BRCA1 safeguards genome integrity by activating chromosome asynapsis checkpoint to eliminate recombination-defective oocytes.

In the meiotic prophase, programmed DNA double-strand breaks are repaired by meiotic recombination. Recombination-defective meiocytes are eliminated to preserve genome integrity in gametes. BRCA1 is a critical protein in somatic homologous recombination, but studies have suggested that BRCA1 is dispensable for meiotic recombination. Here we show that BRCA1 is essential for meiotic recombination. Interestingly, BRCA1 also has a function in eliminating recombination-defective oocytes. Brca1 knockout (KO) rescues the survival of Dmc1 KO oocytes far more efficiently than removing CHK2, a vital component of the DNA damage checkpoint in oocytes. Mechanistically, BRCA1 activates chromosome asynapsis checkpoint by promoting ATR activity at unsynapsed chromosome axes in Dmc1 KO oocytes. Moreover, Brca1 KO also rescues the survival of asynaptic Spo11 KO oocytes. Collectively, our study not only unveils an unappreciated role of chromosome asynapsis in eliminating recombination-defective oocytes but also reveals the dual functions of BRCA1 in safeguarding oocyte genome integrity.

The FHA and BRCT domains recognize ADP-ribosylation during DNA damage response.

Poly-ADP-ribosylation is a unique post-translational modification participating in many biological processes, such as DNA damage response. Here, we demonstrate that a set of Forkhead-associated (FHA) and BRCA1 C-terminal (BRCT) domains recognizes poly(ADP-ribose) (PAR) both in vitro and in vivo. Among these FHA and BRCT domains, the FHA domains of APTX and PNKP interact with iso-ADP-ribose, the linkage of PAR, whereas the BRCT domains of Ligase4, XRCC1, and NBS1 recognize ADP-ribose, the basic unit of PAR. The interactions between PAR and the FHA or BRCT domains mediate the relocation of these domain-containing proteins to DNA damage sites and facilitate the DNA damage response. Moreover, the interaction between PAR and the NBS1 BRCT domain is important for the early activation of ATM during DNA damage response and ATM-dependent cell cycle checkpoint activation. Taken together, our results demonstrate two novel PAR-binding modules that play important roles in DNA damage response.

Zinc Finger Protein 618 Regulates the Function of UHRF2 (Ubiquitin-like with PHD and Ring Finger Domains 2) as a Specific 5-Hydroxymethylcytosine Reader.

5-Hydroxymethylcytosine (5hmC) is an epigenetic modification that is generated by ten-eleven translocation (TET) protein-mediated oxidation of 5-methylcytosine (5mC). 5hmC is associated with transcription regulation and is decreased in many cancers including melanoma. Accumulating evidence has suggested that 5hmC is functionally distinct from 5mC. Ubiquitin-like with PHD and ring finger domains 2 (UHRF2) is the first known specific 5hmC reader that has higher affinity to 5hmC than 5mC, suggesting that UHRF2 might mediate 5hmC's function. Structural analysis has revealed the molecular mechanism of UHRF2-5hmC binding in vitro, but it is not clear how UHRF2 recognizes 5hmC in vivo In this study, we have identified zinc figure protein 618 (ZNF618) as a novel binding partner of UHRF2. ZNF618 specifically interacts with UHRF2 but not its paralog UHRF1. Importantly, ZNF618 co-localizes with UHRF2 at genomic loci that are enriched for 5hmC. The ZNF618 chromatin localization is independent of its interaction with UHRF2 and is through its first two zinc fingers. Instead, ZNF618 regulates UHRF2 chromatin localization. Collectively, our study suggests that ZNF618 is a key protein that regulates UHRF2 function as a specific 5hmC reader in vivo.

Topoisomerase II regulates the maintenance of DNA methylation.

