Homologous recombination (HR) serves to eliminate deleterious lesions, such as double-stranded breaks and interstrand crosslinks, from chromosomes. HR is also critical for the preservation of replication forks, for telomere maintenance, and chromosome segregation in meiosis I. As such, HR is indispensable for the maintenance of genome integrity and the avoidance of cancers in humans. The HR reaction is mediated by a conserved class of enzymes termed recombinases. Two recombinases, Rad51 and Dmc1, catalyze the pairing and shuffling of homologous DNA sequences in eukaryotic cells via a filamentous intermediate on ssDNA called the presynaptic filament. The assembly of the presynaptic filament is a rate-limiting process that is enhanced by recombination mediators, such as the breast tumor suppressor BRCA2. HR accessory factors that facilitate other stages of the Rad51- and Dmc1-catalyzed homologous DNA pairing and strand exchange reaction have also been identified. Recent progress on elucidating the mechanisms of action of Rad51 and Dmc1 and their cohorts of ancillary factors is reviewed here.

Homologous recombination (HR) repairs chromosome damage and is indispensable for tumor suppression in humans. RAD51 mediates the DNA strand-pairing step in HR. RAD51 associated protein 1 (RAD51AP1) is a RAD51-interacting protein whose function has remained elusive. Knockdown of RAD51AP1 in human cells by RNA interference engenders sensitivity to different types of genotoxic stress, and RAD51AP1 is epistatic to the HR protein XRCC3. Moreover, RAD51AP1-depleted cells are impaired for the recombinational repair of a DNA double-strand break and exhibit chromatid breaks both spontaneously and upon DNA-damaging treatment. Purified RAD51AP1 binds both dsDNA and a D loop structure and, only when able to interact with RAD51, greatly stimulates the RAD51-mediated D loop reaction. Biochemical and cytological results show that RAD51AP1 functions at a step subsequent to the assembly of the RAD51-ssDNA nucleoprotein filament. Our findings provide evidence that RAD51AP1 helps maintain genomic integrity via RAD51 recombinase enhancement.

Summary The tumor suppressor BRCA2 is thought to facilitate the handoff of ssDNA from replication protein A (RPA) to the RAD51 recombinase during DNA break and replication fork repair by homologous recombination. However, we find that RPA-RAD51 exchange requires the BRCA2 partner DSS1. Biochemical, structural, and in vivo analyses reveal that DSS1 allows the BRCA2-DSS1 complex to physically and functionally interact with RPA. Mechanistically, DSS1 acts as a DNA mimic to attenuate the affinity of RPA for ssDNA. A mutation in the solvent-exposed acidic domain of DSS1 compromises the efficacy of RPA-RAD51 exchange. Thus, by targeting RPA and mimicking DNA, DSS1 functions with BRCA2 in a two-component homologous recombination mediator complex in genome maintenance and tumor suppression. Our findings may provide a paradigm for understanding the roles of DSS1 in other biological processes.

The HOP2 and MND1 genes are indispensable for meiotic recombination. The products of these genes associate to form a stable heterodimeric complex that binds DNA and stimulates the recombinase activity of Rad51 and Dmc1. Here we conduct molecular studies to delineate the action mechanism of the Hop2-Mnd1 complex. We present evidence to implicate Hop2 as the major DNA-binding subunit and Mnd1 as the prominent Rad51 interaction entity. Hop2-Mnd1 stabilizes the Rad51-single-stranded DNA (ssDNA) nucleoprotein filament, the catalytic intermediate in recombination reactions. We also show that Hop2-Mnd1 enhances the ability of the Rad51-ssDNA nucleoprotein filament to capture duplex DNA, an obligatory step in the formation of the synaptic complex critical for DNA joint formation. Thus, our results unveil a bipartite mechanism of Hop2-Mnd1 in homologous DNA pairing: stabilization of the Rad51 presynaptic filament and duplex DNA capture to enhance synaptic complex formation. HR is best understood in the context of DSB repair. Herein, 3Ј single-stranded DNA (ssDNA) tails derived from the nucleolytic processing of the DSB are engaged by the HR machinery, leading to the targeting and invasion of a homologous chromatid to form a DNA joint called the D-loop. Subsequent steps include DNA synthesis, resolution of DNA intermediates, and ligation (Symington 2002;Sung and Klein 2006). In eukaryotes, the homologous DNA pairing reaction responsible for D-loop formation is catalyzed by one of two recombinase enzymes, Rad51 and Dmc1. Whereas Rad51 is needed for recombination in both mitotic and meiotic cells, the expression of Dmc1 is restricted to meiosis. These two recombinases yield inter-homolog crossovers necessary for bridging the homologous chromosomes to ensure their disjunction in meiosis I (Bishop and Zickler 2004;Neale and Keeney 2006). Both Rad51 and Dmc1 are structurally related to the Escherichia coli recombinase RecA. Like RecA, Rad51 and Dmc1 polymerize on ssDNA in an ATP-dependent manner to form a filamentous structure, referred to as the presynaptic filament. The presynaptic filament engages the duplex DNA partner to yield a ternary complex consisting of the recombinase protein filament, ssDNA, and duplex DNA. DNA homology search leads to the formation of the synaptic complex in which the recombining DNA molecules are aligned in homologous registry, and DNA strand invasion occurs upon the location of a free DNA end in either of the DNA molecules (Radding 1982;Sung and Klein 2006).Genetic studies in Saccharomyces cerevisiae and mice Article is online at http://www.genesdev.org/cgi

