53BP1 Enforces Distinct Pre- and Post-resection Blocks on Homologous Recombination.

53BP1 activity drives genome instability and lethality in BRCA1-deficient mice by inhibiting homologous recombination (HR). The anti-recombinogenic functions of 53BP1 require phosphorylation-dependent interactions with PTIP and RIF1/shieldin effector complexes. While RIF1/shieldin blocks 5'-3' nucleolytic processing of DNA ends, it remains unclear how PTIP antagonizes HR. Here, we show that mutation of the PTIP interaction site in 53BP1 (S25A) allows sufficient DNA2-dependent end resection to rescue the lethality of BRCA1Δ11 mice, despite increasing RIF1 "end-blocking" at DNA damage sites. However, double-mutant cells fail to complete HR, as excessive shieldin activity also inhibits RNF168-mediated loading of PALB2/RAD51. As a result, BRCA1Δ1153BP1S25A mice exhibit hallmark features of HR insufficiency, including premature aging and hypersensitivity to PARPi. Disruption of shieldin or forced targeting of PALB2 to ssDNA in BRCA1D1153BP1S25A cells restores RNF168 recruitment, RAD51 nucleofilament formation, and PARPi resistance. Our study therefore reveals a critical function of shieldin post-resection that limits the loading of RAD51.

Repeat-mediated deletions can be induced by a chromosomal break far from a repeat, but multiple pathways suppress such rearrangements.

Chromosomal deletion rearrangements mediated by repetitive elements often involve repeats separated by several kilobases and sequences that are divergent. While such rearrangements are likely induced by DNA double-strand breaks (DSBs), it has been unclear how the proximity of DSBs relative to repeat sequences affects the frequency of such events. We generated a reporter assay in mouse cells for a deletion rearrangement involving repeats separated by 0.4 Mb. We induced this repeat-mediated deletion (RMD) rearrangement with two DSBs: the 5' DSB that is just downstream from the first repeat and the 3' DSB that is varying distances upstream of the second repeat. Strikingly, we found that increasing the 3' DSB/repeat distance from 3.3 kb to 28.4 kb causes only a modest decrease in rearrangement frequency. We also found that RMDs are suppressed by KU70 and RAD51 and promoted by RAD52, CtIP, and BRCA1. In addition, we found that 1%-3% sequence divergence substantially suppresses these rearrangements in a manner dependent on the mismatch repair factor MSH2, which is dominant over the suppressive role of KU70. We suggest that a DSB far from a repeat can stimulate repeat-mediated rearrangements, but multiple pathways suppress these events.

m6 A deposition is regulated by PRMT1-mediated arginine methylation of METTL14 in its disordered C-terminal region.

The N6-methyladenosine (m6 A) RNA modification serves crucial functions in RNA metabolism; however, the molecular mechanisms underlying the regulation of m6 A are not well understood. Here, we establish arginine methylation of METTL14, a component of the m6 A methyltransferase complex, as a novel pathway that controls m6 A deposition in mammalian cells. Specifically, protein arginine methyltransferase 1 (PRMT1) interacts with, and methylates the intrinsically disordered C terminus of METTL14, which promotes its interaction with RNA substrates, enhances its RNA methylation activity, and is crucial for its interaction with RNA polymerase II (RNAPII). Mouse embryonic stem cells (mESCs) expressing arginine methylation-deficient METTL14 exhibit significantly reduced global m6 A levels. Transcriptome-wide m6 A analysis identified 1,701 METTL14 arginine methylation-dependent m6 A sites located in 1,290 genes involved in various cellular processes, including stem cell maintenance and DNA repair. These arginine methylation-dependent m6 A sites are associated with enhanced translation of genes essential for the repair of DNA interstrand crosslinks; thus, METTL14 arginine methylation-deficient mESCs are hypersensitive to DNA crosslinking agents. Collectively, these findings reveal important aspects of m6 A regulation and new functions of arginine methylation in RNA metabolism.

53BP1 inhibits homologous recombination in Brca1-deficient cells by blocking resection of DNA breaks.

