Thrombin prefers substrates carrying Arg at the site of cleavage (P1) because of the presence of D189 in the primary specificity (S1) pocket but can also cleave substrates carrying Phe at P1. The structural basis of this property is unknown.

The partner and localizer of BRCA2 (PALB2) is a scaffold protein linking BRCA1 with BRCA2 and RAD51 during homologous recombination (HR). PALB2 interaction with DNA strongly enhances HR in cells, while the PALB2 DNA-binding domain (PALB2-DBD) supports DNA strand exchange . We determined that PALB2-DBD is intrinsically disordered beyond a single N-terminal α-helix. Coiled-coil mediated dimerization is stabilized by interaction between intrinsically disordered regions (IDRs) leading to a 2-fold structural compaction. Single-stranded (ss)DNA binding promotes additional structural compaction and protein tetramerization. Using confocal single-molecule FRET, we observed bimodal and oligomerization-dependent compaction of ssDNA bound to PALB2-DBD, suggesting a novel strand exchange mechanism. Bioinformatics analysis and preliminary observations indicate that PALB2 forms protein-nucleic acids condensates. Intrinsically disordered DBDs are prevalent in the human proteome. PALB2-DBD and similar IDRs may use a chaperone-like mechanism to aid formation and resolution of DNA and RNA multichain intermediates during DNA replication, repair and recombination.

The Partner and Localizer of BRCA2 (PALB2) tumor suppressor is a scaffold protein that links BRCA1 with BRCA2 to initiate homologous recombination (HR). PALB2 interaction with DNA strongly enhances HR efficiency. The PALB2 DNA-binding domain (PALB2-DBD) supports DNA strand exchange, a complex multistep reaction supported by only a few protein families such as RecA-like recombinases or Rad52. The mechanisms of PALB2 DNA binding and strand exchange are unknown. We performed circular dichroism, electron paramagnetic spectroscopy, and small-angle X-ray scattering analyses and determined that PALB2-DBD is intrinsically disordered, even when bound to DNA. The intrinsically disordered nature of this domain was further supported by bioinformatics analysis. Intrinsically disordered proteins (IDPs) are prevalent in the human proteome and have many important biological functions. The complexity of the strand exchange reaction significantly expands the functional repertoire of IDPs. The results of confocal single-molecule FRET indicated that PALB2-DBD binding leads to oligomerization-dependent DNA compaction. We hypothesize that PALB2-DBD uses a chaperone-like mechanism to aid formation and resolution of complex DNA and RNA multichain intermediates during DNA replication and repair. Since PALB2-DBD alone or within the full-length PALB2 is predicted to have strong liquid-liquid phase separation (LLPS) potential, protein-nucleic acids condensates are likely to play a role in complex functionality of PALB2-DBD. Similar DNA-binding intrinsically disordered regions may represent a novel class of functional domains that evolved to function in eukaryotic nucleic acid metabolism complexes.

The tumor suppressor protein partner and localizer of BRCA2 (PALB2) orchestrates the interactions between breast cancer susceptibility proteins 1 and 2 (BRCA1, -2) that are critical for genome stability, homologous recombination (HR) and DNA repair. PALB2 mutations predispose patients to a spectrum of cancers, including breast and ovarian cancers. PALB2 localizes HR machinery to chromatin and links it with transcription through multiple DNA and protein interactions. This includes its interaction with MRG15 (Morf-related gene on chromosome 15), which is part of many transcription complexes, including the HAT-associated and the HDAC-associated complexes. This interaction is critical for PALB2 localization in actively transcribed genes, where transcription/replication conflicts lead to frequent replication stress and DNA breaks. We solved the crystal structure of the MRG15 MRG domain bound to the PALB2 peptide and investigated the effect of several PALB2 mutations, including patient-derived variants. PALB2 interacts with an extended surface of the MRG that is known to interact with other proteins. This, together with a nanomolar affinity, suggests that the binding of MRG15 partners, including PALB2, to this region is mutually exclusive. Breast cancer-related mutations of PALB2 cause only minor attenuation of the binding affinity. New data reveal the mechanism of PALB2-MRG15 binding, advancing our understanding of PALB2 function in chromosome maintenance and tumorigenesis.

Long interspersed nuclear element-1 (L1) is an autonomous non-LTR retrotransposon comprising ∼20% of the human genome. L1 self-propagation causes genomic instability and is strongly associated with aging, cancer and other diseases. The endonuclease domain of L1’s ORFp2 protein (L1-EN) initiates de novo L1 integration by nicking the consensus sequence 5′-TTTTT/AA-3′. In contrast, related nucleases including structurally conserved apurinic/apyrimidinic endonuclease 1 (APE1) are non-sequence specific. To investigate mechanisms underlying sequence recognition and catalysis by L1-EN, we solved crystal structures of L1-EN complexed with DNA substrates. This showed that conformational properties of the preferred sequence drive L1-EN’s sequence-specificity and catalysis. Unlike APE1, L1-EN does not bend the DNA helix, but rather causes ‘compression’ near the cleavage site. This provides multiple advantages for L1-EN’s role in retrotransposition including facilitating use of the nicked poly-T DNA strand as a primer for reverse transcription. We also observed two alternative conformations of the scissile bond phosphate, which allowed us to model distinct conformations for a nucleophilic attack and a transition state that are likely applicable to the entire family of nucleases. This work adds to our mechanistic understanding of L1-EN and related nucleases and should facilitate development of L1-EN inhibitors as potential anticancer and antiaging therapeutics.