BRCA2 likely exerts its tumor suppressor function by enhancing the efficiency of the homology-directed repair of injured chromosomes. To help define the DNA repair role of BRCA2, we expressed and purified a polypeptide, BRC3/4-DBD, that harbors its BRC3 and BRC4 repeats and DNA binding domain. BRC3/4-DBD interacted with hRad51 and bound DNA with a distinct preference for single-stranded (ss) DNA. Importantly we demonstrated by biochemical means and electron microscopy that BRC3/4-DBD nucleates hRad51 onto ssDNA and acts as a recombination mediator in enabling hRad51 to utilize replication protein A-coated ssDNA as recombination substrate. These functions of BRC3/4-DBD required both the BRC repeats and the BRCA2 DNA binding domain. The results thus clarify the role of BRCA2 in Rad51-dependent DNA recombination and repair, and the experimental strategies described herein should be valuable for systematically deciphering this BRCA2 function. Homologous recombination (HR)3 helps eliminate DNA breaks, cross-links, and other deleterious lesions from chromosomes. A failure in HR leads to cancer formation (1, 2), which aptly underscores the importance of delineating the mechanism of the HR machinery. HR is best understood in the context of DNA double strand break repair wherein the break is processed nucleolytically to yield ssDNA, which serves as the nucleation site for the Rad51 recombinase. Polymerization of Rad51 onto the ssDNA results in a helical protein filament called the presynaptic filament. The presynaptic filament harbors a binding site for duplex DNA and provides the catalytic center for the search of homology in the duplex and the formation of a joint between the ssDNA and duplex substrates. Assembly of the presynaptic filament is slow, rendering it prone to interference by the single strand binding factor RPA. In addition, the timely delivery of Rad51 to the single-stranded recombination substrate is complicated by a high affinity of this recombinase for duplex DNA. In Saccharomyces cerevisiae, several HR factors, termed recombination mediators, act to promote the assembly of the Rad51 presynaptic filament (3, 4).Mutations in BRCA2 account for a significant portion of familial breast and ovarian cancers (5) and can lead to the cancer-prone syndrome Fanconi anemia (2). BRCA2 mutant cells exhibit hypersensitivity to genotoxic agents and HR deficiency (1, 6, 7). BRCA2 harbors eight copies of a Rad51 binding motif called the BRC repeat (8 -10). In addition, the carboxyl terminus of BRCA2 contains a distinct Rad51 binding domain, and phosphorylation of this domain by cyclin-dependent kinases appears to modulate the interaction with Rad51 (11). Crystallographic and biochemical characterization of mouse Brca2 has revealed a DNA binding domain that comprises three oligonucleotide/oligosaccharide binding (OB) folds (12).Several lines of evidence suggest that BRCA2 functions as a recombination mediator. First, as noted above, BRCA2 interacts with Rad51 (10) and possesses ssDNA binding activity (12), characteristics...