The maintenance of DNA methylation in nascent DNA is a critical event for numerous biological processes. Following DNA replication, DNMT1 is the key enzyme that strictly copies the methylation pattern from the parental strand to the nascent DNA. However, the mechanism underlying this highly specific event is not thoroughly understood. In this study, we identified topoisomerase IIα (TopoIIα) as a novel regulator of the maintenance DNA methylation. UHRF1, a protein important for global DNA methylation, interacts with TopoIIα and regulates its localization to hemimethylated DNA. TopoIIα decatenates the hemimethylated DNA following replication, which might facilitate the methylation of the nascent strand by DNMT1. Inhibiting this activity impairs DNA methylation at multiple genomic loci. We have uncovered a novel mechanism during the maintenance of DNA methylation.

MRE11 is essential for the long-term viability of undifferentiated spermatogonia.

In the meiotic prophase, programmed SPO11-linked DNA double-strand breaks (DSBs) are repaired by homologous recombination (HR). The MRE11-RAD50-NBS1 (MRN) complex is essential for initiating DNA end resection, the first step of HR. However, residual DNA end resection still occurs in Nbs1 knockout (KO) spermatocytes for unknown reasons. Here, we show that DNA end resection is completely abolished in Mre11 KO spermatocytes. In addition, Mre11 KO, but not Nbs1 KO, undifferentiated spermatogonia are rapidly exhausted due to DSB accumulation, proliferation defects, and elevated apoptosis. Cellular studies reveal that a small amount of MRE11 retained in the nucleus of Nbs1 KO cells likely underlies the differences between Mre11 and Nbs1 KO cells. Taken together, our study not only demonstrates an irreplaceable role of the MRE11 in DNA end resection at SPO11-linked DSBs but also unveils a unique function of MRE11 in maintaining the long-term viability of undifferentiated spermatogonia.

Cerebellar defects in Pdss2 conditional knockout mice during embryonic development and in adulthood.

PDSS2 is a gene that encodes one of the two subunits of trans-prenyl diphosphate synthase that is essential for ubiquinone biosynthesis. It is known that mutations in PDSS2 can cause primary ubiquinone deficiency in humans and a similar disease in mice. Cerebellum is the most often affected organ in ubiquinone deficiency, and cerebellar atrophy has been diagnosed in many infants with this disease. In this study, two Pdss2 conditional knockout mouse lines directed by Pax2-cre and Pcp2-cre were generated to investigate the effect of ubiquinone deficiency on cerebellum during embryonic development and in adulthood, respectively. The Pdss2(f/-); Pax2-cre mouse recapitulates some symptoms of ubiquinone deficiency in infants, including severe cerebellum hypoplasia and lipid accumulation in skeletal muscles at birth. During early cerebellum development (E12.5-14.5), Pdss2 knockout initially causes the delay of radial glial cell growth and neuron progenitor migration, so the growth of mutant cerebellum is retarded. During later development (E15.5-P0), increased ectopic apoptosis of neuroblasts and impaired cell proliferation result in the progression of cerebellum hypoplasia in the mutant. Thus, the mutant cerebellum contains fewer neurons at birth, and the cells are disorganized. The developmental defect of mutant cerebellum does not result from reduced Fgf8 expression before E12.5. Electron microscopy reveals mitochondrial defects and increased autophagic-like vacuolization that may arise in response to abnormal mitochondria in the mutant cerebellum. Nevertheless, the mutant mice die soon after birth probably due to cleft palate and micrognathia, which may result from Pdss2 knockout caused by ectopic Pax2-cre expression in the first branchial arch. On the other hand, the Pdss2(f/-); Pcp2-cre mouse is healthy at birth but gradually loses cerebellar Purkinje cells and develops ataxia-like symptoms at 9.5 months; thus this conditional knockout mouse may serve as a model for ubiquinone deficiency in adult patients. In conclusion, this study provides two mouse models of Pdss2 based ubiquinone deficiency. During cerebellum development, Pdss2 knockout results in severe cerebellum hypoplasia by impairing cell migration and eliciting ectopic apoptosis, whereas Pdss2 knockout in Purkinje cells at postnatal stages leads to the development of cerebellar ataxia.

Meiotic recombination: insights into its mechanisms and its role in human reproduction with a special focus on non-obstructive azoospermia.