Defective DNA repair by homologous recombination (HR) is thought to be a major contributor to tumorigenesis in individuals carrying Brca1 mutations. Here, we show that DNA breaks in Brca1-deficient cells are aberrantly joined into complex chromosome rearrangements by a process dependent on the nonhomologous end-joining (NHEJ) factors 53BP1 and DNA ligase 4. Loss of 53BP1 alleviates hypersensitivity of Brca1 mutant cells to PARP inhibition and restores error-free repair by HR. Mechanistically, 53BP1 deletion promotes ATM-dependent processing of broken DNA ends to produce recombinogenic single-stranded DNA competent for HR. In contrast, Lig4 deficiency does not rescue the HR defect in Brca1 mutant cells but prevents the joining of chromatid breaks into chromosome rearrangements. Our results illustrate that HR and NHEJ compete to process DNA breaks that arise during DNA replication and that shifting the balance between these pathways can be exploited to selectively protect or kill cells harboring Brca1 mutations.

Resolution of sequence divergence for repeat-mediated deletions shows a polarity that is mediated by MLH1.

Repeat-mediated deletions (RMDs) are a type of chromosomal rearrangement between two homologous sequences that causes loss of the sequence between the repeats, along with one of the repeats. Sequence divergence between repeats suppresses RMDs; the mechanisms of such suppression and of resolution of the sequence divergence remains poorly understood. We identified RMD regulators using a set of reporter assays in mouse cells that test two key parameters: repeat sequence divergence and the distances between one repeat and the initiating chromosomal break. We found that the mismatch repair factor MLH1 suppresses RMDs with sequence divergence in the same pathway as MSH2 and MSH6, and which is dependent on residues in MLH1 and its binding partner PMS2 that are important for nuclease activity. Additionally, we found that the resolution of sequence divergence in the RMD product has a specific polarity, where divergent bases that are proximal to the chromosomal break end are preferentially removed. Moreover, we found that the domain of MLH1 that forms part of the MLH1-PMS2 endonuclease is important for polarity of resolution of sequence divergence. We also identified distinctions between MLH1 versus TOP3α in regulation of RMDs. We suggest that MLH1 suppresses RMDs with sequence divergence, while also promoting directional resolution of sequence divergence in the RMD product.

Microarray screening reveals two non-conventional SUMO-binding modules linked to DNA repair by non-homologous end-joining.

SUMOylation is critical for numerous cellular signalling pathways, including the maintenance of genome integrity via the repair of DNA double-strand breaks (DSBs). If misrepaired, DSBs can lead to cancer, neurodegeneration, immunodeficiency and premature ageing. Using systematic human proteome microarray screening combined with widely applicable carbene footprinting, genetic code expansion and high-resolution structural profiling, we define two non-conventional and topology-selective SUMO2-binding regions on XRCC4, a DNA repair protein important for DSB repair by non-homologous end-joining (NHEJ). Mechanistically, the interaction of SUMO2 and XRCC4 is incompatible with XRCC4 binding to three other proteins important for NHEJ-mediated DSB repair. These findings are consistent with SUMO2 forming a redundant NHEJ layer with the potential to regulate different NHEJ complexes at distinct levels including, but not limited to, XRCC4 interactions with XLF, LIG4 and IFFO1. Regulation of NHEJ is not only relevant for carcinogenesis, but also for the design of precision anti-cancer medicines and the optimisation of CRISPR/Cas9-based gene editing. In addition to providing molecular insights into NHEJ, this work uncovers a conserved SUMO-binding module and provides a rich resource on direct SUMO binders exploitable towards uncovering SUMOylation pathways in a wide array of cellular processes.

Arginine regulates HSPA5/BiP translation through ribosome pausing in triple-negative breast cancer cells.