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FANCI is integral to the Fanconi anemia (FA) pathway of DNA damage repair. Upon the occurrence of DNA damage, FANCI becomes monoubiquitinated on Lys-523 and relocalizes to chromatin, where it functions with monoubiquitinated FANCD2 to facilitate DNA repair. We show that FANCI and its C-terminal fragment possess a DNA binding activity that prefers branched structures. We also demonstrate that FANCI can be ubiquitinated on Lys-523 by the UBE2T-FANCL pair in vitro. These findings should facilitate future efforts directed at elucidating molecular aspects of the FA pathway.Fanconi anemia (FA) 4 is characterized by developmental defects, bone marrow failure, and a strong predisposition to cancer. FA cells exhibit exquisite sensitivity to DNA cross-linking agents and marked genomic instability, indicative of a failure to repair damaged DNA (1-3). Thirteen FA proteins have been identified, of which eight, FANC-A, -B, -C, -E, -F, -G, -L, and -M, form part of a nuclear core complex that is required to monoubiquitinate two other FA proteins, FANCD2 and FANCI. When monoubiquitinated, FANCD2 and FANCI become chromatin-associated in foci that contain various factors, including the RAD51 recombinase BRCA2 (also known as FANCD1) and PALB2 (also called FANCN), which mediate DNA repair via RAD51-catalyzed homologous recombination (4).Monoubiquitination of FANCD2 appears to be a key event for proper repair of exogenous DNA damage but also occurs during an unperturbed S phase, likely in response to stalled replication forks (4 -7). FANCD2 monoubiquitination depends on the E3 ligase activity of FANCL (8) and on the E2 ubiquitin-conjugating enzyme, UBE2T (9). In vitro, FANCL and UBE2T can monoubiquitinate chicken FANCD2 (10).FANCI was identified recently as a target protein for the ATM/ATR kinase. FANCI is also monoubiquitinated, in a manner that is dependent on the FA core complex (11). In cells, a fraction of FANCD2 and FANCI associates in a complex. Moreover, the amount and monoubiquitination of these two FA proteins are co-dependent in human cells, i.e. the quantity and monoubiquitination of FANCD2 are diminished in FANCI-deficient cells and vice versa (11)(12)(13)(14). These observations suggest that FANCI and FANCD2 form a complex integral to cellular DNA repair capacity. Mutating the ubiquitinated target lysine of FANCI (Lys-523) renders cells sensitive to DNA damage and impairs the assembly of DNA damage-induced nuclear foci of FANCD2 and FANCI (11,14). Herein, we document studies that reveal several biochemical attributes of FANCI, including DNA binding, and its monoubiquitination, that are relevant for understanding the biological role of this key FA protein. EXPERIMENTAL PROCEDURESCloning for Protein Expression-Oligonucleotides and detailed procedures used for cloning are provided in supplemental Table 1 and supplemental Materials and Methods, respectively. The cDNA for human FANCI (isoform 1, KIAA1794) in the pCMV6-XL4 vector was subcloned into pFastBacHT(B) (Invitrogen) to add an N-terminal His 6 epitope to the FANCI pro...

Despite their commercial importance, there are relatively few facile methods for genomic manipulation of the lactic acid bacteria. Here, the lactococcal group II intron, Ll.ltrB, was targeted to insert efficiently into genes encoding malate decarboxylase (mleS) and tetracycline resistance (tetM) within the Lactococcus lactis genome. Integrants were readily identified and maintained in the absence of a selectable marker. Since splicing of the Ll.ltrB intron depends on the intron-encoded protein, targeted invasion with an intron lacking the intron open reading frame disrupted TetM and MleS function, and MleS activity could be partially restored by expressing the intron-encoded protein in trans. Restoration of splicing from intron variants lacking the intron-encoded protein illustrates how targeted group II introns could be used for conditional expression of any gene. Furthermore, the modified Ll.ltrB intron was used to separately deliver a phage resistance gene (abiD) and a tetracycline resistance marker (tetM) into mleS, without the need for selection to drive the integration or to maintain the integrant. Our findings demonstrate the utility of targeted group II introns as a potential food-grade mechanism for delivery of industrially important traits into the genomes of lactococci.The lactic acid bacteria (LAB) are a broad group of grampositive bacteria that possess similar morphological, metabolic, and physiological characteristics (33). The LAB have been the subject of considerable research and commercial development, given their significance in fermentation, bioprocessing, agriculture, food, and medicine (12, 13). An impediment to fundamental studies on the LAB is the lack of facile genetic tools for manipulation of chromosomal genes (19). Moreover, public concern over the use of genetically engineered cultures for food production has prompted a search for "self-cloning" methods, whereby genetic manipulation is achieved using DNA solely from food-grade microorganisms, preferably from within the same genus (6).Mobile group II introns are catalytic RNA elements present in a wide range of prokaryotic and eukaryotic organisms (18). Some of these introns can mobilize autonomously at a high frequency to allelic sites in a process known as homing (3). Mobile group II introns possess an intron-encoded protein (IEP) that has reverse transcriptase, RNA splicing ("maturase"), and DNA endonuclease activities (3). Mobility initiates when the IEP helps the intron RNA fold into the catalytically active RNA structure to promote splicing, resulting in ligated exons and an intron lariat-IEP ribonucleoprotein (RNP) complex. The RNP complex recognizes specific DNA target sites and promotes integration by reverse splicing of the intron RNA directly into one strand of the target DNA. The IEP then cleaves the opposite strand and uses it as a primer for target DNA-primed reverse transcription of the inserted intron RNA (16,34,36,37). The resulting cDNA copy of the intron is integrated into genomic DNA by cellular recombination or rep...