Structural insights into the promiscuous DNA binding and broad substrate selectivity of fowlpox virus resolvase.

Fowlpox virus resolvase (Fpr) is an endonuclease that cleaves a broad range of branched DNA structures, including the Holliday junction (HJ), with little sequence-specificity. To better understand the mechanisms underlying its relaxed substrate specificity, we determined the crystal structures of Fpr and that in a novel complex with HJ at 3.1-Å resolution. In the Fpr-HJ complex, two Fpr dimers use several distinct regions to interact with different DNA structural motifs, showing versatility in DNA-binding. Biochemical and solution NMR data support the existence of non-canonical modes of HJ interaction in solution. The binding of Fpr to various DNA motifs are mediated by its flat DNA-binding surface, which is centered on a short loop spanning K61 to I72 and flanked by longer α-helices at the outer edges, and basic side grooves near the dimer interface. Replacing the Fpr loop K61~I72 with a longer loop from Thermus thermophilus RuvC (E71~A87) endows Fpr with an enhanced selectivity toward HJ cleavage but with a target sequence preference distinct from that of RuvC, highlighting a unique role of this loop region in Fpr-HJ interaction. Our work helps explain the broad substrate selectivity of Fpr and suggests a possible mode of its association with poxvirus hairpin telomeres.

Binding of FANCI-FANCD2 Complex to RNA and R-Loops Stimulates Robust FANCD2 Monoubiquitination.

Fanconi anemia (FA) is characterized by developmental abnormalities, bone marrow failure, and cancer predisposition. FA cells are hypersensitive to DNA replicative stress and accumulate co-transcriptional R-loops. Here, we use the Damage At RNA Transcription assay to reveal colocalization of FANCD2 with R-loops in a highly transcribed genomic locus upon DNA damage. We further demonstrate that highly purified human FANCI-FANCD2 (ID2) complex binds synthetic single-stranded RNA (ssRNA) and R-loop substrates with high affinity, preferring guanine-rich sequences. Importantly, we elucidate that human ID2 binds an R-loop structure via recognition of the displaced ssDNA and ssRNA but not the RNA:DNA hybrids. Finally, a series of RNA and R-loop substrates are found to strongly stimulate ID2 monoubiquitination, with activity corresponding to their binding affinity. In summary, our results support a mechanism whereby the ID2 complex suppresses the formation of pathogenic R-loops by binding ssRNA and ssDNA species, thereby activating the FA pathway.

Importance of homo-dimerization of Fanconi-associated nuclease 1 in DNA flap cleavage.

Fanconi-associated nuclease 1 (FAN1) removes interstrand DNA crosslinks (ICLs) through its DNA flap endonuclease and exonuclease activities. Crystal structures of human and bacterial FAN1 bound to a DNA flap have been solved. The Pseudomonas aeruginosa bacterial FAN1 and human FAN1 (hFAN1) missing a flexible loop are monomeric, while intact hFAN1 is homo-dimeric in structure. Importantly, the monomeric and dimeric forms of FAN1 exhibit very different DNA binding modes. Here, we interrogate the functional differences between monomeric and dimeric forms of FAN1 and provide an explanation for the discrepancy in oligomeric state between the two hFAN1 structures. Specifically, we show that the flexible loop in question is needed for hFAN1 dimerization. While monomeric and dimeric bacterial or human FAN1 proteins cleave a short 5' flap strand with similar efficiency, optimal cleavage of a long 5' flap strand is contingent upon protein dimerization. Our study therefore furnishes biochemical evidence for a role of hFAN1 homodimerization in biological processes that involve 5' DNA Flap cleavage.

BRCA1-BARD1 promotes RAD51-mediated homologous DNA pairing.

The tumour suppressor complex BRCA1-BARD1 functions in the repair of DNA double-stranded breaks by homologous recombination. During this process, BRCA1-BARD1 facilitates the nucleolytic resection of DNA ends to generate a single-stranded template for the recruitment of another tumour suppressor complex, BRCA2-PALB2, and the recombinase RAD51. Here, by examining purified wild-type and mutant BRCA1-BARD1, we show that both BRCA1 and BARD1 bind DNA and interact with RAD51, and that BRCA1-BARD1 enhances the recombinase activity of RAD51. Mechanistically, BRCA1-BARD1 promotes the assembly of the synaptic complex, an essential intermediate in RAD51-mediated DNA joint formation. We provide evidence that BRCA1 and BARD1 are indispensable for RAD51 stimulation. Notably, BRCA1-BARD1 mutants with weakened RAD51 interactions show compromised DNA joint formation and impaired mediation of homologous recombination and DNA repair in cells. Our results identify a late role of BRCA1-BARD1 in homologous recombination, an attribute of the tumour suppressor complex that could be targeted in cancer therapy.

