article: BReast Cancer Associated proteins 1 and 2 (BRCA1, −2) and Partner and Localizer of BRCA2 (PALB2) protein are tumour suppressors linked to a spectrum of malignancies, including breast cancer and Fanconi anemia. PALB2 coordinates functions of BRCA1 and BRCA2 during homology-directed repair (HDR) and interacts with several chromatin proteins. In addition to protein scaffold function, PALB2 binds DNA. The functional role of this interaction is poorly understood. We identified a major DNA-binding site of PALB2, mutations in which reduce RAD51 foci formation and the overall HDR efficiency in cells by 50%. PALB2 N-terminal DNA-binding domain (N-DBD) stimulates the function of RAD51 recombinase. Surprisingly, it possesses the strand exchange activity without RAD51. Moreover, N-DBD stimulates the inverse strand exchange and can use 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.
  Breast cancer associated proteins 1 and 2 (BRCA1, −2) regulate an efficient non-mutagenic pathway of chromosome break repair and are described as guardians of chromosomal integrity (Venkitaraman, 2014). They initiate RAD51-mediated homologous recombination (HR) (Davies et al. , 2001; Moynahan et al. , 2001; Sharan et al. , 1997; Venkitaraman, 2000) and facilitate restart of stalled replication (Badie et al. , 2010; Lomonosov et al. , 2003; Schlacher et al. , 2011). BRCA2 belongs to a ubiquitous family of Recombination Mediator Proteins (RMPs), which stimulate formation of recombinase filament on single-stranded (ss) DNA protected by ssDNA-binding proteins, like SSB and RPA (Beernink and Morrical, 1999; Cox, 2007; Kowalczykowski, 2005). The Partner and Localizer of BRCA2 (PALB2) protein was discovered as a protein forming a complex with BRCA2 and regulating BRCA2 activity (Xia et al. , 2006). Like BRCA proteins, PALB2 is an essential mammalian protein linked to a similar spectrum of cancers and Fanconi anemia (Ducy et al. , 2019; Pauty et al. , 2014; Xia et al. , 2007). The PALB2 C-terminal WD40 domain interacts with BRCA2 (Oliver et al. , 2009; Xia et al. , 2006) while the N-terminus forms a complex with BRCA1 (Zhang et al. , 2009a; Zhang et al. , 2009b). The latter localizes at double-stranded DNA break (DSB) sites at earlier stage of repair, inhibiting an alternative pathway of non-homologous end joining and initiating homology-directed repair (HDR) through recruitment of PALB2/BRCA2/RAD51 (Prakash et al. , 2015). PALB2 is often described as the hub for a network of tumor suppressors (Park et al. , 2014b; Sy et al. , 2009b). In addition to BRCA1 and −2 interactions, it contains a chromatin-association motif (ChAM) that interacts with histones H3 and H2B (Bleuyard et al. , 2012). PALB2 binds MRG15 protein, a component of histone acetyltransferase-deacetylase complexes (Hayakawa et al. , 2010; Sy et al. , 2009a); RAD51 and its paralogs RAD51C, RAD51AP1 and XRCC3 (Dray et al. , 2010; Park et al. , 2014a); translesion DNA polymerase η (Polη) during recombination-associated DNA synthesis (Buisson et al. , 2014); an oxidative stress response protein KEAP1 (Ma et al. , 2012); and RNF168 ubiquitin ligase (Luijsterburg et al. , 2017). PALB2 is ubiquitinylated in G1 phase of the cell cycle by KEAP1 and CUL3, leading to its degradation, and, thereby, restraining its activity in S/G2 (Orthwein et al. , 2015). Furthermore, PALB2 is an RMP itself as it promotes the assembly of RAD51-ssDNA presynaptic nucleofilaments in the absence of BRCA2 in vitro (Buisson et al. , 2010; Dray et al. , 2010). PALB2 recruits Polη to DSB sites and stimulates recombination-associated DNA synthesis by Polη (Buisson et al. , 2014). Apart from protein-protein interaction domains, BRCA1, −2 and PALB2 proteins also contain DNA binding domains (DBDs), the function of which is poorly understood (Buisson et al. , 2010; Dray et al. , 2010; Paull