BACKGROUND: Meiosis is an essential stage in the life cycle of sexually reproducing species, underlying formation of haploid gametes and serving as the basis of genetic diversity. A central mechanism of meiosis is recombination between homologous chromosomes, during which programmed DNA double-strand breaks (DSBs) are sequentially repaired to form the crossovers essential for faithful chromosomal segregation. Aberrant meiotic recombination often leads to gametogenic failure or produces aneuploid gametes resulting in subfertility or infertility, miscarriage or birth defects. OBJECTIVE AND RATIONALE: The goal of this review was to characterize the molecular mechanisms of meiotic recombination and related human infertility disorders, particularly male infertility caused by non-obstructive azoospermia (NOA). SEARCH METHODS: Our search included PubMed database articles, focusing mainly on English-language publications dated between January 2016 and February 2022. The search term 'meiosis' was combined with the following keywords: meiotic initiation, chromosome pairing, homologous recombination, chromosome axis, DSB, DSB repair, crossover, meiotic sex chromosome inactivation, meiotic checkpoints, meiotic arrest, NOA, premature ovarian insufficiency (POI) or premature ovarian failure, treatment and cancer. In addition, references within these articles were used to identify additional studies. OUTCOMES: The preliminary search generated ∼3500 records. The majority of articles were identified as meeting abstracts or duplicates, contained non-English text or provided insufficient data and were therefore eliminated. A total of 271 articles associated with meiotic recombination were included in the final analysis. This review provides an overview of molecules and mechanisms involved in meiotic recombination processes, specifically meiosis-specific chromosome structures, DSB formation, homology search, formation of recombination intermediates and crossover formation. The cumulative results suggest that meiosis is regulated sequentially by a series of meiotic recombination genes and proteins. Importantly, mutations in these genes often affect meiotic progression, activating meiotic checkpoints, causing germ cell arrest and leading to subfertility or infertility. At least 26 meiotic recombination-related genes have been reported to be mutated in NOA in men, and 10 of these genes are mutated in POI in women. This suggests that variants of meiotic recombination-related genes can cause human subfertility or infertility, especially NOA. WIDER IMPLICATIONS: Understanding the processes of homologous chromosome pairing, recombination and timely resolution of homologous chromosomes may provide guidance for the analysis of potential monogenetic causes of human subfertility or infertility and the development of personalized treatments. In clinical practice, we can develop a meiotic recombination-related gene panel to screen for gene mutations in individuals with subfertility or infertility. Testicular sperm extraction should not be recommended when an NOA-affected individual carries definite disease-causing mutations of a meiotic gene, so as to avoid the unnecessary invasive diagnosis. Risk of ovarian dysfunction should be evaluated if a woman carries meiotic recombination-related gene mutations. It may be possible to improve or restore fertility through manipulation of meiotic recombination-related genes in the future.

BRCA1 and homologous recombination: implications from mouse embryonic development.

As an important player in DNA damage response, BRCA1 maintains genomic stability and suppresses tumorigenesis by promoting DNA double-strand break (DSB) repair through homologous recombination (HR). Since the cloning of BRCA1 gene, many Brca1 mutant alleles have been generated in mice. Mice carrying homozygous Brca1 mutant alleles are embryonic lethal, suggesting that BRCA1's functions are important for embryonic development. Studies of embryonic development in Brca1 mutant mice not only reveal the physiological significance of BRCA1's known function in HR, but also lead to the discovery of BRCA1's new function in HR: regulation of DSB repair pathway choice.

NBS1 is required for SPO11-linked DNA double-strand break repair in male meiosis.