Novel TDP2-ubiquitin interactions and their importance for the repair of topoisomerase II-mediated DNA damage.

Tyrosyl DNA phosphodiesterase 2 (TDP2) is a multifunctional protein implicated in DNA repair, signal transduction and transcriptional regulation. In its DNA repair role, TDP2 safeguards genome integrity by hydrolyzing 5'-tyrosyl DNA adducts formed by abortive topoisomerase II (Top2) cleavage complexes to allow error-free repair of DNA double-strand breaks, thereby conferring cellular resistance against Top2 poisons. TDP2 consists of a C-terminal catalytic domain responsible for its phosphodiesterase activity, and a functionally uncharacterized N-terminal region. Here, we demonstrate that this N-terminal region contains a ubiquitin (Ub)-associated (UBA) domain capable of binding multiple forms of Ub with distinct modes of interactions and preference for either K48- or K63-linked polyUbs over monoUb. The structure of TDP2 UBA bound to monoUb shows a canonical mode of UBA-Ub interaction. However, the absence of the highly conserved MGF motif and the presence of a fourth α-helix make TDP2 UBA distinct from other known UBAs. Mutations in the TDP2 UBA-Ub binding interface do not affect nuclear import of TDP2, but severely compromise its ability to repair Top2-mediated DNA damage, thus establishing the importance of the TDP2 UBA-Ub interaction in DNA repair. The differential binding to multiple Ub forms could be important for responding to DNA damage signals under different contexts or to support the multi-functionality of TDP2.

Promotion of presynaptic filament assembly by the ensemble of S. cerevisiae Rad51 paralogues with Rad52.

The conserved budding yeast Rad51 paralogues, including Rad55, Rad57, Csm2 and Psy3 are indispensable for homologous recombination (HR)-mediated chromosome damage repair. Rad55 and Rad57 are associated in a heterodimer, while Csm2 and Psy3 form the Shu complex with Shu1 and Shu2. Here we show that Rad55 bridges an interaction between Csm2 with Rad51 and Rad52 and, using a fully reconstituted system, demonstrate that the Shu complex synergizes with Rad55-Rad57 and Rad52 to promote nucleation of Rad51 on single-stranded DNA pre-occupied by replication protein A (RPA). The csm2-F46A allele is unable to interact with Rad55, ablating the ability of the Shu complex to enhance Rad51 presynaptic filament assembly in vitro and impairing HR in vivo. Our results reveal that Rad55-Rad57, the Shu complex and Rad52 act as a functional ensemble to promote Rad51-filament assembly, which has important implications for understanding the role of the human RAD51 paralogues in Fanconi anaemia and cancer predisposition.

Structure of Est3 reveals a bimodal surface with differential roles in telomere replication.

Telomerase is essential for continuous cellular proliferation. Substantial insights have come from studies of budding yeast telomerase, which consists of a catalytic core in association with two regulatory proteins, ever shorter telomeres 1 and 3 (Est1 and Est3). We report here a high-resolution structure of the Est3 telomerase subunit determined using a recently developed strategy that combines minimal NMR experimental data with Rosetta de novo structure prediction algorithms. Est3 adopts an overall protein fold which is structurally similar to that adopted by the shelterin component TPP1. However, the characteristics of the surface of the experimentally determined Est3 structure are substantially different from those predicted by prior homology-based models of Est3. Structure-guided mutagenesis of the complete surface of the Est3 protein reveals two adjacent patches on a noncanonical face of the protein that differentially mediate telomere function. Mapping these two patches on the Est3 structure defines a set of shared features between Est3 and HsTPP1, suggesting an analogous multifunctional surface on TPP1.

Dissecting protein function: an efficient protocol for identifying separation-of-function mutations that encode structurally stable proteins.

