ATM suppresses c-Myc overexpression in the mammary epithelium in response to estrogen.

ATM gene mutation carriers are predisposed to estrogen-receptor-positive breast cancer (BC). ATM prevents BC oncogenesis by activating p53 in every cell; however, much remains unknown about tissue-specific oncogenesis after ATM loss. Here, we report that ATM controls the early transcriptional response to estrogens. This response depends on topoisomerase II (TOP2), which generates TOP2-DNA double-strand break (DSB) complexes and rejoins the breaks. When TOP2-mediated ligation fails, ATM facilitates DSB repair. After estrogen exposure, TOP2-dependent DSBs arise at the c-MYC enhancer in human BC cells, and their defective repair changes the activation profile of enhancers and induces the overexpression of many genes, including the c-MYC oncogene. CRISPR/Cas9 cleavage at the enhancer also causes c-MYC overexpression, indicating that this DSB causes c-MYC overexpression. Estrogen treatment induced c-Myc protein overexpression in mammary epithelial cells of ATM-deficient mice. In conclusion, ATM suppresses the c-Myc-driven proliferative effects of estrogens, possibly explaining such tissue-specific oncogenesis.

Triple-helix potential of the mouse genome.

Certain DNA sequences, including mirror-symmetric polypyrimidine•polypurine runs, are capable of folding into a triple-helix­containing non­B-form DNA structure called H-DNA. Such H-DNA­forming sequences occur frequently in many eukaryotic genomes, including in mammals, and multiple lines of evidence indicate that these motifs are mutagenic and can impinge on DNA replication, transcription, and other aspects of genome function. In this study, we show that the triplex-forming potential of H-DNA motifs in the mouse genome can be evaluated using S1-sequencing (S1-seq), which uses the single-stranded DNA (ssDNA)­specific nuclease S1 to generate deep-sequencing libraries that report on the position of ssDNA throughout the genome. When S1-seq was applied to genomic DNA isolated from mouse testis cells and splenic B cells, we observed prominent clusters of S1-seq reads that appeared to be independent of endogenous double-strand breaks, that coincided with H-DNA motifs, and that correlated strongly with the triplex-forming potential of the motifs. Fine-scale patterns of S1-seq reads, including a pronounced strand asymmetry in favor of centrally positioned reads on the pyrimidine-containing strand, suggested that this S1-seq signal is specific for one of the four possible isomers of H-DNA (H-y5). By leveraging the abundance and complexity of naturally occurring H-DNA motifs across the mouse genome, we further defined how polypyrimidine repeat length and the presence of repeat-interrupting substitutions modify the structure of H-DNA. This study provides an approach for studying DNA secondary structure genome-wide at high spatial resolution.

Meiosis: Disentangling polyploid chromosomes with supercharged crossover interference.

Meiosis depends on the cell's ability to match each chromosome to its homolog in a strictly pairwise fashion. A new study describes an elegant mechanism that tetraploid Arabidopsis arenosa plants evolved to faithfully connect and segregate pairs of homologous chromosomes.

Mechanistic Insight into Crossing over during Mouse Meiosis.

Crossover recombination is critical for meiotic chromosome segregation, but how mammalian crossing over is accomplished is poorly understood. Here, we illuminate how strands exchange during meiotic recombination in male mice by analyzing patterns of heteroduplex DNA in recombinant molecules preserved by the mismatch correction deficiency of Msh2-/- mutants. Surprisingly, MSH2-dependent recombination suppression was not evident. However, a substantial fraction of crossover products retained heteroduplex DNA, and some provided evidence of MSH2-independent correction. Biased crossover resolution was observed, consistent with asymmetry between DNA ends in earlier intermediates. Many crossover products yielded no heteroduplex DNA, suggesting dismantling by D-loop migration. Unlike the complexity of crossovers in yeast, these simple modifications of the original double-strand break repair model-asymmetry in recombination intermediates and D-loop migration-may be sufficient to explain most meiotic crossing over in mice while also addressing long-standing questions related to Holliday junction resolution.

Cyclin B3 promotes anaphase I onset in oocyte meiosis.