BACKGROUND: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with a high mortality rate due to a lack of therapeutic targets. Many TNBC cells are reliant on extracellular arginine for survival and express high levels of binding immunoglobin protein (BiP), a marker of metastasis and endoplasmic reticulum (ER) stress response. METHODS: In this study, the effect of arginine shortage on BiP expression in the TNBC cell line MDA-MB-231 was evaluated. Two stable cell lines were generated in MDA-MB-231 cells: the first expressed wild-type BiP, and the second expressed a mutated BiP free of the two arginine pause-site codons, CCU and CGU, termed G-BiP. RESULTS: The results showed that arginine shortage induced a non-canonical ER stress response by inhibiting BiP translation via ribosome pausing. Overexpression of G-BiP in MDA-MB-231 cells promoted cell resistance to arginine shortage compared to cells overexpressing wild-type BiP. Additionally, limiting arginine led to decreased levels of the spliced XBP1 in the G-BiP overexpressing cells, potentially contributing to their improved survival compared to the parental WT BiP overexpressing cells. CONCLUSION: In conclusion, these findings suggest that the downregulation of BiP disrupts proteostasis during arginine shortage-induced non-canonical ER stress and plays a key role in cell growth inhibition, indicating BiP as a target of codon-specific ribosome pausing upon arginine shortage.

RNF8 has both KU-dependent and independent roles in chromosomal break repair.

Chromosomal double strand breaks (DSBs) can initiate several signaling events, such as ubiquitination, however the precise influence of such signaling on DSB repair outcomes remains poorly understood. With an RNA interference screen, we found that the E3 ubiquitin ligase RNF8 suppresses a deletion rearrangement mediated by canonical non-homologous end joining (C-NHEJ). We also found that RNF8 suppresses EJ without insertion/deletion mutations, which is a hallmark of C-NHEJ. Conversely, RNF8 promotes alternative EJ (ALT-EJ) events involving microhomology that is embedded from the edge of the DSB. These ALT-EJ events likely require limited end resection, whereas RNF8 is not required for single-strand annealing repair involving extensive end resection. Thus, RNF8 appears to specifically facilitate repair events requiring limited end resection, which we find is dependent on the DSB end protection factor KU. However, we also find that RNF8 is important for homology-directed repair (HDR) independently of KU, which appears linked to promoting PALB2 function. Finally, the influence of RNF8 on EJ is distinct from 53BP1 and the ALT-EJ factor, POLQ. We suggest that RNF8 mediates both ALT-EJ and HDR, but via distinct mechanisms, since only the former is dependent on KU.

Distinct roles of RAD52 and POLQ in chromosomal break repair and replication stress response.

Disrupting either the DNA annealing factor RAD52 or the A-family DNA polymerase POLQ can cause synthetic lethality with defects in BRCA1 and BRCA2, which are tumor suppressors important for homology-directed repair of DNA double-strand breaks (DSBs), and protection of stalled replication forks. A likely mechanism of this synthetic lethality is that RAD52 and/or POLQ are important for backup pathways for DSB repair and/or replication stress responses. The features of DSB repair events that require RAD52 vs. POLQ, and whether combined disruption of these factors causes distinct effects on genome maintenance, have been unclear. Using human U2OS cells, we generated a cell line with POLQ mutations upstream of the polymerase domain, a RAD52 knockout cell line, and a line with combined disruption of both genes. We also examined RAD52 and POLQ using RNA-interference. We find that combined disruption of RAD52 and POLQ causes at least additive hypersensitivity to cisplatin, and a synthetic reduction in replication fork restart velocity. We also examined the influence of RAD52 and POLQ on several DSB repair events. We find that RAD52 is particularly important for repair using ≥ 50 nt repeat sequences that flank the DSB, and that also involve removal of non-homologous sequences flanking the repeats. In contrast, POLQ is important for repair events using 6 nt (but not ≥ 18 nt) of flanking repeats that are at the edge of the break, as well as oligonucleotide microhomology-templated (i.e., 12-20 nt) repair events requiring nascent DNA synthesis. Finally, these factors show key distinctions with BRCA2, regarding effects on DSB repair events and response to stalled replication forks. These findings indicate that RAD52 and POLQ have distinct roles in genome maintenance, including for specific features of DSB repair events, such that combined disruption of these factors may be effective for genotoxin sensitization and/or synthetic lethal strategies.

The canonical non-homologous end joining factor XLF promotes chromosomal deletion rearrangements in human cells.

