RECOMBINATION REPAIR

 

Recombination repair (RR) fixes double strand breaks (DSBs) in DNA that are cause by chemical or physical damage.  In the absence of repair, a single DSB can provoke cell death. Genome instability resulting from defects in RR have been linked to hereditary breast cancer (BRCA1/2) as well as hematopoietic and other solid tumors (Ataxia telangiectasia mutated, ATM; Nijmegen breakage syndrome, NBS; Fanconia anemia, FANC; Bloom’s syndrome, BLM) among others.  RR engenders a complex cascade of responses that include cellular signaling integrated with the physical processes of DSB repair.  RR can result in high fidelity homologous recombination repair (HRR) as well as lower fidelity non-homologous end joining (NHEJ).  Our group focuses on HRR where the repair event uses homologous chromosomal sequences to bridge the DSB.

 


Figure 1. A Simplified Model for Homologous Recombination DSB Repair on Chromatin DNA.

Chromosomes where HRR occurs are composed of protein-DNA composites (chromatin) that compact ~ 1 meter of DNA into a cellular nucleus that is less than 10 micrometer in diameter.  The most obvious repeating units of chromatin are nucleosomes composed of ~147 bp of duplex DNA wrapped around an octamer core containing dimers of four histones (H2A, H2B, H3 and H4).  HRR biochemical reactions must manage the disrupted chromatin on the broken donor DNA in order to search and pair with the assembled chromatin of a bridging homologous acceptor DNA.  Deficiencies in any one of the multiple enzymatic steps will affect the outcome of RR and ultimately affect genome stability (Fig. 1).  Our group has focused on understanding the biophysical processes associated with the formation and activities of the HRR protein complex, particularly on well-defined chromatin substrates.  

 

MRE11/RAD50/NBS1 (MRN) or the Ku70/80 heterodimer are among the first to arrive at a DSB. These sensors induce an ATM-dependent damage signaling response that includes cell cycle arrest by a protein phosphorylation cascade.  A repair complex is then formed on the broken end of the donor strand.  It appears MRN enhances the formation of an HHR repair complex while Ku70/80 enhances the formation of an NHEJ repair complex. 

 

Remarkably, very little is known about the detailed biophysical events associated with HRR on chromatin.  This is largely because the fundamental repair reactions have not been reconstituted with well-defined chromatin, nor has there been any attempt to accurately account for the disposition of individual nucleosomes that structure the chromatin of the donor and/or the acceptor DNAs during HRR. It is likely that in both cases a chromatin-remodeling step is required that may be enhanced by RAD51 (dsDNA filament forming activity), RAD54, and/or the INO80 chromatin remodeling machinery.  After strand resection by ExoI/MRE11, the RAD51 protein forms a ssDNA filament that is capable of searching and pairing with the homologous chromatin of the acceptor DNA.  It is unknown whether this process occurs by an identical iterative A:T flipping process similar to naked DNA?  To form a plectonemic strand exchange intermediate, chromatin remodeling must occur surrounding the acceptor-pairing region.  Remodeling candidates include RAD51 (dsDNA filament forming activity), RAD54, and/or INO80.

 

Questions:

 

1. What are the kinetics and mechanism of the RAD51 homology search, strand exchange and remodeling function(s) within chromatin?

A number of pioneering studies have demonstrated that RAD51 with RAD54 can perform homologous pairing and strand exchange with DNA substrates containing nucleosomes. DNA wrapped around a histone octamer core contains multiple contacts and presents additional topological problems to the HRR homology search and ultimately to the strand exchange process. The mechanism, kinetics, and fate of the nucleosomes on the donor and acceptor strands are largely unknown. We would like to answer the following questions: i) Is the RAD51 homology search within nucleosomes similar to the homology search on naked DNA (A:T flipping) and is it influences by histone PTMs?, ii) Does RAD54 alter the RAD51 homology search and/or strand exchange and is it influences by histone PTMs?, iii) Does RAD54 alter RAD51 nucleosome disassembly and is it influences by histone PTMs?, iv) What is the fate of the nucleosome located within the acceptor homologous DNA region and is this fate influenced by RAD54 and or histone PTMs?, and v) Do nucleosomes containing histone PTMs on the donor DNA influence RAD51-RAD54 activities?

 

2. How does the ensemble interplay between RAD51-paralogs and INO80 on the RAD51-RAD54 homology search, strand exchange, and remodeling function(s) within chromatin?

The RAD51 paralogs (RAd51B, RAD51C, RAd51D, XRCC2 and XRCC3) and INO80 have been biochemically and genetically implicated in DSB repair. We have found that the RAD51B-RAD51C paralog complex stabilizes the RAD51 ssDNA to dissociation by BLM. Other studies from our group have suggested that the RAD51D-XRCC2 paralog complex influences RAD51 ADP/ATP processing. The function of the remaining RAD51 paralog complexes in HRR is largely unknown. One possibility is that they may influence the stability of the RAD51 dsDNA NPF and nucleosome stability. The role of the INO80 chromatin-remodeling complex in DSB repair is largely unknown and could be involved in nucleosome remodeling before, during or after homologous pairing and strand exchange. We would like to answer the following questions: i) Do any of the four known RAD51 paralog complexes (RAD51B-RAD51C, RAD51D/XRCC2, Rad51C-XRCC3, and RAD51B-RAD51C-RAD51D-XRCC2) influence the RAD51-RAD54 homology search, strand exchange or remodeling activities on defined chromatin? and ii.) Does INO80 influence the RAD51-RAD54 homology search, strand exchange or remodeling activities?