Meiosis poses unique challenges because two rounds of chromosome segregation must be executed without intervening DNA replication. Mammalian cells express numerous temporally regulated cyclins, but how these proteins collaborate to control meiosis remains poorly understood. Here, we show that female mice genetically ablated for cyclin B3 are viable-indicating that the protein is dispensable for mitotic divisions-but are sterile. Mutant oocytes appear normal until metaphase I but then display a highly penetrant failure to transition to anaphase I. They arrest with hallmarks of defective anaphase-promoting complex/cyclosome (APC/C) activation, including no separase activity, high CDK1 activity, and high cyclin B1 and securin levels. Partial APC/C activation occurs, however, as exogenously expressed APC/C substrates can be degraded. Cyclin B3 forms active kinase complexes with CDK1, and meiotic progression requires cyclin B3-associated kinase activity. Cyclin B3 homologues from frog, zebrafish, and fruit fly rescue meiotic progression in cyclin B3-deficient mouse oocytes, indicating conservation of the biochemical properties and possibly cellular functions of this germline-critical cyclin.

Shu complex SWS1-SWSAP1 promotes early steps in mouse meiotic recombination.

The DNA-damage repair pathway homologous recombination (HR) requires factors that promote the activity of strand-exchange protein RAD51 and its meiosis-specific homolog DMC1. Here we show that the Shu complex SWS1-SWSAP1, a candidate for one such HR regulator, is dispensable for mouse viability but essential for male and female fertility, promoting the assembly of RAD51 and DMC1 on early meiotic HR intermediates. Only a fraction of mutant meiocytes progress to form crossovers, which are crucial for chromosome segregation, demonstrating crossover homeostasis. Remarkably, loss of the DNA damage checkpoint kinase CHK2 rescues fertility in females without rescuing crossover numbers. Concomitant loss of the BRCA2 C terminus aggravates the meiotic defects in Swsap1 mutant spermatocytes, suggesting an overlapping role with the Shu complex during meiotic HR. These results demonstrate an essential role for SWS1-SWSAP1 in meiotic progression and emphasize the complex interplay of factors that ensure recombinase function.

A Case of Gastric Metastatic Melanoma 15 Years after the Initial Diagnosis of Cutaneous Melanoma.

Melanoma is the most common cancer to metastasize to the gastrointestinal tract; however, metastasis to the stomach is a rare occurrence. We present the case of a patient with a history of melanoma of the chest wall 15 years prior to presentation who initially presented to the hospital with sepsis but was later found to have metastatic melanoma in the gastric cardia. This case illustrates the rare occurrence of metastatic melanoma to the stomach which occurred 15 years after the initial skin diagnosis of melanoma was made, its endoscopic appearance, and how the nonspecific symptoms frequently lead to a delayed diagnosis or one that is not made at all until after autopsy. For these reasons, endoscopy should be promptly performed if there is a suspicion of gastrointestinal metastatic melanoma.

p53 and TAp63 participate in the recombination-dependent pachytene arrest in mouse spermatocytes.

To protect germ cells from genomic instability, surveillance mechanisms ensure meiosis occurs properly. In mammals, spermatocytes that display recombination defects experience a so-called recombination-dependent arrest at the pachytene stage, which relies on the MRE11 complex-ATM-CHK2 pathway responding to unrepaired DNA double-strand breaks (DSBs). Here, we asked if p53 family members-targets of ATM and CHK2-participate in this arrest. We bred double-mutant mice combining a mutation of a member of the p53 family (p53, TAp63, or p73) with a Trip13 mutation. Trip13 deficiency triggers a recombination-dependent response that arrests spermatocytes in pachynema before they have incorporated the testis-specific histone variant H1t into their chromatin. We find that deficiency for either p53 or TAp63, but not p73, allowed spermatocytes to progress further into meiotic prophase despite the presence of numerous unrepaired DSBs. Even so, the double mutant spermatocytes apoptosed at late pachynema because of sex body deficiency; thus p53 and TAp63 are dispensable for arrest caused by sex body defects. These data affirm that recombination-dependent and sex body-deficient arrests occur via genetically separable mechanisms.

Distinct DNA-binding surfaces in the ATPase and linker domains of MutLγ determine its substrate specificities and exert separable functions in meiotic recombination and mismatch repair.