DNA double-strand breaks (DSBs) pose a serious threat to genomic stability. Paradoxically, hundreds of programed DSBs are generated by SPO11 in meiotic prophase, which are exclusively repaired by homologous recombination (HR) to promote obligate crossover between homologous chromosomes. In somatic cells, MRE11-RAD50-NBS1 (MRN) complex-dependent DNA end resection is a prerequisite for HR repair, especially for DSBs that are covalently linked with proteins or chemicals. Interestingly, all meiotic DSBs are linked with SPO11 after being generated. Although MRN complex's function in meiotic DSB repair has been established in lower organisms, the role of MRN complex in mammalian meiotic DSB repair is not clear. Here, we show that MRN complex is essential for repairing meiotic SPO11-linked DSBs in male mice. In male germ cells, conditional inactivation of NBS1, a key component of MRN complex, causes dramatic reduction of DNA end resection and defective HR repair in meiotic prophase. NBS1 loss severely disrupts chromosome synapsis, generates abnormal chromosome structures, and eventually leads to meiotic arrest and male infertility in mice. Unlike in somatic cells, the recruitment of NBS1 to SPO11-linked DSB sites is MDC1-independent but requires other phosphorylated proteins. Collectively, our study not only reveals the significance of MRN complex in repairing meiotic DSBs but also discovers a unique mechanism that recruits MRN complex to SPO11-linked DSB sites.

The RNF20/40 complex regulates p53-dependent gene transcription and mRNA splicing.

p53 is a key transcription factor to regulate gene transcription. However, the molecular mechanism of chromatin-associated p53 on gene transcription remains elusive. Here, using unbiased protein affinity purification, we found that the RNF20/40 complex associated with p53 on the chromatin. Further analyses indicated that p53 mediated the recruitment of the RNF20/40 complex to p53 target gene loci including p21 and PUMA loci and regulated the transcription of p21 and PUMA via the RNF20/40 complex-dependent histone H2B ubiquitination (ubH2B). Lacking the RNF20/40 complex suppressed not only ubH2B but also the generation of the mature mRNA of p21 and PUMA. Moreover, ubH2B was recognized by the ubiquitin-binding motif of pre-mRNA processing splicing factor 8 (PRPF8), a subunit in the spliceosome, and PRPF8 was required for the maturation of the mRNA of p21 and PUMA. Our study unveils a novel p53-dependent pathway that regulates mRNA splicing for tumor suppression.

Aberrantly expressed HORMAD1 disrupts nuclear localization of MCM8-MCM9 complex and compromises DNA mismatch repair in cancer cells.

HORMAD1 is a meiosis-specific protein that promotes synapsis and recombination of homologous chromosomes in meiotic prophase. Originally identified as a cancer/testis antigen, HORMAD1 is also aberrantly expressed in several cancers. However, the functions of HORMAD1 in cancer cells are still not clear. Here, we show that HORMAD1 is aberrantly expressed in a wide variety of cancers and compromises DNA mismatch repair in cancer cells. Mechanistically, HORMAD1 interacts with MCM8-MCM9 complex and prevents its efficient nuclear localization. As a consequence, HORMAD1-expressing cancer cells have reduced MLH1 chromatin binding and DNA mismatch repair defects. Consistently, HORMAD1 expression is associated with increased mutation load and genomic instability in many cancers. Taken together, our study provides mechanistic insights into HORMAD1's functions in cancer cells, which can potentially be exploited for targeted therapy of HORMAD1-expressing cancers.

53BP1 loss rescues embryonic lethality but not genomic instability of BRCA1 total knockout mice.

BRCA1 is critical for DNA double-strand break (DSB) repair by homologous recombination (HR). BRCA1 deficient mice are embryonic lethal. Previous studies have shown that 53BP1 knockout (KO) rescues embryonic lethality of BRCA1 hypomorphic mutant mice by restoring HR. Here, we show that 53BP1 KO can partially rescue embryonic lethality of BRCA1 total KO mice, but HR is not restored in BRCA1-53BP1 double knockout (DKO) mice. As a result, BRCA1-53BP1 DKO cells are extremely sensitive to PARP inhibitors (PARPi). In addition to HR deficiency, BRCA1-53BP1 DKO cells have elevated microhomology-mediated end joining (MMEJ) activity and G2/M cell cycle checkpoint defects, causing severe genomic instability in these cells. Interestingly, BRCA1-53BP1 DKO mice rapidly develop thymic lymphoma that is 100% penetrant, which is not observed in any BRCA1 mutant mice rescued by 53BP1 KO. Taken together, our study reveals that 53BP1 KO can partially rescue embryonic lethality caused by complete BRCA1 loss without rescuing HR-related defects. This finding suggests that loss of 53BP1 can support the development of cancers with silenced BRCA1 expression without causing PARPi resistance.