Clastogen exposure can result in chromosomal rearrangements, including large deletions and inversions that are associated with cancer development. To examine such rearrangements in human cells, here we developed a reporter assay based on endogenous genes on chromosome 12. Using the RNA-guided nuclease Cas9, we induced two DNA double-strand breaks, one each in the GAPDH and CD4 genes, that caused a deletion rearrangement leading to CD4 expression from the GAPDH promoter. We observed that this GAPDH-CD4 deletion rearrangement activates CD4+ cells that can be readily detected by flow cytometry. Similarly, double-strand breaks in the LPCAT3 and CD4 genes induced an LPCAT3-CD4 inversion rearrangement resulting in CD4 expression. Studying the GAPDH-CD4 deletion rearrangement in multiple cell lines, we found that the canonical non-homologous end joining (C-NHEJ) factor XLF promotes these rearrangements. Junction analysis uncovered that the relative contribution of C-NHEJ appears lower in U2OS than in HEK293 and A549 cells. Furthermore, an ATM kinase inhibitor increased C-NHEJ-mediated rearrangements only in U2OS cells. We also found that an XLF residue that is critical for an interaction with the C-NHEJ factor X-ray repair cross-complementing 4 (XRCC4), and XRCC4 itself are each important for promoting both this deletion rearrangement and end joining without insertion/deletion mutations. In summary, a reporter assay based on endogenous genes on chromosome 12 reveals that XLF-dependent C-NHEJ promotes deletion rearrangements in human cells and that cell type-specific differences in the contribution of C-NHEJ and ATM kinase inhibition influence these rearrangements.

Electrophilic fatty acids impair RAD51 function and potentiate the effects of DNA-damaging agents on growth of triple-negative breast cells.

Homologous recombination (HR)-directed DNA double-strand break (DSB) repair enables template-directed DNA repair to maintain genomic stability. RAD51 recombinase (RAD51) is a critical component of HR and facilitates DNA strand exchange in DSB repair. We report here that treating triple-negative breast cancer (TNBC) cells with the fatty acid nitroalkene 10-nitro-octadec-9-enoic acid (OA-NO2) in combination with the antineoplastic DNA-damaging agents doxorubicin, cisplatin, olaparib, and γ-irradiation (IR) enhances the antiproliferative effects of these agents. OA-NO2 inhibited IR-induced RAD51 foci formation and enhanced H2A histone family member X (H2AX) phosphorylation in TNBC cells. Analyses of fluorescent DSB reporter activity with both static-flow cytometry and kinetic live-cell studies enabling temporal resolution of recombination revealed that OA-NO2 inhibits HR and not nonhomologous end joining (NHEJ). OA-NO2 alkylated Cys-319 in RAD51, and this alkylation depended on the Michael acceptor properties of OA-NO2 because nonnitrated and saturated nonelectrophilic analogs of OA-NO2, octadecanoic acid and 10-nitro-octadecanoic acid, did not react with Cys-319. Of note, OA-NO2 alkylation of RAD51 inhibited its binding to ssDNA. RAD51 Cys-319 resides within the SH3-binding site of ABL proto-oncogene 1, nonreceptor tyrosine kinase (ABL1), so we investigated the effect of OA-NO2-mediated Cys-319 alkylation on ABL1 binding and found that OA-NO2 inhibits RAD51-ABL1 complex formation both in vitro and in cell-based immunoprecipitation assays. The inhibition of the RAD51-ABL1 complex also suppressed downstream RAD51 Tyr-315 phosphorylation. In conclusion, RAD51 Cys-319 is a functionally significant site for adduction of soft electrophiles such as OA-NO2 and suggests further investigation of lipid electrophile-based combinational therapies for TNBC.

Contribution of canonical nonhomologous end joining to chromosomal rearrangements is enhanced by ATM kinase deficiency.