Mlh1-Mlh3 (MutLγ) is a mismatch repair factor with a central role in formation of meiotic crossovers, presumably through resolution of double Holliday junctions. MutLγ has DNA-binding, nuclease, and ATPase activities, but how these relate to one another and to in vivo functions are unclear. Here, we combine biochemical and genetic analyses to characterize Saccharomyces cerevisiae MutLγ. Limited proteolysis and atomic force microscopy showed that purified recombinant MutLγ undergoes ATP-driven conformational changes. In vitro, MutLγ displayed separable DNA-binding activities toward Holliday junctions (HJ) and, surprisingly, single-stranded DNA (ssDNA), which was not predicted from current models. MutLγ bound DNA cooperatively, could bind multiple substrates simultaneously, and formed higher-order complexes. FeBABE hydroxyl radical footprinting indicated that the DNA-binding interfaces of MutLγ for ssDNA and HJ substrates only partially overlap. Most contacts with HJ substrates were located in the linker regions of MutLγ, whereas ssDNA contacts mapped within linker regions as well as the N-terminal ATPase domains. Using yeast genetic assays for mismatch repair and meiotic recombination, we found that mutations within different DNA-binding surfaces exert separable effects in vivo. For example, mutations within the Mlh1 linker conferred little or no meiotic phenotype but led to mismatch repair deficiency. Interestingly, mutations in the N-terminal domain of Mlh1 caused a stronger meiotic defect than mlh1Δ, suggesting that the mutant proteins retain an activity that interferes with alternative recombination pathways. Furthermore, mlh3Δ caused more chromosome missegregation than mlh1Δ, whereas mlh1Δ but not mlh3Δ partially alleviated meiotic defects of msh5Δ mutants. These findings illustrate functional differences between Mlh1 and Mlh3 during meiosis and suggest that their absence impinges on chromosome segregation not only via reduced formation of crossovers. Taken together, our results offer insights into the structure-function relationships of the MutLγ complex and reveal unanticipated genetic relationships between components of the meiotic recombination machinery.

Pelvic fracture pattern predicts the need for hemorrhage control intervention-Results of an AAST multi-institutional study.

BACKGROUND: Early identification of patients with pelvic fractures at risk of severe bleeding requiring intervention is critical. We performed a multi-institutional study to test our hypothesis that pelvic fracture patterns predict the need for a pelvic hemorrhage control intervention. METHODS: This prospective, observational, multicenter study enrolled patients with pelvic fracture due to blunt trauma. Inclusion criteria included shock on admission (systolic blood pressure <90 mm Hg or heart rate >120 beats/min and base deficit >5, and the ability to review pelvic imaging). Demographic data, open pelvic fracture, blood transfusion, pelvic hemorrhage control intervention (angioembolization, external fixator, pelvic packing, and/or REBOA [resuscitative balloon occlusion of the aorta]), and mortality were recorded. Pelvic fracture pattern was classified according to Young-Burgess in a blinded fashion. Predictors of pelvic hemorrhage control intervention and mortality were analyzed by univariate and multivariate regression analyses. RESULTS: A total of 163 patients presenting in shock were enrolled from 11 Level I trauma centers. The most common pelvic fracture pattern was lateral compression I, followed by lateral compression I, and vertical shear. Of the 12 patients with an anterior-posterior compression III fracture, 10 (83%) required a pelvic hemorrhage control intervention. Factors associated with the need for pelvic fracture hemorrhage control intervention on univariate analysis included vertical shear pelvic fracture pattern, increasing age, and transfusion of blood products. Anterior-posterior compression III fracture patterns and open pelvic fracture predicted the need for pelvic hemorrhage control intervention on multivariate analysis. Overall in-hospital mortality for patients admitted in shock with pelvic fracture was 30% and did not differ based on pelvic fracture pattern on multivariate analysis. CONCLUSION: Blunt trauma patients admitted in shock with anterior-posterior compression III fracture patterns or patients with open pelvic fracture are at greatest risk of bleeding requiring pelvic hemorrhage control intervention. LEVEL OF EVIDENCE: Prognostic/epidemiologic study, level III.

Current management of hemorrhage from severe pelvic fractures: Results of an American Association for the Surgery of Trauma multi-institutional trial.

