Maintaining genome integrity is crucial for cell survival and genome instability is one of the hallmarks of cancer. The genomes of many living organisms including humans are composed of different numbers of DNA molecules that are folded into structures called chromosomes. Each chromosome represents one double stranded DNA molecule. Most of our cells harbor two sets of each chromosome, which are denoted as homologous chromosomes (Figure 1, left panel). The homologous chromosome pairs can contain identical or different versions (alleles) of the genes that they carry since they are inherited from each parent. In this sense homologous chromosomes are genetically non-identical.
Most of the cells in our body are constantly renewed and replaced in a process that involves cellular division. During cell division the genome needs to be duplicated so that each of the newly formed daughter cells receives a genome copy. The process of DNA duplication is called DNA replication. During replication, each of the two strands of the DNA double helix serves as a template for the synthesis of a new complementary DNA strand. These chromosome copies that are produced during cell division are named sister chromatids (Figure 1, right panel), and they are found transiently in the cells just before the division. Unlike the homologous chromosomes, the alleles of the genes in the sister chromatids are genetically identical since they are produced by DNA replication.
Figure 1: Schematic representation of the homologous chromosomes and the sister chromatids produced during cellular division. Created with BioRender.com
During DNA replications, as well as under the influence of diverse environmental and endogenous agents, lesions constantly occur on DNA. These DNA “injuries” can trigger mutations, compromise the genome integrity, or even cause cell death. DNA double-strand breaks (DSBs) are one of the most deleterious types of DNA lesions that can lead to gross chromosome rearrangements. Similar rearrangements are very common in cancer. One of the major mechanisms for DNA DSBs repair is homologous recombination (HR), a process explained below. Mutations in different genes from the HR pathway have been associated with diseases like cancer of Fanconi anemia, as well as to hypersensitivity to DNA-damaging agents which increase mutation accumulation.
One of the versions of HR involves the use of the intact sister chromatid as donor of information. Since the sister chromatid has an identical sequence to the damaged DNA molecule, this repair system faithfully restores the genetic information and is considered as being error free. Homologous recombination can also occur between the homologous chromosome pairs in the case of non-dividing cells. In HR, the DNA DSB ends are processed and a long single-stranded DNA (ssDNA) overhang is left to serve as platform for the assembly of the protein machinery mediating the repair (Figure 1, a). This ssDNA is coated by the protein RPA to prevent degradation and folding. Subsequently RPA is replaced by RAD51, into a structure termed the presynaptic nucleoprotein filament. This filament is capable of searching for a homologous undamaged DNA molecule that will serve as a template for repair. This exchange between RPA and RAD51 on ssDNA in the presynaptic filament assembly is facilitated by mediators of HR. One of those mediators is a complex of four proteins that are paralogues of RAD51: the BCDX2 complex. However, the exact mechanism of action of the BCDX2 complex in HR remained elusive.
Figure 2. Double-strand breaks (DSBs) can be repaired by several homologous recombination (HR)-mediated pathways. Represented is a simplified version of the “synthesis-dependent strand annealing HR pathway”. In all pathways, the repair is initiated by resection of a DSB to provide 3′ single-stranded DNA (ssDNA) overhangs (a and b). The ssRNA is rapidly coated with RPA to prevent damage and folding (c). RPA is exchanged with RAD51 with the help of HR mediator complexes, which are composed from different RAD51 paralogues, one of which is four protein complex BCDX2 (d). RAD51 coated DNA can search for the non-damaged homologous chromosome / sister chromatid pair and invade that intact DNA duplex (e). After strand invasion and “copying” of the information from the intact DNA on one of the damaged strands from the blue DNA duplex, the reaction can proceed to hybridization to the ssDNA on the other break end of the blue DNA duplex, followed by DNA synthesis (f) and restoring of blue DNA molecule (g). Created with BioRender.com
Recent studies from Columbia postdoc Aviv Meir and colleagues revealed the structure of the human BCDX2 complex. Cryogenic electron microscopy, a technique that allows the high-resolution structure determination of biomolecules in solution, was used to resolve both the free and single-strand DNA-bound states of BCDX2. This provided the first structural information of one of the RAD51 paralogues complexes. This structural information provides insight into how the complex assembles and disassembles, which in turn is linked to the regulation of its function. The scientists also discovered by single molecule analysis that the association of BCDX2 with RPA–ssDNA enhances the rate of assembly of the RAD51–ssDNA filament. In humans, BCDX2 binds the RPA–ssDNA prior to the arrival of RAD51 and then promotes the RAD51 filament assembly. This novel mode of action for the proteins of the BCDX2 complex is different from what was previously observed in other organisms, where BCDX2 only transiently associates with the ssDNA. The work by Dr. Meir and colleagues, recently published in the prestigious journal Nature, not only elucidates how BCDX2 mediates RPA–RAD51 exchange on ssDNA but also provides a foundation for deciphering how alterations in BCDX2 subunits that were found in patients with cancer can impact genome repair and can lead to the pathogenesis. This valuable information opens the doors for future targeting of those “defectuous” BCDX2 parts for therapeutic developments.
Reviewed by: Maaike Schilperoort, Apurva Limaye, Giulia Mezzadri , Trang Nguyen