A likely mechanism of chromosomal rearrangement formation involves joining the ends from two different chromosomal double-strand breaks (DSBs). These events could potentially be mediated by either of two end-joining (EJ) repair pathways [canonical nonhomologous end joining (C-NHEJ) or alternative end joining (ALT-EJ)], which cause distinct rearrangement junction patterns. The relative role of these EJ pathways during rearrangement formation has remained controversial. Along these lines, we have tested whether the DNA damage response mediated by the Ataxia Telangiectasia Mutated (ATM) kinase may affect the relative influence of C-NHEJ vs. ALT-EJ on rearrangement formation. We developed a reporter in mouse cells for a 0.4-Mbp deletion rearrangement that is formed by EJ between two DSBs induced by the Cas9 endonuclease. We found that disruption of the ATM kinase causes an increase in the frequency of the rearrangement as well as a shift toward rearrangement junctions that show hallmarks of C-NHEJ. Furthermore, ATM suppresses rearrangement formation in an experimental condition, in which C-NHEJ is the predominant EJ repair event (i.e., expression of the 3' exonuclease Trex2). Finally, several C-NHEJ factors are required for the increase in rearrangement frequency caused by inhibition of the ATM kinase. We also examined ATM effectors and found that H2AX shows a similar influence as ATM, whereas the influence of ATM on this rearrangement seems independent of 53BP1. We suggest that the contribution of the C-NHEJ pathway to the formation of a 0.4-Mbp deletion rearrangement is enhanced in ATM-deficient cells.

Regulation of Single-Strand Annealing and its Role in Genome Maintenance.

Single-strand annealing (SSA) is a DNA double-strand break (DSB) repair pathway that uses homologous repeats to bridge DSB ends. SSA involving repeats that flank a single DSB causes a deletion rearrangement between the repeats, and hence is relatively mutagenic. Nevertheless, this pathway is conserved, in that SSA events have been found in several organisms. In this review, we describe the mechanism of SSA and its regulation, including the cellular conditions that may favor SSA versus other DSB repair events. We will also evaluate the potential contribution of SSA to cancer-associated genome rearrangements, and to DSB-induced gene targeting.

Tetratricopeptide repeat factor XAB2 mediates the end resection step of homologous recombination.

We examined the influence of the tetratricopeptide repeat factor XAB2 on chromosomal break repair, and found that XAB2 promotes end resection that generates the 3' ssDNA intermediate for homologous recombination (HR). Namely, XAB2 is important for chromosomal double-strand break (DSB) repair via two pathways of HR that require end resection as an intermediate step, end resection of camptothecin (Cpt)-induced DNA damage, and RAD51 recruitment to ionizing radiation induced foci (IRIF), which requires end resection. Furthermore, XAB2 mediates specific aspects of the DNA damage response associated with end resection proficiency: CtIP hyperphosphorylation induced by Cpt and BRCA1 IRIF. XAB2 also promotes histone acetylation events linked to HR proficiency. From truncation mutation analysis, the capacity for XAB2 to promote HR correlates with its ability to form a complex with ISY1 and PRP19, which show a similar influence as XAB2 on HR. This XAB2 complex localizes to punctate structures consistent with interchromatin granules that show a striking adjacent-localization to the DSB marker γH2AX. In summary, we suggest that the XAB2 complex mediates DNA damage response events important for the end resection step of HR, and speculate that its adjacent-localization relative to DSBs marked by γH2AX is important for this function.

DNA damage response factors from diverse pathways, including DNA crosslink repair, mediate alternative end joining.