BACKGROUND: There is no consensus as to the optimal treatment paradigm for patients presenting with hemorrhage from severe pelvic fracture. This study was established to determine the methods of hemorrhage control currently being used in clinical practice. METHODS: This prospective, observational multi-center study enrolled patients with pelvic fracture from blunt trauma. Demographic data, admission vital signs, presence of shock on admission (systolic blood pressure < 90 mm Hg or heart rate > 120 beats per minute or base deficit < -5), method of hemorrhage control, transfusion requirements, and outcome were collected. RESULTS: A total of 1,339 patients with pelvic fracture were enrolled from 11 Level I trauma centers. Fifty-seven percent of the patients were male, with a mean ± SD age of 47.1 ± 21.6 years, and Injury Severity Score (ISS) of 19.2 ± 12.7. In-hospital mortality was 9.0 %. Angioembolization and external fixator placement were the most common method of hemorrhage control used. A total of 128 patients (9.6%) underwent diagnostic angiography with contrast extravasation noted in 63 patients. Therapeutic angioembolization was performed on 79 patients (5.9%). There were 178 patients (13.3%) with pelvic fracture admitted in shock with a mean ± SD ISS of 28.2 ± 14.1. In the shock group, 44 patients (24.7%) underwent angiography to diagnose a pelvic source of bleeding with contrast extravasation found in 27 patients. Thirty patients (16.9%) were treated with therapeutic angioembolization. Resuscitative endovascular balloon occlusion of the aorta was performed on five patients in shock and used by only one of the participating centers. Mortality was 32.0% for patients with pelvic fracture admitted in shock. CONCLUSION: Patients with pelvic fracture admitted in shock have high mortality. Several methods were used for hemorrhage control with significant variation across institutions. The use of resuscitative endovascular balloon occlusion of the aorta may prove to be an important adjunct in the treatment of patients with severe pelvic fracture in shock; however, it is in the early stages of evaluation and not currently used widely across trauma centers. LEVEL OF EVIDENCE: Prognostic study, level II; therapeutic study, level III.

The ATM signaling cascade promotes recombination-dependent pachytene arrest in mouse spermatocytes.

Most mutations that compromise meiotic recombination or synapsis in mouse spermatocytes result in arrest and apoptosis at the pachytene stage of the first meiotic prophase. Two main mechanisms are thought to trigger arrest: one independent of the double-strand breaks (DSBs) that initiate meiotic recombination, and another activated by persistent recombination intermediates. Mechanisms underlying the recombination-dependent arrest response are not well understood, so we sought to identify factors involved by examining mutants deficient for TRIP13, a conserved AAA+ ATPase required for the completion of meiotic DSB repair. We find that spermatocytes with a hypomorphic Trip13 mutation (Trip13mod/mod) arrest with features characteristic of early pachynema in wild type, namely, fully synapsed chromosomes without incorporation of the histone variant H1t into chromatin. These cells then undergo apoptosis, possibly in response to the arrest or in response to a defect in sex body formation. However, TRIP13-deficient cells that additionally lack the DSB-responsive kinase ATM progress further, reaching an H1t-positive stage (i.e., similar to mid/late pachynema in wild type) despite the presence of unrepaired DSBs. TRIP13-deficient spermatocytes also progress to an H1t-positive stage if ATM activity is attenuated by hypomorphic mutations in Mre11 or Nbs1 or by elimination of the ATM-effector kinase CHK2. These mutant backgrounds nonetheless experience an apoptotic block to further spermatogenic progression, most likely caused by failure to form a sex body. DSB numbers are elevated in Mre11 and Nbs1 hypomorphs but not Chk2 mutants, thus delineating genetic requirements for the ATM-dependent negative feedback loop that regulates DSB numbers. The findings demonstrate for the first time that ATM-dependent signaling enforces the normal pachytene response to persistent recombination intermediates. Our work supports the conclusion that recombination defects trigger spermatocyte arrest via pathways than are genetically distinct from sex body failure-promoted apoptosis and confirm that the latter can function even when recombination-dependent arrest is inoperative. Implications of these findings for understanding the complex relationships between spermatocyte arrest and apoptosis are discussed.

The Rad50 hook domain regulates DNA damage signaling and tumorigenesis.

The Mre11 complex (Mre11, Rad50, and Nbs1) is a central component of the DNA damage response (DDR), governing both double-strand break repair and DDR signaling. Rad50 contains a highly conserved Zn(2+)-dependent homodimerization interface, the Rad50 hook domain. Mutations that inactivate the hook domain produce a null phenotype. In this study, we analyzed mutants with reduced hook domain function in an effort to stratify hook-dependent Mre11 complex functions. One of these alleles, Rad50(46), conferred reduced Zn(2+) affinity and dimerization efficiency. Homozygous Rad50(46/46) mutations were lethal in mice. However, in the presence of wild-type Rad50, Rad50(46) exerted a dominant gain-of-function phenotype associated with chronic DDR signaling. At the organismal level, Rad50(+/46) exhibited hydrocephalus, liver tumorigenesis, and defects in primitive hematopoietic and gametogenic cells. These outcomes were dependent on ATM, as all phenotypes were mitigated in Rad50(+/46) Atm(+/-) mice. These data reveal that the murine Rad50 hook domain strongly influences Mre11 complex-dependent DDR signaling, tissue homeostasis, and tumorigenesis.