et al. , 2001; Pellegrini et al. , 2002). Recently, studies of BRCA1/BARD1 complex interaction with DNA and RAD51 led to the discovery of the BRCA1/BARD1 role in RAD51-mediated strand invasion and D-loop formation (Zhao et al. , 2017). Most missense mutations in the BRCA2 DBD are pathogenic (Guidugli et al. , 2013; Wu et al. , 2005). Disruption of DNA binding in a BRCA2 truncation variant lacking the PALB2-binding motif leads to a significant HDR reduction (Siaud et al. , 2011). However, deletion of the BRCA2 DBD has a negligible effect when interaction with PALB2 is preserved, highlighting the functional importance of the previously reported DNA-binding property of PALB2 (Buisson et al. , 2010; Dray et al. , 2010). Two truncation fragments of PALB2, T1 (residues 1–200) and T3 (residues 372–561), bind DNA (Buisson et al. , 2010). While the precise mechanism of DNA interaction and its functional role remain unknown, several reports indirectly support its importance in DNA repair. For example, PALB2 truncation of 500 amino acids situated between the BRCA1 and BRCA2 binding motifs and containing both DBDs does not support BRCA2 and RAD51 foci formation in cells during DNA damage (Sy et al. , 2009b). Since both the BRCA1-binding N-terminal and the BRCA2-interacting WD40 C-terminal domains are retained in this mutant, the results points to the potential importance of DBDs in PALB2 function. The importance of DNA binding was demonstrated for other RMPs, including bacterial RecFOR (Korolev, 2017; Morimatsu and Kowalczykowski, 2003; Ryzhikov et al. , 2014; Sakai and Cox, 2009; Umezu et al. , 1993) and eukaryotic RAD52 (Arai et al. , 2011; Seong et al. , 2008). In the current study, we identified a major DBD of PALB2 (N-DBD) and specific amino acids involved in DNA binding. Mutations of only four amino acids significantly reduce RAD51 foci formation and the efficiency of HDR in a model cell system. PALB2 N-DBD by itself stimulates RAD51 strand exchange reactions. Surprisingly, we found that the PALB2 N-DBD supports both forward and inverse strand exchange even in the absence of RAD51 and can use RNA as a substrate. Altogether, our data reveal a novel activity of PALB2 and highlight the importance of PALB2 DNA binding in chromosome maintenance.
  We identified major DNA-binding residues of PALB2 and demonstrated their critical role for HDR in cells. PALB2 is described as a scaffold protein linking BRCA1 with BRCA2 during HDR and interacting with many other chromatin proteins. However, the truncation variant with the preserved BRCA1 and BRCA2 binding motifs but without the middle portion of the protein, which contains DBDs, does not support BRCA2 and RAD51 recruitment to DSBs (Sy et al. , 2009b). Recent studies suggest an alternative BRCA1-independent recruitment of PALB2 to DSB through direct interaction with RNF168 (Luijsterburg et al. , 2017; Zong et al. , 2019). However, this interaction is mediated by the C-terminal WD40 domain, which is preserved in the described above truncation mutant. A critical role of PALB2 DNA binding was also suggested by studies of the BRCA2 (Siaud et al. , 2011), where the ‘miniBRCA2’ construct, including only DBDs with two BRC repeats, was 3–4 times less efficient than ‘midiBRCA2’ which includes PALB2 interaction motif. Moreover, interaction with PALB2 alleviates the requirement of BRCA2 DNA binding, including a deletion of the entire BRCA2 DBD. Here, we demonstrate that mutation of only four DNA-binding residues of PALB2 reduces both RAD51 foci and overall HDR efficiency by 50% in the presence of endogenous BRCA1 and BRCA2. Even at 8 hr post-IR siRNA PALB2 cells complemented with PALB2-146AAAA still display a defect in HR. We suggest that the mutant display a failure to repair a subset of DSBs due to its defective DNA binding. Therefore, PALB2 interaction with DNA is