formed (but not for pHDON). These DSBs were dependent on both replication and transcription because they were eliminated in an induced promoter or HU treated cells (a replication inhibitor). The DNA lesion was confirmed to be a DSB by running on a gel and monitoring the fast migration of the single stranded plasmid compared to the circular one. Therefore this group was able to conclude that naturally backtracked ECs cause DSBs due to codirectional collisions with replisome. They also demonstrated the important role the ribosome (i,e translation) plays in suppressing RNAp backtracking and by that eliminating DSBs: conversion of the untranslated region (UTR) to open reading frame (ORF) eliminated the effect of GreA/B deficiency because of the “pushing” effect of the ribosome on RNAp. The location of the DSB was determined by comparing footprints of the transcription bubble with those of DSB and found that the pattern was very similar which means the position of the DSB matches the position of the Nun-arrested ECs. To confirm that EC indeed can backtrack, the authors cloned and purified a 6His-taged RNAp and let it transcribe a DNA sequence containing pL-nut-RBS-UTR to find that EC actually halted at position +298. When they removed free dNTP and this DNA-RNAp complex was treated with GreA or B, the size of Gre cleavage product was consistent with backtracking of about 15 nucleotides.

Why do we get DSB upon transcription-replication collision? When the EC and replisome collide the RNAp might dislodge leaving the elongated RNA to anneal with the DNA forming an RNA: DNA hybrid which is called the R loop. In the case of backtracking ECs this loop might be large and therefore more stable. In the case of codirectional collision, these R loops provide a 3’OH terminus that could serve as primer for DNA replication. This switch from transcription to replication leads to a break in the leading strand that if not fixed could lead to DSB. To insure that the R loop formation indeed is the cause of DSB, pCODIR, Gre deficient cells were transformed with physiological level RNase H expression and high RNase expression. Only at high RNase expression were DSBs significantly reduced, from which the authors conclude that R loops are the cause of DSBs and that physiological expression levels of RNase are not enough to eliminate the R loops when the anti-backtracking mechanisms are compromised. In support of this mechanism, mutation in RNAp that forms a less stable complex with the DNA and therefore reduces the collisions with replisomes, as well as one that the RNAp ignore an arrest site, like rpoB*35, also suppress the formation of DSB because they avoid the pausing and the backtracking of RNAp.


Do anti-backtracking mechanisms contribute to genome stability? Treating the cells with sub-lethal concentration of Cm slows down the ribosome. This is predicted to increase backtracking and as a consequence increase DSB. Similarly treating the cells with BCM is expected to increase DSBs. However treatment of WT cells with Cm or BCM only reduced DSBs slightly. However, treatment of ∆greB cells with Cm or BCM reduced DSBs a lot. Over expression of GreB largely eliminated DSBs. The authors also checked whether these results would also apply to a non-plasmid system (like the E.Coli chromosome). They did that by visualizing DSBs using pulse-field gel electrophoresis and saw two bands. One band corresponding to the intact circular DNA stays at origin, and the other that corresponds to linear DNA (because of DSB) is seen in the middle of the gel. DNA isolated from WT and ∆gre cells stayed at the origin. DNA isolated form ∆gre, Cm treated cells showed DSBs, these DSBs were eliminated with high expression of RNase or rpoB*35 allele. Monitoring the SOS response with recA-gfp fusion was another way to monitor the anti-backtracking effect on genome stability. Treating cells with Cm induced SOS response (fluorescence). ∆gre cells treated with Cm gave an even higher fluorescence signal. And treating ∆gre cells with Cm and BCM, at a concentration that had almost no effect when administered by itself, induced a yet higher fluorescence signal. GreB over expression suppressed hyperinduction of SOS response by Cm.


Summary

The ribosome is the primarily sensor of cellular metabolism and various stresses. In this study we see how factors that affect the ribosome movement affect genome stability. We can see how modulating the rate of ribosome movement by rare codons, for example, can effect genome stability. When translation in insufficient, cells utilize different mechanisms to prevent ECs backtracking such as: 1) Rho factor, that suppress backtracking by terminating transcription, 2) Mfd, which disrupt ECs arrest, and 3) GreA/GreB factors, which suppress DSBs by restarting backtracked ECs.


References

1. Dutta, D., et al., Linking RNA polymerase backtracking to genome instability in E. coli. Cell, 2011. 146(4): p. 533-43.

2. Korzheva, N. and A. Mustaev, Transcription elongation complex: structure and function. Curr Opin Microbiol, 2001. 4(2): p. 119-25.

3. Shaevitz, J.W., et al., Backtracking by single RNA polymerase molecules observed at near-base-pair resolution. Nature, 2003. 426(6967): p. 684-7.

4. Guerin, M., M. Leng, and A.R. Rahmouni, High resolution mapping of E.coli transcription elongation complex in situ reveals protein interactions with the non-transcribed strand. EMBO J, 1996. 15(19): p. 5397-407.

5. Proshkin, S., et al., Cooperation between translating ribosomes and RNA polymerase in transcription elongation. Science, 2010. 328(5977): p. 504-8.

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