Mouse BAZ1A (ACF1) is dispensable for double-strand break repair but is essential for averting improper gene expression during spermatogenesis.

ATP-dependent chromatin remodelers control DNA access for transcription, recombination, and other processes. Acf1 (also known as BAZ1A in mammals) is a defining subunit of the conserved ISWI-family chromatin remodelers ACF and CHRAC, first purified over 15 years ago from Drosophila melanogaster embryos. Much is known about biochemical properties of ACF and CHRAC, which move nucleosomes in vitro and in vivo to establish ordered chromatin arrays. Genetic studies in yeast, flies and cultured human cells clearly implicate these complexes in transcriptional repression via control of chromatin structures. RNAi experiments in transformed mammalian cells in culture also implicate ACF and CHRAC in DNA damage checkpoints and double-strand break repair. However, their essential in vivo roles in mammals are unknown. Here, we show that Baz1a-knockout mice are viable and able to repair developmentally programmed DNA double-strand breaks in the immune system and germ line, I-SceI endonuclease-induced breaks in primary fibroblasts via homologous recombination, and DNA damage from mitomycin C exposure in vivo. However, Baz1a deficiency causes male-specific sterility in accord with its high expression in male germ cells, where it displays dynamic, stage-specific patterns of chromosomal localization. Sterility is caused by pronounced defects in sperm development, most likely a consequence of massively perturbed gene expression in spermatocytes and round spermatids in the absence of BAZ1A: the normal spermiogenic transcription program is largely intact but more than 900 other genes are mis-regulated, primarily reflecting inappropriate up-regulation. We propose that large-scale changes in chromatin composition that occur during spermatogenesis create a window of vulnerability to promiscuous transcription changes, with an essential function of ACF and/or CHRAC chromatin remodeling activities being to safeguard against these alterations.

Homeostatic control of recombination is implemented progressively in mouse meiosis.

Humans suffer from high rates of fetal aneuploidy, often arising from the absence of meiotic crossover recombination between homologous chromosomes. Meiotic recombination is initiated by double-strand breaks (DSBs) generated by the SPO11 transesterase. In yeast and worms, at least one buffering mechanism, crossover homeostasis, maintains crossover numbers despite variation in DSB numbers. We show here that mammals exhibit progressive homeostatic control of recombination. In wild-type mouse spermatocytes, focus numbers for early recombination proteins (RAD51, DMC1) were highly variable from cell to cell, whereas foci of the crossover marker MLH1 showed little variability. Furthermore, mice with greater or fewer copies of the Spo11 gene--with correspondingly greater or fewer numbers of early recombination foci--exhibited relatively invariant crossover numbers. Homeostatic control is enforced during at least two stages, after the formation of early recombination intermediates and later while these intermediates mature towards crossovers. Thus, variability within the mammalian meiotic program is robustly managed by homeostatic mechanisms to control crossover formation, probably to suppress aneuploidy. Meiotic recombination exemplifies how order can be progressively implemented in a self-organizing system despite natural cell-to-cell disparities in the underlying biochemical processes.

ATM controls meiotic double-strand-break formation.

In many organisms, developmentally programmed double-strand breaks (DSBs) formed by the SPO11 transesterase initiate meiotic recombination, which promotes pairing and segregation of homologous chromosomes. Because every chromosome must receive a minimum number of DSBs, attention has focused on factors that support DSB formation. However, improperly repaired DSBs can cause meiotic arrest or mutation; thus, having too many DSBs is probably as deleterious as having too few. Only a small fraction of SPO11 protein ever makes a DSB in yeast or mouse and SPO11 and its accessory factors remain abundant long after most DSB formation ceases, implying the existence of mechanisms that restrain SPO11 activity to limit DSB numbers. Here we report that the number of meiotic DSBs in mouse is controlled by ATM, a kinase activated by DNA damage to trigger checkpoint signalling and promote DSB repair. Levels of SPO11-oligonucleotide complexes, by-products of meiotic DSB formation, are elevated at least tenfold in spermatocytes lacking ATM. Moreover, Atm mutation renders SPO11-oligonucleotide levels sensitive to genetic manipulations that modulate SPO11 protein levels. We propose that ATM restrains SPO11 via a negative feedback loop in which kinase activation by DSBs suppresses further DSB formation. Our findings explain previously puzzling phenotypes of Atm-null mice and provide a molecular basis for the gonadal dysgenesis observed in ataxia telangiectasia, the human syndrome caused by ATM deficiency.