critical for recruitment of the BRCA2 and RAD51 to DSB sites and efficient DNA repair in cells. Secondly, we demonstrate that the PALB2 N-DBD stimulates RAD51-mediated strand exchange in vitro. N-DBD encompasses the RAD51 binding site, therefore, it can recruit RAD51 to ssDNA to initiate nucleation of nucleoprotein filament. DNA-binding of N-DBD plays a critical role in this process, as the DNA-binding mutant (146AAAA) retains interaction with RAD51 (Figure 3—figure supplement 3) but is unable to stimulate RAD51 binding to ssDNA (Figure 5—figure supplement 1). Alternatively, the PALB2 N-DBD can support a DNA conformation favourable for RAD51 binding to ssDNA or/and nucleation of RAD51 filament, similarly to the prokaryotic RecOR proteins (Bell et al. , 2012; Ryzhikov et al. , 2014; Sakai and Cox, 2009). The most unexpected finding is the ability of PALB2 to stimulate strand exchange between homologous ss- and dsDNA fragments in the absence of recombinase. This process is protein-specific and is not a consequence of simple DNA melting and reannealing of separated strands in solution, since the PALB2 N-DBD does not unwind the DNA helix and promotes DNA annealing. PALB2-mediated strand exchange differs from that of supported by RAD51, as it does not require ATP binding and hydrolysis and it is less efficient and not unidirectional. This reaction resembles that of the RAD52 (Mazina et al. , 2017). In the cited manuscript, RAD52 promotes inverse reaction much more efficiently than forward and seemingly faster than that by PALB2 in our hands. However, the substrates used in RAD52 studies differ from those used in the current work by the presence of the ssDNA tail in dsDNA. The reaction with a no-tail substrate appears to be less efficient than the one supported by PALB2. Another distinction from RAD52 is the initial reaction rate, which was much faster in case of inverse reaction even with no-tail substrate for RAD52. PALB2-mediated reactions are slower with no difference between forward and inverse reactions. Proteins supporting strand exchange, such as RecA and RAD52, share several common features. They interact with both ss- and dsDNA through distinct sites located next to each other, they form oligomeric structures, such as recombinase-DNA filament or Rad52 ring structure, and they distort the dsDNA helix to initiate strand exchange with the bound complementary ssDNA. RecA stretches dsDNA (Chen et al. , 2008; Leger et al. , 1998), while Rad52 bends the DNA helix bound to the toroidal oligomeric ring (Brouwer et al. , 2017). The PALB2 N-DBD interacts with both ss- and dsDNA. Previously, we demonstrated that PALB2 immobilized on ssDNA beads efficiently pulls down non-homologous dsDNA (Buisson et al. , 2010). It is unclear whether ss- and dsDNA substrates are bound to the same sites of different subunits of a PALB2 oligomer or two different DNA binding sites on the same monomer. The presence of at least two other minor DNA-binding sites in the N-DBD suggests such a possibility. Higher affinity towards longer ssDNA (Figure 1) and the FRET experiment (Figure 8—figure supplement 1B) support a model of wrapping long flexible ssDNA around an oligomer. In contrast, interaction with dsDNA is less length dependent (Figure 1). We can speculate that binding of dsDNA to more than one monomer in PALB2 oligomer can trigger DNA helix distortion. Thus, PALB2 shares several specific structural and DNA-interaction features with both RecA/RAD51 and Rad52 proteins and supports a protein-specific strand exchange reaction. At the same time, PALB2 does not support D-loop formation with supercoiled dsDNA opposite to a full length PALB2. This may be a consequence of the inability to efficiently bind and distort supercoiled dsDNA plasmid through the secondary DNA-binding site. It is important to note one distinct feature