Alternative end joining (Alt-EJ) chromosomal break repair involves bypassing classical non-homologous end joining (c-NHEJ), and such repair causes mutations often with microhomology at the repair junction. Since the mediators of Alt-EJ are not well understood, we have sought to identify DNA damage response (DDR) factors important for this repair event. Using chromosomal break reporter assays, we surveyed an RNAi library targeting known DDR factors for siRNAs that cause a specific decrease in Alt-EJ, relative to an EJ event that is a composite of Alt-EJ and c-NHEJ (Distal-EJ between two tandem breaks). From this analysis, we identified several DDR factors that are specifically important for Alt-EJ relative to Distal-EJ. While these factors are from diverse pathways, we also found that most of them also promote homologous recombination (HR), including factors important for DNA crosslink repair, such as the Fanconi Anemia factor, FANCA. Since bypass of c-NHEJ is likely important for both Alt-EJ and HR, we disrupted the c-NHEJ factor Ku70 in Fanca-deficient mouse cells and found that Ku70 loss significantly diminishes the influence of Fanca on Alt-EJ. In contrast, an inhibitor of poly ADP-ribose polymerase (PARP) causes a decrease in Alt-EJ that is enhanced by Ku70 loss. Additionally, the helicase/nuclease DNA2 appears to have distinct effects from FANCA and PARP on both Alt-EJ, as well as end resection. Finally, we found that the proteasome inhibitor Bortezomib, a cancer therapeutic that has been shown to disrupt FANC signaling, causes a significant reduction in both Alt-EJ and HR, relative to Distal-EJ, as well as a substantial loss of end resection. We suggest that several distinct DDR functions are important for Alt-EJ, which include promoting bypass of c-NHEJ and end resection.

EXO1 is critical for embryogenesis and the DNA damage response in mice with a hypomorphic Nbs1 allele.

The maintenance of genome stability is critical for the suppression of diverse human pathologies that include developmental disorders, premature aging, infertility and predisposition to cancer. The DNA damage response (DDR) orchestrates the appropriate cellular responses following the detection of lesions to prevent genomic instability. The MRE11 complex is a sensor of DNA double strand breaks (DSBs) and plays key roles in multiple aspects of the DDR, including DNA end resection that is critical for signaling and DNA repair. The MRE11 complex has been shown to function both upstream and in concert with the 5'-3' exonuclease EXO1 in DNA resection, but it remains unclear to what extent EXO1 influences DSB responses independently of the MRE11 complex. Here we examine the genetic relationship of the MRE11 complex and EXO1 during mammalian development and in response to DNA damage. Deletion of Exo1 in mice expressing a hypomorphic allele of Nbs1 leads to severe developmental impairment, embryonic death and chromosomal instability. While EXO1 plays a minimal role in normal cells, its loss strongly influences DNA replication, DNA repair, checkpoint signaling and damage sensitivity in NBS1 hypomorphic cells. Collectively, our results establish a key role for EXO1 in modulating the severity of hypomorphic MRE11 complex mutations.

Myelodysplasia-associated mutations in serine/arginine-rich splicing factor SRSF2 lead to alternative splicing of CDC25C.

BACKGROUND: Serine-arginine rich splicing factor 2 (SRSF2) is a protein known for its role in RNA splicing and genome stability. It has been recently discovered that SRSF2, along with other splicing regulators, is frequently mutated in patients with myelodysplastic syndrome (MDS). The most common MDS mutations in SRSF2 occur at proline 95; the mutant proteins are shown to have different RNA binding preferences, which may contribute to splicing changes detected in mutant cells. However, the influence of these SRSF2 MDS-associated mutations on specific splicing events remains poorly understood. RESULTS: A tetracycline-inducible TF-1 erythroleukemia cell line was transduced with retroviruses to create cell lines expressing HA-tagged wildtype SRSF2, SRSF2 with proline 95 point mutations found in MDS, or SRSF2 with a deletion of one of the four major domains of the protein. Effects of these mutants on apoptosis and specific alternative splicing events were evaluated. Cells were also treated with DNA damaging drugs for comparison. MDS-related P95 point mutants of SRSF2 were expressed and phosphorylated at similar levels as wildtype SRSF2. However, cells expressing mutant SRSF2 exhibited higher levels of apoptosis than cells expressing wildtype SRSF2. Regarding alternative splicing events, in nearly all examined cases, SRSF2 P95 mutants acted in a similar fashion as the wildtype SRSF2. However, cells expressing SRSF2 P95 mutants had a percent increase in the C5 spliced isoform of cell division cycle 25C (CDC25C). The same alternative splicing of CDC25C was detected by treating cells with DNA damaging drugs, such as cisplatin, camptothecin, and trichostatin A at appropriate dosage. However, unlike DNA damaging drugs, SRSF2 P95 mutants did not activate the Ataxia telangiectasia mutated (ATM) pathway. CONCLUSION: SRSF2 P95 mutants lead to alternative splicing of CDC25C in a manner that is not dependent on the DNA damage response.