Expression of arf tumor suppressor in spermatogonia facilitates meiotic progression in male germ cells.

The mammalian Cdkn2a (Ink4a-Arf) locus encodes two tumor suppressor proteins (p16(Ink4a) and p19(Arf)) that respectively enforce the anti-proliferative functions of the retinoblastoma protein (Rb) and the p53 transcription factor in response to oncogenic stress. Although p19(Arf) is not normally detected in tissues of young adult mice, a notable exception occurs in the male germ line, where Arf is expressed in spermatogonia, but not in meiotic spermatocytes arising from them. Unlike other contexts in which the induction of Arf potently inhibits cell proliferation, expression of p19(Arf) in spermatogonia does not interfere with mitotic cell division. Instead, inactivation of Arf triggers germ cell-autonomous, p53-dependent apoptosis of primary spermatocytes in late meiotic prophase, resulting in reduced sperm production. Arf deficiency also causes premature, elevated, and persistent accumulation of the phosphorylated histone variant H2AX, reduces numbers of chromosome-associated complexes of Rad51 and Dmc1 recombinases during meiotic prophase, and yields incompletely synapsed autosomes during pachynema. Inactivation of Ink4a increases the fraction of spermatogonia in S-phase and restores sperm numbers in Ink4a-Arf doubly deficient mice but does not abrogate γ-H2AX accumulation in spermatocytes or p53-dependent apoptosis resulting from Arf inactivation. Thus, as opposed to its canonical role as a tumor suppressor in inducing p53-dependent senescence or apoptosis, Arf expression in spermatogonia instead initiates a salutary feed-forward program that prevents p53-dependent apoptosis, contributing to the survival of meiotic male germ cells.

The E3 ubiquitin ligase Cullin 4A regulates meiotic progression in mouse spermatogenesis.

The Cullin-RING ubiquitin-ligase CRL4 controls cell cycle and DNA damage checkpoint response and ensures genomic integrity. Inactivation of the Cul4 component of the CRL4 E3 ligase complex in Caenorhabditis elegans by RNA interference results in massive mitotic DNA re-replication in the blast cells, largely due to failed degradation of the DNA licensing protein, CDT-1, and premature spermatogenesis. Here we show that inactivation of Cul4a by gene-targeting in mice only affected male but not female fertility. This male infertility phenotype resulted from a combination of decreased spermatozoa number, reduced sperm motility and defective acrosome formation. Agenesis of the mutant germ cells was accompanied by increased cell death in pachytene/diplotene cells with markedly elevated levels of phospho-p53 and CDT-1. Despite apparent normal assembly of synaptonemal complexes and DNA double strand break repair, dissociation of MLH1, a component of the late recombination nodule, was delayed in Cul4a -/- diplotene spermatocytes, which potentially led to subsequent disruptions in meiosis II and spermiogenesis. Together, our study revealed an indispensable role for Cul4a during male germ cell meiosis.

Molecular basis for enhancement of the meiotic DMC1 recombinase by RAD51 associated protein 1 (RAD51AP1).

Homologous recombination is needed for meiotic chromosome segregation, genome maintenance, and tumor suppression. RAD51AP1 (RAD51 associated protein 1) has been shown to interact with and enhance the recombinase activity of RAD51. Accordingly, genetic ablation of RAD51AP1 leads to enhanced sensitivity to and also chromosome aberrations upon DNA damage, demonstrating a role for RAD51AP1 in mitotic homologous recombination. Here we show physical association of RAD51AP1 with the meiosis-specific recombinase DMC1 and a stimulatory effect of RAD51AP1 on the DMC1-mediated D-loop reaction. Mechanistic studies have revealed that RAD51AP1 enhances the ability of the DMC1 presynaptic filament to capture the duplex-DNA partner and to assemble the synaptic complex, in which the recombining DNA strands are homologously aligned. We also provide evidence that functional cooperation is dependent on complex formation between DMC1 and RAD51AP1 and that distinct epitopes in RAD51AP1 mediate interactions with RAD51 and DMC1. Finally, we show that RAD51AP1 is expressed in mouse testes, and that RAD51AP1 foci colocalize with a subset of DMC1 foci in spermatocytes. These results suggest that RAD51AP1 also serves an important role in meiotic homologous recombination.