of the PALB2 N-DBD: the secondary structure prediction (Figure 2—figure supplement 1) suggests a different folding of the N-DBD fragment than that of RecA-like domains or a Rad52. The latter proteins are formed by α/β sandwich folds, while PALB2 N-DBD folding is predicted to be composed of only α-helices, which may form helical bundle-like structure similar to that of Hop2-Mnd1 (Kang et al. , 2015). Therefore, PALB2 N-DBD represents a novel structural fold that supports strand exchange. While the functional significance of this property remains to be investigated, it further supports the involvement of PALB2 in specific DNA transactions during HDR, similar to the involvement of BRCA1/BARD1 in D-loop formation (Zhao et al. , 2017). It was shown that both PALB2 and BRCA2 stimulate Polη DNA synthesis within a D-loop substrate in vitro through the recruitment of the polymerase to the invading strand in the D-loop (Buisson et al. , 2014). Interestingly, DNA synthesis was more efficient in the presence of PALB2 than BRCA2, while both proteins were shown be equally efficient in recruiting polymerase to DSB sites. PALB2 strand exchange may contribute to other steps of HDR such as second-end capture (Mazloum and Holloman, 2009; McIlwraith and West, 2008; Nimonkar et al. , 2009). Interestingly, PALB2 (FANCN) and BRCA2 (FANCD) are involved in replication-dependant removal of interstrand DNA crosslinks associated with Fanconi anemia (Howlett et al. , 2002; Moldovan and D' Andrea, 2009; Xia et al. , 2007). The strand exchange function of PALB2 may also be important for alternative DNA repair pathways. Indeed, PALB2 supports strand exchange not only with ssDNA, but with ssRNA substrates, and can be involved in transcription-initiated DNA repair. This hypothesis seems particularly attractive in light of the PALB2 interaction with MRG15 and its localization at the sites of actively transcribed genes Bleuyard et al. (2017).
  Target amino acids were mutated by site directed mutagenesis using Stratagene QuikChange protocol. Single, double, triple and four residues mutants were generated by single stranded synthesis (Supplementary file 1B). PCR samples were subjected to DpnI digestion at 37°C for 6 hr and annealed gradually by reducing temperature from 95°C to 37°C for an hour with a 1°C drop per minute. DpnI treated PCR samples were transformed into chemically competent OmniMAX cells (ThermoFischer). Mutations were confirmed by sequencing and plasmids were transformed into BL21 (DE) cells. Mutant proteins were expressed and purified exactly as described for wild type fragments. Fluorescence anisotropy experiments were carried out at room temperature with 5 nM fluorescein (6FAM) -labelled DNA substrates (Supplementary file 1C) using a Synergy four plate reader (BioTek). Titration with protein was performed by serially diluting protein in 40 μL of assay buffer (20 mM Tris-acetate pH 7. 0,100 mM NaCl, 5% glycerol, 1 mM TCEP and 10% DMSO) from 5000 nM to 0. 3 nM and incubating with DNA substrate for 15 min at RT. Fluorescence anisotropy was measured by excitation at 485/20 nm and by monitoring emission 528/20 nm at room temperature using Gen5. 0 (BioTek) software. An equilibrium dissociation constant was calculated with Prism software using one to one binding scheme, P+[D]=[DP], where D is DNA and P is PALB2. Anisotropy data were fitted by a non-linear regression analysis of Prism software using standard four-parameter logistic equation to identify Kdy=ymin+ (ymax-ymin1+10logEC50-X×n) Where, ymin and ymax is the minimum and maximum anisotropy values, X represents the log concentration of protein, n represents Hillslope, and EC50 is equal to KD. R2 is determined by the prism software by computing the sum of the squares of the distances of the points from the best-fit curve determined by nonlinear regression model. DNA annealing assays were performed with Cy5-labelled ss35 and compl IOWA-labelled ss35 (100 nM, Supplementary file 1D). The protein at 1,2 and 4 μM concentrations was mixed and incubated with Cy5-labelled ssDNA for 5 min at 37°C in 40 μL reaction buffer (40 mM HEPES pH 7. 