Homologous recombination: from model organisms to human disease

Recent experiments show that properly controlled recombination between homologous DNA molecules is essential for the maintenance of genome stability and for the prevention of tumorigenesis

Revealing the Competition between Peeled-Ssdna, Melting Bubbles and S-DNA during DNA Overstretching using Fluorescence Microscopy

Understanding the structural changes occurring in double-stranded (ds)DNA during mechanical strain is essential to build a quantitative picture of how proteins interact and modify DNA. However, the elastic response of dsDNA to tension is only well-understood for forces < 65 pN. Above this force, torsionally unconstrained dsDNA gains ∼70% of its contour length, a process known as overstretching. The structure of overstretched DNA has proved elusive, resulting in a rich and controversial debate in recent years. At the centre of the debate is the question of whether overstretching yields a base-paired elongated structure, known as S-DNA, or instead forms single-stranded (ss)DNA via base-pair cleavage. Here, we show clearly, using a combination of fluorescence microscopy and optical tweezers, that both S-DNA and base-pair melted structures can exist, often concurrently, during overstretching. The balance between the two models is affected strongly by temperature and ionic strength. Moreover, we reveal, for the first time, that base-pair melting can proceed via two entirely different processes: progressive strand unpeeling from a free end in the backbone, or by the formation of ‘bubbles' of ssDNA, nucleating initially in AT-rich regions. We demonstrate that the mechanism of base-pair melting is governed by DNA topology: strand unpeeling is favored when there are free ends in the DNA backbone. Our studies settle a long running debate, and unite the contradictory dogmas of DNA overstretching. These findings have important implications for both medical and biological sciences. Force-induced melting transitions (yielding either peeled-ssDNA or melting bubbles) may play active roles in DNA replication and damage repair. Further, the ability to switch easily from DNA containing melting bubbles to S-DNA may be particularly advantageous in the cell, for instance during the formation of RNA within transcription bubbles. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved

Revealing the Competition between Peeled-Ssdna, Melting Bubbles and S-DNA during DNA Overstretching using Fluorescence Microscopy

Understanding the structural changes occurring in double-stranded (ds)DNA during mechanical strain is essential to build a quantitative picture of how proteins interact and modify DNA. However, the elastic response of dsDNA to tension is only well-understood for forces < 65 pN. Above this force, torsionally unconstrained dsDNA gains ∼70% of its contour length, a process known as overstretching. The structure of overstretched DNA has proved elusive, resulting in a rich and controversial debate in recent years. At the centre of the debate is the question of whether overstretching yields a base-paired elongated structure, known as S-DNA, or instead forms single-stranded (ss)DNA via base-pair cleavage. Here, we show clearly, using a combination of fluorescence microscopy and optical tweezers, that both S-DNA and base-pair melted structures can exist, often concurrently, during overstretching. The balance between the two models is affected strongly by temperature and ionic strength. Moreover, we reveal, for the first time, that base-pair melting can proceed via two entirely different processes: progressive strand unpeeling from a free end in the backbone, or by the formation of ‘bubbles' of ssDNA, nucleating initially in AT-rich regions. We demonstrate that the mechanism of base-pair melting is governed by DNA topology: strand unpeeling is favored when there are free ends in the DNA backbone. Our studies settle a long running debate, and unite the contradictory dogmas of DNA overstretching. These findings have important implications for both medical and biological sciences. Force-induced melting transitions (yielding either peeled-ssDNA or melting bubbles) may play active roles in DNA replication and damage repair. Further, the ability to switch easily from DNA containing melting bubbles to S-DNA may be particularly advantageous in the cell, for instance during the formation of RNA within transcription bubbles. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved

Human Rad51 filaments on double- and single-stranded DNA: correlating regular and irregular forms with recombination function

Recombinase proteins assembled into helical filaments on DNA are believed to be the catalytic core of homologous recombination. The assembly, disassembly and dynamic rearrangements of this structure must drive the DNA strand exchange reactions of homologous recombination. The sensitivity of eukaryotic recombinase activity to reaction conditions in vitro suggests that the status of bound nucleotide cofactors is important for function and possibly for filament structure. We analyzed nucleoprotein filaments formed by the human recombinase Rad51 in a variety of conditions on double-stranded and single-stranded DNA by scanning force microscopy. Regular filaments with extended double-stranded DNA correlated with active in vitro recombination, possibly due to stabilizing the DNA products of these assays. Though filaments formed readily on single-stranded DNA, they were very rarely regular structures. The irregular structure of filaments on single-stranded DNA suggests that Rad51 monomers are dynamic in filaments and that regular filaments are transient. Indeed, single molecule force spectroscopy of Rad51 filament assembly and disassembly in magnetic tweezers revealed protein association and disassociation from many points along the DNA, with kinetics different from those of RecA. The dynamic rearrangements of proteins and DNA within Rad51 nucleoprotein filaments could be key events driving strand exchange in homologous recombination

Two distinct conformational states define the interaction of human RAD51-ATP with single-stranded DNA.

An essential mechanism for repairing DNA double-strand breaks is homologous recombination (HR). One of its core catalysts is human RAD51 (hRAD51), which assembles as a helical nucleoprotein filament on single-stranded DNA, promoting DNA-strand exchange. Here, we study the interaction of hRAD51 with single-stranded DNA using a single-molecule approach. We show that ATP-bound hRAD51 filaments can exist in two different states with different contour lengths and with a free-energy difference of ~4 kBT per hRAD51 monomer. Upon ATP hydrolysis, the filaments convert into a disassembly-competent ADP-bound configuration. In agreement with the single-molecule analysis, we demonstrate the presence of two distinct protomer interfaces in the crystal structure of a hRAD51-ATP filament, providing a structural basis for the two conformational states of the filament. Together, our findings provide evidence that hRAD51-ATP filaments can exist in two interconvertible conformational states, which might be functionally relevant for DNA homology recognition and strand exchange

Domain swapping between FEN-1 and XPG defines regions in XPG that mediate nucleotide excision repair activity and substrate specificity

FEN-1 and XPG are members of the FEN-1 family of structure-specific nucleases, which share a conserved active site. FEN-1 plays a central role in DNA replication, whereas XPG is involved in nucleotide excision repair (NER). Both FEN-1 and XPG are active on flap structures, but only XPG cleaves bubble substrates. The spacer region of XPG is dispensable for nuclease activity on flap substrates but is required for NER activity and for efficient processing of bubble substrates. Here, we inserted the spacer region of XPG between the nuclease domains of FEN-1 to test whether this domain would be sufficient to confer XPG-like substrate specificity and NER activity on a related nuclease. The resulting FEN-1-XPG hybrid protein is active on flap and, albeit at low levels, on bubble substrates. Like FEN-1, the activity of FEN-1-XPG was stimulated by a double-flap substrate containing a 1-nt 3′ flap, whereas XPG does not show this substrate preference. Although no NER activity was detected in vitro, the FEN-1-XPG hybrid displays substantial NER activity in vivo. Hence, insertion of the XPG spacer region into FEN-1 results in a hybrid protein with biochemical properties reminiscent of both nucleases, including partial NER activity