Mutations that confer the loss of a single biochemical property (separation-of-function mutations) can often uncover a previously unknown role for a protein in a particular biological process. However, most mutations are identified based on loss-of-function phenotypes, which cannot differentiate between separation-of-function alleles vs. mutations that encode unstable/unfolded proteins. An alternative approach is to use overexpression dominant-negative (ODN) phenotypes to identify mutant proteins that disrupt function in an otherwise wild-type strain when overexpressed. This is based on the assumption that such mutant proteins retain an overall structure that is comparable to that of the wild-type protein and are able to compete with the endogenous protein (Herskowitz 1987). To test this, the in vivo phenotypes of mutations in the Est3 telomerase subunit from Saccharomyces cerevisiae were compared with the in vitro secondary structure of these mutant proteins as analyzed by circular-dichroism spectroscopy, which demonstrates that ODN is a more sensitive assessment of protein stability than the commonly used method of monitoring protein levels from extracts. Reverse mutagenesis of EST3, which targeted different categories of amino acids, also showed that mutating highly conserved charged residues to the oppositely charged amino acid had an increased likelihood of generating a severely defective est3(-) mutation, which nevertheless encoded a structurally stable protein. These results suggest that charge-swap mutagenesis directed at a limited subset of highly conserved charged residues, combined with ODN screening to eliminate partially unfolded proteins, may provide a widely applicable and efficient strategy for generating separation-of-function mutations.

Partners in crime: the TGFß and MAPK pathways in cancer progression.

The TGFß and Ras-MAPK pathways play critical roles in cell development and cell cycle regulation, as well as in tumor formation and metastasis. In the absence of cellular transformation, these pathways operate in opposition to one another, where TGFß maintains an undifferentiated cell state and suppresses proliferation, while Ras-MAPK pathways promote proliferation, survival and differentiation. However, in colorectal and pancreatic cancers, the opposing pathways' mechanisms are simultaneously activated in order to promote cancer progression and metastasis. Here, we highlight the roles of the TGFß and Ras-MAPK pathways in normal and malignant states, and provide an explanation for how the concomitant activation of these pathways drives tumor biology. Finally, we survey potential therapeutic targets in these pathways.

Drosophila polypyrimidine tract-binding protein is necessary for spermatid individualization.

Although mammalian polypyrimidine tract-binding (PTB) protein functions in most or all cell types to regulate a wide spectrum of transcripts, Drosophila PTB encodes an abundant male germline-specific mRNA isoform (dmPTB) whose expression correlates with male fertility. The biological function of this isoform is unknown. Using selection-amplification, we show that mammalian and Drosophila PTB have similar RNA sequence preference, suggesting that cell-specific expression rather than unique RNA-binding properties account for the sex-specific function of dmPTB. We also show that the dmPTB protein isoform expressed in the male germline is by far the most abundant isoform, and reduction of its levels correlates with male sterility. Finally, we show that dmPTB expression is necessary for proper spermatid individualization, the terminal step necessary for production of motile sperm. Loss of dmPTB results in severe disruption of the actin cones of the spermatid individualization complex. This represents a cytological defect resulting from PTB loss. We discuss the basis for functional differences between mammalian and Drosophila PTB orthologs.

Investigating the role of the Est3 protein in yeast telomere replication.

The Est3 subunit of yeast telomerase, which adopts a predicted OB-fold, is essential for telomere replication. To assess the possible contributions that Est3 might make to enzyme catalysis, we compared telomerase activity from wild type and est3-Delta strains of Saccharomyces castellii, which revealed that loss of the Est3 subunit results in a 2- to 3-fold decline in nucleotide addition. This effect was not primer-specific, based on assessment of a panel of primers that spanned the template of the S. castellii telomerase RNA. Furthermore, using nuclear magnetic resonance chemical shift perturbation, no chemical shift change was observed at any site in the protein upon addition of single-stranded DNA, arguing against a role for Est3 in recognition of telomeric substrates by telomerase. Addition of exogenous Est3 protein, including mutant Est3 proteins that are severely impaired for telomere replication in vivo, fully restored activity in est3-Delta telomerase reactions. Thus, Est3 performs an in vivo regulatory function in telomere replication, which is distinct from any potential contribution that Est3 might make to telomerase activity.