Highly efficient deletion method for the engineering of plasmid DNA with single-stranded oligonucleotides.

The lamda phage Red recombination system has been used to modify plasmid, bacterial artificial chromosome (BAC), and chromosomal DNA in a highly precise and versatile manner Linear double-stranded DNA fragments or synthetic single-stranded oligonucleotides (SSOs) with short flanking homologies (<50 bp) to the target loci can be used as substrates to direct changes, including point mutations, insertions, and deletions. In attempts to explore mechanistic bases under this recombination process, we and others have previously identified factors that influence SSO-mediated single base substitutions. In this report, we focus our study on SSO-mediated deletion on plasmids. We found that SSOs as short as 63 bp were sufficient to mediate deletion as long as 2 kb with efficiency higher than 1%. Strand bias was consistently observed, and SSOs with sequences identical to the nascent lagging strand during replication always resulted in higher efficiency. Unlike SSO-mediated single nucleotide substitution, homology on each side of SSO flanking the fragment to be deleted was important for successful deletion, and abolishing the host methyl-directed mismatch repair (MMR) system did not lead to detectable changes in deletion efficiency. Finally, we showed that by optimizing its design, SSO-mediated deletion was efficient enough to make it possible to manipulate plasmids without selectable markers.

The involvement of replication in single stranded oligonucleotide-mediated gene repair.

Targeted gene repair mediated by single-stranded oligonucleotides (SSOs) has great potential for use in functional genomic studies and gene therapy. Genetic changes have been created using this approach in a number of prokaryotic and eukaryotic systems, including mouse embryonic stem cells. However, the underlying mechanisms remain to be fully established. In one of the current models, the 'annealing-integration' model, the SSO anneals to its target locus at the replication fork, serving as a primer for subsequent DNA synthesis mediated by the host replication machinery. Using a lambda-Red recombination-based system in the bacterium Escherichia coli, we systematically examined several fundamental premises that form the mechanistic basis of this model. Our results provide direct evidence strongly suggesting that SSO-mediated gene repair is mechanistically linked to the process of DNA replication, and most likely involves a replication intermediate. These findings will help guide future experiments involving SSO-mediated gene repair in mammalian and prokaryotic cells, and suggest several mechanisms by which the efficiencies may be reliably and substantially increased.

UHRF2 regulates local 5-methylcytosine and suppresses spontaneous seizures.

The 5-methylcytosine (5mC) modification regulates multiple cellular processes and is faithfully maintained following DNA replication. In addition to DNA methyltransferase (DNMT) family proteins, ubiquitin-like PHD and ring finger domain-containing protein 1 (UHRF1) plays an important role in the maintenance of 5mC levels. Loss of UHRF1 abolishes 5mC in cells and leads to embryonic lethality in mice. Interestingly, UHRF1 has a paralog, UHRF2, that has similar sequence and domain architecture, but its biologic function is not clear. Here, we have generated Uhrf2 knockout mice and characterized the role of UHRF2 in vivo. Uhrf2 knockout mice are viable, but the adult mice develop frequent spontaneous seizures and display abnormal electrical activities in brain. Despite no global DNA methylation changes, 5mC levels are decreased at certain genomic loci in the brains of Uhrf2 knockout mice. Therefore, our study has revealed a unique role of UHRF2 in the maintenance of local 5mC levels in brain that is distinct from that of its paralog UHRF1.

The adamantane-derived bananins are potent inhibitors of the helicase activities and replication of SARS coronavirus.

Bananins are a class of antiviral compounds with a unique structural signature incorporating a trioxa-adamantane moiety covalently bound to a pyridoxal derivative. Six members of this class of compounds: bananin, iodobananin, vanillinbananin, ansabananin, eubananin, and adeninobananin were synthesized and tested as inhibitors of the SARS Coronavirus (SCV) helicase. Bananin, iodobananin, vanillinbananin, and eubananin were effective inhibitors of the ATPase activity of the SCV helicase with IC50 values in the range 0.5-3 microM. A similar trend, though at slightly higher inhibitor concentrations, was observed for inhibition of the helicase activities, using a FRET-based fluorescent assay. In a cell culture system of SCV, bananin exhibited an EC50 of less than 10 microM and a CC50 of over 300 microM. Kinetics of inhibition are consistent with bananin inhibiting an intracellular process or processes involved in SCV replication.