5,20 mM NaCl and 1 mM TCEP). Reactions were initiated by addition of compl IOWA-labelled ss35 (100 nM) in 40 μL of reaction buffer. Decrease in Cy5 fluorescence was monitored by measuring fluorescence at 680 nm by excitation at 635 nm on a Synergy four plate reader (BioTek). DNA strand exchange assays (80 μl) were performed with 35 bp dsDNA obtained by annealing of 5’-Cy5- and 3’-IOWA-labelled compl strands (Supplementary file 1D) and a 90mer ssDNA (ss90) with a region homologous to the plus strand. Alternatively, FAM/Dabsyl 49 bp DNA was used. For the forward reaction, ss90 (100 nM) was incubated with 2 μM (or as mentioned in the figure legends) protein for 10 min in 40 μL reaction buffer (40 mM HEPES pH 7. 5,20 mM NaCl, 5 mM MgCl2,1 mM TCEP and 0. 02% Tween 20) at 37°C. Strand exchange was initiated by addition of 100 nM Cy5/IOWA-dsDNA35 (or FAM/Dabsyl-dsDNA49), the plate was immediately placed in plate reader and the intensity of Cy5 (or FAM) fluorescence was measured at 30 s intervals for 1 hr with excitation at 635 nm and emission at 680 nm. For reactions with RecA and Rad51, an ATP regeneration system (2 mM ATP, 3 mM phosphoenol pyruvate and 30 U of pyruvate kinase) was used (Sigma-Aldrich, USA). Experiments without RecA and RAD51 were performed without ATP. For the inverse reaction, protein was incubated with Cy5/IOWA-dsDNA35 (or FAM/Dabsyl-dsDNA49) substrate and reaction was initiated by addition of ss90. The strand exchange assay with ssRNA substrate was performed as described above using a 60 ribonucleotide RNA (Supplementary file 1D) compl to that of 35 bp DNA. Alternatively, Cy3- and Cy5-labelled DNA oligonucleotides were used to prepare dsDNA substrate and the products were analysed by EMSA PAGE (below). Fluorescent-labelled DNA products of strand exchange reactions were also analysed on EMSA PAGE. After fluorescence measurement on plate reader, the final reaction mix (80 μl) products were deproteinated by incubation with proteinase K (0. 5 mg/ml) with 0. 5 mM EDTA and 1% w/v SDS for 20 min at 37°C and the DNA fragments were separated on 10% PAGE gel in TBE buffer. The gel was imaged using a Typhoon 9400 image scanner (GE) and analysed with ImageJ software. FRET assay was performed in 96 well plate format. 100 nM of dual labelled dT70 or ss40 (Cy5 at 5’ end and Cy3 at 3’ end) was dispensed into 80 μL assay buffer identical to the buffer in the strand exchange assay (Supplementary file 1D). Excitation was at 540/25 nm bandpass. Emission for both Cy3 at 590/35 nm bandpass and Cy5 at 680/30 nm were measured. PALB2 was added with final concentration between 4000 and 15. 6 nM and incubated for 10 min. FRET efficiency was calculated by using the formula FRET=1. 51×Icy51. 51×Icy5+0. 669×Icy3, where 1. 51 and 0. 669 are correction factors for fraction of Cy5 and Cy3 intensities in Cy3 and Cy5 channels respectively. Protein concentrations were as described in the figure legends. D-loop buffer (25 mM Tris-acetate pH 7. 5,100 µg/mL bovine serum albumin, 2 mM CaCl2,2 mM ATP, 1 mM DTT), containing 1 µM of radiolabelled oligonucleotide JYM1413 (homologous sequence to the plasmid pPB4. 3, Supplementary file 1C) was incubated for 5 min at 37°C with the indicated concentration of RAD51 or PALB2-T1 fragment. CsCl- purified pPB4. 3 replicative form I DNA (300 µM) was added and the reaction and incubated for 5 min. Finally, the reaction was stopped with the addition of 0,6% SDS, 20 mM Tris-HCl pH 7. 5,20 mM MgCl2 and 2 mg/mL proteinase K following by incubation for 30 min at 37°C. Labelled DNA products were analysed by electrophoresis through a 0. 