 

New Technologies:

 

Single molecule and bulk analysis of nucleosome remodeling – Using a quantitative FRET system, we can distinguishing nucleosome sliding along the DNA from nucleosomes that are disassembled (unwrapped) during a remodeling process (Fig. 2A). This system is amenable to both single molecule as well as bulk analysis. The system uses the Cy3 and Cy5 FRET pairs attached to the DNA within the nucleosome localization sequence (NLS; 601 or 5S-rDNA) and to the histone octamer. The precise position of the wrapped NLS compared to Cys residues on the two histone H2A [H2A(C119)] peptides within the octamer was determined from the nucleosome crystal structure. We linked the Cy5 to H2A(C119) using maleimide chemistry and then reconstituted nucleosomes with an NLS-containing DNA labeled with Cy3 at the entry-exit region (Fig. 2A, middle) or dyad region (Fig. 2A, bottom). Significant FRET between the Cy3-Cy5 pairs in both nucleosome constructs was detected: with the dyad label displaying more FRET efficiency that the entry-exit label (compare Fig. 2B and 8C, zero time).

 


Figure 2. RAD51 Unwraps Nucleosomes.
(A) Illustration of the mechanism of nucleosome sliding or unwrapping (top) and the predicted sliding or unwrapping FRET kinetics between the histone octamer labeled with Cy3 and the nucleosome DNA that is labeled with Cy5 in the entry-exit (middle) or dyad (bottom) regions. Cartoon shows unwrapping only . (B) Representative kinetic FRET traces of nucleosome DNA labeled in the entry-exit region at multiple RAD51 concentrations (C) Representative kinetic FRET traces of nucleosome DNA labeled in the dyad region at multiple RAD51 concentrations. Note the slower kinetics of FRET decay (koff) between entry-exit and dyad labeled nucleosomes; establishing an unwrapping mechanism. (D) Normalized FRET at equilibrium versus RAD51 concentration using two different nucleosome localization sequences (601 and 5S rDNA). Standard of deviation from at least three independent experiments is shown for each point. The S0.5•unwrapping = 300 nM.

In this illustration we determined movement of octamer relative to the NLS label by bulk stop-flow kinetic analysis.  If a nucleosome remodeler “slides” the octamer along the DNA (and eventually off an open end), then both the entry-exit and dyad regions will move simultaneously relative to the NLS and the kinetics of FRET decay will be identical (Fig 8A, middle and bottom).  In contrast, if the NLS is “unwrapped” from the octamer, then the unwrapping will likely start at the entry-exit region and progress through the dyad region; insuring that the kinetics of FRET decay at the entry-exit region will be faster than the FRET decay of the dyad region (Fig. 2A, middle and bottom).  We found that WT RAD51 catalyzes ATP-dependent unwrapping of nucleosomes (Fig. 2B and 2C, compare the kinetics of FRET decay).  Importantly, the level of FRET decay at equilibrium was dependent on protein concentration.   Plotting the normalized equilibrium FRET with RAD51 concentration determined the half unwrapping disassembly activity  (S0.5•unwrapping = 300 nM).  Because the cellular concentration of RAD51 is ~6mM, there appears to be sufficient cellular protein to perform efficient nucleosome remodeling.  Stoichiometric analysis suggests that complete unwrapping requires ~1/2 of the NLS DNA be occupied by a RAD51 NPF, consistent with studies that have shown octamer dissociation when the dyad region is unable to form normal contacts.   Moreover, a DNA tail outside the NLS is not required to provoke disassembly, suggesting that RAD51 does not need to initiate the NPF on adjacent DNA.  These observations have important implications when considering the biophysical role(s) of RAD51 in HRR.

 

Engineering Histones – While amino acid substitutions have been used to mimic a number of histone modifications, they often do not reproduce the precise molecular configuration of specific PTMs.  Expressed Protein Ligation (EPL) and Native Chemical Ligation (NCL) combine the control of peptide synthesis with protein engineering approaches derived from inteins.  EPL and NCL utilize chemical peptide ligation in which one peptide contains an N-terminal Cys and the other peptide contains a C-terminal thio-ester.  Desulferization following peptide ligation may be used to generate an Ala from the Cys.  Thus, any combination of synthetic and/or intein-based peptides that contains an Ala or Cys at the ligation site may be used to create a native full-length protein.  Peptides containing any combination of well-defined histone PTMs may be routinely synthesized and combined with ECL or NCL peptides to generate full-length histones.  These studies have been performed in collaboration with Dr. Jenifer Ottesen (Chemistry and Biochemistry).

 

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