An RNF168 fragment defective for focal accumulation at DNA damage is proficient for inhibition of homologous recombination in BRCA1 deficient cells.

The E3 ubiquitin ligase RNF168 is a DNA damage response (DDR) factor that promotes monoubiquitination of H2A/H2AX at K13/15, facilitates recruitment of other DDR factors (e.g. 53BP1) to DNA damage, and inhibits homologous recombination (HR) in cells deficient in the tumor suppressor BRCA1. We have examined the domains of RNF168 important for these DDR events, including chromosomal HR that is induced by several nucleases (I-SceI, CAS9-WT and CAS9-D10A), since the inducing nuclease affects the relative frequency of distinct repair outcomes. We found that an N-terminal fragment of RNF168 (1-220/N221*) efficiently inhibits HR induced by each of these nucleases in BRCA1 depleted cells, and promotes recruitment of 53BP1 to DNA damage and H2AX monoubiquitination at K13/15. Each of these DDR events requires a charged residue in RNF168 (R57). Notably, RNF168-N221* fails to self-accumulate into ionizing radiation induced foci (IRIF). Furthermore, expression of RNF168 WT and N221* can significantly bypass the role of another E3 ubiquitin ligase, RNF8, for inhibition of HR in BRCA1 depleted cells, and for promotion of 53BP1 IRIF. We suggest that the ability for RNF168 to promote H2A/H2AX monoubiquitination and 53BP1 IRIF, but not RNF168 self-accumulation into IRIF, is important for inhibition of HR in BRCA1 deficient cells.

Visualization of local DNA unwinding by Mre11/Rad50/Nbs1 using single-molecule FRET.

The Mre11/Rad50/Nbs1 (MRN) complex initiates and coordinates DNA repair and signaling events at double-strand breaks. The interaction between MRN and DNA ends is critical for the recruitment of DNA-processing enzymes, end tethering, and activation of the ATM protein kinase. Here we visualized MRN binding to duplex DNA molecules using single-molecule FRET, and found that MRN unwinds 15-20 base pairs at the end of the duplex, holding the branched structure open for minutes at a time in an ATP-dependent reaction. A Rad50 catalytic domain mutant that is specifically deficient in this ATP-dependent opening is impaired in DNA end resection in vitro and in resection-dependent repair of breaks in human cells, demonstrating the importance of MRN-generated single strands in the repair of DNA breaks.

HP1 promotes tumor suppressor BRCA1 functions during the DNA damage response.

The DNA damage response (DDR) involves both the control of DNA damage repair and signaling to cell cycle checkpoints. Therefore, unraveling the underlying mechanisms of the DDR is important for understanding tumor suppression and cellular resistance to clastogenic cancer therapeutics. Because the DDR is likely to be influenced by chromatin regulation at the sites of DNA damage, we investigated the role of heterochromatin protein 1 (HP1) during the DDR process. We monitored double-strand breaks (DSBs) using the γH2AX foci marker and found that depleting cells of HP1 caused genotoxic stress, a delay in the repair of DSBs and elevated levels of apoptosis after irradiation. Furthermore, we found that these defects in repair were associated with impaired BRCA1 function. Depleting HP1 reduced recruitment of BRCA1 to DSBs and caused defects in two BRCA1-mediated DDR events: (i) the homologous recombination repair pathway and (ii) the arrest of cell cycle at the G2/M checkpoint. In contrast, depleting HP1 from cells did not affect the non-homologous end-joining (NHEJ) pathway: instead it elevated the recruitment of the 53BP1 NHEJ factor to DSBs. Notably, all three subtypes of HP1 seemed to be almost equally important for these DDR functions. We suggest that the dynamic interaction of HP1 with chromatin and other DDR factors could determine DNA repair choice and cell fate after DNA damage. We also suggest that compromising HP1 expression could promote tumorigenesis by impairing the function of the BRCA1 tumor suppressor.