8% TAE1X/agarose gel, ran at 65V for 1 hr, dried onto DE81 filter paper at 85°C, and visualized by autoradiography. HeLa cells were seeded on glass coverslips in 6-well plates at 225 000 cells per well. Knockdown of PALB2 was performed 18 hr later with 50 nM PALB2 siRNA (Supplementary file 1E) using Lipofectamine RNAiMAX (Invitrogen). After 5 hr, cells were subjected to double thymidine block. Briefly, cells were treated with 2 mM thymidine for 18 hr and released after changing the media. After a release of 9 hr, PALB2 silenced cells were complemented using transfection with the indicated YFP constructs using Lipofectamine 2000. Cells were then treated with 2 mM thymidine for 17 hr and protected from light from this point on. After 2 hr of release from the second block, cells were X-irradiated with 2 Gy and processed for immunofluorescence 1 hr, 2 hr, 4 hr, 6 hr and 8 hr post-irradiation. All immunofluorescence dilutions were prepared in PBS and incubations performed at room temperature with intervening washes in PBS. Cell fixation was carried out by incubation with 4% paraformaldehyde for 10 min followed by 100% ice-cold methanol for 5 min at −20°C. Cells were then permeabilized in 0. 2% Triton X-100 for 5 min and quenched using 0. 1% sodium borohydride for 5 min. After blocking for 1 hr in a solution containing 10% goat serum and 1% BSA, cells were incubated for 1 hr with primary antibodies to RAD51 (B-bridge International, #70–001) and to cyclin A (BD Biosciences, # 611268) diluted in 1% BSA. Secondary antibodies, Alexa Fluor 568 goat anti-rabbit (Invitrogen, #A-11011) and Alexa Fluor 647 goat anti-mouse (Invitrogen, #A-21235), were used in PBS containing 1% BSA for 1 hr. Nuclei were stained for 10 min with 1 μg/mL 4,6-diamidino-2-phenylindole (DAPI) prior to mounting onto slides with 90% glycerol containing 1 mg/ml paraphenylenediamine anti-fade reagent. Z-stack images were acquired at 63X magnification on a Leica DM6000 microscope, then deconvoluted and analysed for RAD51 foci formation with Volocity software v6. 0. 1 (Perkin-Elmer Improvision). The number of RAD51 foci per cyclin A-positive cells (n = 300), among the transfected population, was manually scored and reported in a scatter dot plot representing the SEM. An Anova test (Kruskal-Wallis-test for multiple comparison) was performed followed by a non-parametric Mann-Whitney test. U2OS cells were seeded in 6-well plates. Knockdown of PALB2 (Buisson et al. , 2017) was performed 6–8 hr later using Lipofectamine RNAiMAX (Invitrogen). Twenty-four hours post-transfection, 1. 5−2 × 106 cells were pelleted for each condition and resuspended in 100 μL complete nucleofector solution (SE Cell Line 4D-Nucleofector X Kit, Lonza) to which 1 μg of pCR2. 1-mClover-LMNAdonor, 1 μg pX330-LMNAgRNA, 0. 1 μg of iRFP670 and 1 μg of pcDNA3 empty vector or the Flag-PALB2 constructs, and 20 nM of each siRNA were added. Once transferred to a 100 μl Lonza certified cuvette, cells were transfected using the 4D-Nucleofector X-unit, program CM-104 and transferred to a 10 cm dish. After 48 hr, cells were trypsinized and plated onto glass coverslips. Expression of the mClover was assayed the next day by fluorescence microscopy (63X), that is 72 hr post-nucleofection. Data are represented as mean percentages of mClover-positive cells over the iRFP-positive population from five independent experiments (total n > 100 iRFP-positive cells) and reported in a scatter dot plot representing SEM, and a classical one-way Anova test was performed. HEK293T were plated to 80% confluency and transfected with YFP-CTL, YFP-PALB2 or YFP-PALB2-146AAAA vector using Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific). 24 hr after, cells were irradiated with 5 Gy and processed for GFP-Trap Pulldown 2 hr post-irradiation. Briefly, cells were washed twice with ice cold PBS and lysed in lysis buffer (50 mM Tris-HCl, pH 7. 