Identification of factors influencing strand bias in oligonucleotide-mediated recombination in Escherichia coli.

Recombinogenic engineering methodology, also known as recombineering, utilizes homologous recombination to create targeted changes in cellular DNA with great specificity and flexibility. In Escherichia coli, the Red recombination system from bacteriophage lambda has been used successfully to modify both plasmid and chromosomal DNA in a highly efficient manner, using either a linear double-stranded DNA fragment or a synthetic single-stranded oligonucleotide (SSO). The current model for Red/SSO-mediated recombination involves the SSO first annealing to a transient, single-stranded region of DNA before being incorporated into the chromosome or plasmid target. It has been observed previously, in both eukaryotes and prokaryotes, that mutations in the two strands of the DNA double helix are 'corrected' by complementary SSOs with differing efficiencies. Here we investigate further the factors that influence the strand bias as well as the overall efficiency of Red/SSO-mediated recombination in E.coli. We show that the direction of DNA replication and the nature of the SSO-encoded mismatch are the main factors dictating the recombinational strand bias. However, the influence that the SSO-encoded mismatch exerts upon the recombinational strand bias is abolished in E.coli strains that are defective in mismatch repair (MMR). This reflects the fact that different base-base mispairs are corrected by the mutS/H/L-dependent MMR pathway with differing efficiencies. Furthermore, our data indicate that transcription has negligible influence on the strand bias. These results demonstrate for the first time that the interplay between DNA replication and MMR has a major effect on the efficiency and strand bias of Red/SSO-mediated recombination in E.coli.

Ubiquitylation of Rad51d Mediated by E3 Ligase Rnf138 Promotes the Homologous Recombination Repair Pathway.

Ubiquitylation has an important role as a signal transducer that regulates protein function, subcellular localization, or stability during the DNA damage response. In this study, we show that Ring domain E3 ubiquitin ligases RNF138 is recruited to DNA damage site quickly. And the recruitment is mediated through its Zinc finger domains. We further confirm that RNF138 is phosphorylated by ATM at Ser124. However, the phosphorylation was dispensable for recruitment to the DNA damage site. Our findings also indicate that RAD51 assembly at DSB sites following irradiation is dramatically affected in RNF138-deficient cells. Hence, RNF138 is likely involved in regulating homologous recombination repair pathway. Consistently, efficiency of homologous recombination decreased observably in RNF138-depleted cells. In addition, RNF138-deficient cell is hypersensitive to DNA damage insults, such as IR and MMS. And the comet assay confirmed that RNF138 directly participated in DNA damage repair. Moreover, we find that RAD51D directly interacted with RNF138. And the recruitment of RAD51D to DNA damage site is delayed and unstable in RNF138-depleted cells. Taken together, these results suggest that RNF138 promotes the homologous recombination repair pathway.

CHFR is important for the survival of male premeiotic germ cells.

DNA damage response is required for male fertility. DNA damage repair mediates recombination between homologous chromosomes in meiotic prophase, which is essential for proper chromosome segregation during meiotic division. Interestingly, some DNA damage response proteins are also required for the survival of premeiotic germ cells, but their roles in these cells are still unclear. CHFR was recently shown to participate in DNA damage response, but it remains to be established if CHFR is required for male fertility. In this study, we characterized Chfr knockout male mice and found that around 30% of them were infertile. The onset of spermatogenesis was delayed and there was significant increase in apoptosis in premeiotic germ cells. This resulted in complete loss of germ cells in testes in 3 months and azoospermia in these mice. We further demonstrated that ATM activation was compromised in the testes of these mice. Therefore, CHFR is important for the survival of male premeiotic germ cells, which is likely through maintaining genomic stability in spermatogonial stem cells.