DNA Repair

Recombination Mediator Proteins (RMPs)

RMPs are important for genome stability in all organisms. Mutations in human RMPs, such as BRCA1, BRCA2 and PALB2, are associated with cancer. In response to DNA damage or stalled replication, RMPs initiate homologous recombination (HR) by stimulating formation of RecA-like recombinase filaments on single-stranded (ss) DNA protected by ssDNA binding protein (SSB or RPA). RMPs are involved in other steps of DNA processing during replication restart and repair. Eukaryotic RMPs include such proteins as BRCA2, Rad52, and PALB2. The long term goal of our research is to understand the mechanism of macromolecular interactions regulating interplay between replication, recombination, and repair.

Two major questions are of a particular interest for such RMPs:

  1. How do RMPs overcome the inhibitory effect of the strongest DNA binding proteins, SSB or RPA, while promoting the binding of RecA-like recombinase to ssDNA?
  2. How does the same RMP perform two alternative reactions: recombinase loading and strand annealing?
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RecO

Structural studies of RecO from D. radiodurans (Dr) and E. coli (Ec) revealed similar structures in spite of a lack of conserved motifs and low sequence homology. The structure of EcRecO was solved in complex with the C-terminus of SSB. Together with biochemical studies, the structure supports the hypothesis that RMPs do not remove SSB from ssDNA but form productive complexes during both processes of recombination initiation and strand annealing. Curiously, SSB-Ct binding site is not conserved in the D. radiodurans homolog.

In collaboration with Dr. M. Glickman from Sloan-Kettering Institute, who identified the novel RecO-dependent DNA annealing pathway in mycobacteria, we studied mycobacterial RecFOR. Mycobaterium smegmatis (Ms) RecO does not interact with SSB-Ct during annealing or recombination initiation. Like DrRecO, it possesses a 4xCys zinc-finger domain. This domain is essential for DNA binding by RecO alone and for DNA annealing, while it is dispensable for interaction with DNA in the presence of RecR and for the initiation of HR. Thus, MsRecO represents a unique opportunity to uncouple two reactions common for most RMPs: RecA-loading and annealing. It also gives us the ability to study the functional role of DNA annealing in vivo.

EcRecO_SSB-Ct

Our results suggest a mechanism by which RMPs initiate repair of DSB and stalled replication in response to DNA damage without interfering with DNA replication under normal conditions. RMPs form reversible complexes with SSB/ssDNA, which promote recombinase filament formation, in case of RecOR, or annealing, in case of RecO, in the presence of corresponding substrates.

 

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RecF

The mechanism of RecF function is the least understood. RecF is suggested to play a regulatory role is HR initiation at the boundaries of the ssDNA gap. Several alternative functions were also suggested.

RecF possesses highly conserved sequence motives of ABC-type ATPase. Similar features are found in membrane transport proteins, DNA repair proteins, like Rad50, and the chromosome maintenance proteins, cohesin and condensin. These proteins form ATP-dependent dimers, which dissociate  upon ATP hydrolysis triggered by interactions with other macromolecules.

The crystal structure of RecF revealed a high similarity with the head domain of Rad50, rendering RecF as an excellent minimal structural model to study the mechanism of allosteric regulations in this class of proteins.

Biochemical studies revealed ATP binding, dimerization, DNA-binding of RecF and its interaction with RecR, as well as a surprising luck of specificity for ds/ssDNA junction structures. We suggest a dynamic model of RecF regulation where transient protein-protein interactions define the regulatory mechanism of RecF in recombination initiation.

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PALB2

 

Breast cancer associated proteins 1 and 2 (BRCA1, -2) and partner and localizer of BRCA2 (PALB2) protein are tumor suppressors linked to a spectrum of malignancies, including breast cancer and Fanconi anemia. They stimulate RAD51 recombinase during homology-directed repair (HDR). Along with being a hub for a protein interaction network, PALB2 interacts with DNA. The mechanism of PALB2 DNA binding and its function are poorly understood.  We identified a major DNA-binding site in PALB2, mutation of which reduces the RAD51 foci formation and the overall HDR efficiency in cells by 50%. PALB2 N-terminal DNA-binding domain (N-DBD) stimulates the RAD51 strand exchange reaction. Surprisingly, it promotes the strand exchange without RAD51. Moreover, N-DBD stimulates the inverse strand exchange and can use both DNA and RNA substrates. Our data reveal a versatile DNA interaction property of PALB2 and demonstrate a critical role of PALB2 DNA binding for chromosome repair in cells.

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  • N-terminal DNA-binding domain of PALB2 (PALB2-DBD) is a new recombinase. It stimulates recombination by RAD51 and supports strand exchange even without RAD51.

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Mutation of several DNA biding residues in PALB2-DBD reduces HR in cells by 50%

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