5,150 mM NaCl, 0,5% NP40, containing PMSF, Aprotinin, Leupeptin, NaF, and Na2VO4). Lysates were sonicated (three times 10 s at 30% amplitude on ice) and centrifuged at 16000 g, for 30 min at 4°C. Protein lysates (3 mg) were incubated with GFP-Trap beads (Chromotek) for 1 hr at 4°C. Beads were washed three times with lysis buffer (without NP40) and bound proteins were resuspended in 35 µL of Laemmli SDS-sample buffer and heated to 95°C for 10 min. Samples were run on NuPAGE 4–12% bis-Tris Protein Gels (Invitrogen) in NuPAGE MOPS SDS Running Buffer according to the manufacturer’s protocol and transferred to Nitrocellulose membrane (Amersham) using XCell II Blot Module (Invitrogen) in 20% methanol transfer buffer. Immunoblots were performed using the following antibodies: anti-RAD51 (14B4) (Novus Biologicals, #NB100-148) and anti-GFP (Roche, #11814460001). peYFP-C1-PALB2 was modified to be resistant to PALB2 siRNA by Q5 Site-Directed Mutagenesis Kit (NEB, E0554) using primers JYM3892/3893 (Supplementary file 1E). The resulting siRNA-resistant construct was then used as a template to generate the mutant construct YFP-PALB2 146AAAA with the primers JYM3909/JYM3910. Flag-tagged PALB2 146AAAA mutant was also obtained via site-directed mutagenesis on pcDNA3-Flag PALB2 (Pauty et al. , 2017). U2OS (HTB-96) and HEK293T (CRL-11268) were purchased from ATCC and HeLa were authenticated using Short Tandem Repeat (STR) analysis by ATCC services (100% match). All the cells lines used were uninfected with Mycoplasma, as routinely verified using the e-Myco Mycoplasma kit from FroggaBio.
summary: DNA in a cell is under constant stress from environmental factors, such as ultraviolet light, or from damage caused by the replication process. These sources of stress can cause breaks in the genome, which if left unrepaired can lead to cancer or cell death. One of the most accurate ways to repair a broken fragment of DNA is through recombination – whereby an undamaged copy of the sequence is located in another DNA molecule and used as a template to replace the missing fragment. DNA recombination is regulated by more than a dozen proteins that help recruit the enzyme RAD51 to sites of DNA damage, and trigger its search for complementary sequences of DNA. A molecule known as PALB2 binds to these DNA repair proteins and coordinates their activity. If PALB2, or these other proteins become mutated, this can increase the risk cancerous growths in various tissues, including the breasts and ovaries. Having a better understanding of how this group of proteins control the repair process could therefore improve prognosis and advance cancer treatments. Now, Deveryshetty et al. have discovered a new and unexpected role for PALB2 within the recombination pathway. As well as binding to other repair proteins, PALB2 interacts directly with DNA, and this interaction was found to be an important part of the repair process. Even in the absence of RAD51, PALB2 was still able to recombine short fragments of DNA sequence. PALB2 achieves this by initiating recombination using single strands of DNA or a DNA-like molecule known as RNA. This latter property may be particularly important if the molecular machines needed to replicate DNA and synthesize RNA collide on the same DNA molecule. This new role for PALB2 could lead to the discovery of other DNA repair mechanisms, and could be used to predict which PALB2 mutations are more likely to cause cancer. Patients who are at greater risk of cancer could then be treated with more advanced therapies, in order to increase their chances of recovery.
keywords: biochemistry and chemical biology
  cancer biology
title: Novel RNA and DNA strand exchange activity of the PALB2 DNA binding domain and its critical role for DNA repair in cells