Promotion of BRCA2-Dependent Homologous Recombination by DSS1 via RPA Targeting and DNA Mimicry.

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.

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.

Mechanistic insights into RAD51-associated protein 1 (RAD51AP1) action in homologous DNA repair.

Homologous recombination catalyzed by the RAD51 recombinase is essential for maintaining genome integrity upon the induction of DNA double strand breaks and other DNA lesions. By enhancing the recombinase activity of RAD51, RAD51AP1 (RAD51-associated protein 1) serves a key role in homologous recombination-mediated chromosome damage repair. We show here that RAD51AP1 harbors two distinct DNA binding domains that are both needed for maximal protein activity under physiological conditions. We have finely mapped the two DNA binding domains in RAD51AP1 and generated mutant variants that are impaired in either or both of the DNA binding domains. Examination of these mutants reveals that both domains are indispensable for RAD51AP1 function in cells. These and other results illuminate the mechanistic basis of RAD51AP1 action in homologous DNA repair.

Characterization of the C-terminal DNA-binding/DNA endonuclease region of a group II intron-encoded protein.

Group II intron retrohoming occurs by a mechanism in which the intron RNA reverse splices directly into one strand of a double-stranded DNA target site, while the intron-encoded reverse transcriptase uses a C-terminal DNA endonuclease activity to cleave the opposite strand and then uses the cleaved 3' end as a primer for reverse transcription of the inserted intron RNA. Here, we characterized the C-terminal DNA-binding/DNA endonuclease region of the LtrA protein encoded by the Lactococcus lactis Ll.LtrB intron. This C-terminal region consists of an upstream segment that contributes to DNA binding, followed by a DNA endonuclease domain that contains conserved sequence motifs characteristic of H-N-H DNA endonucleases, interspersed with two pairs of conserved cysteine residues. Atomic emission spectroscopy of wild-type and mutant LtrA proteins showed that the DNA endonuclease domain contains a single tightly bound Mg(2+) ion at the H-N-H active site. Although the conserved cysteine residue pairs could potentially bind Zn(2+), the purified LtrA protein is active despite the presence of only sub-stoichiometric amounts of Zn(2+), and the addition of exogenous Zn(2+) inhibits the DNA endonuclease activity. Multiple sequence alignments identified features of the DNA-binding region and DNA endonuclease domain that are conserved in LtrA and related group II intron proteins, and their functional importance was demonstrated by unigenic evolution analysis and biochemical assays of mutant LtrA protein with alterations in key amino acid residues. Notably, deletion of the DNA endonuclease domain or mutations in its conserved sequence motifs strongly inhibit reverse transcriptase activity, as well as bottom-strand cleavage, while retaining other activities of the LtrA protein. A UV-cross-linking assay showed that these DNA endonuclease domain mutations do not block DNA primer binding and thus likely inhibit reverse transcriptase activity either by affecting the positioning of the primer or the conformation of the reverse transcriptase domain.

Mobility of the Sinorhizobium meliloti group II intron RmInt1 occurs by reverse splicing into DNA, but requires an unknown reverse transcriptase priming mechanism.

The mobile group II introns characterized to date encode ribonucleoprotein complexes that promote mobility by a major retrohoming mechanism in which the intron RNA reverse splices directly into the sense strand of a double-stranded DNA target site, while the intron-encoded reverse transcriptase/maturase cleaves the antisense strand and uses it as primer for reverse transcription of the inserted intron RNA. Here, we show that the Sinorhizobium meliloti group II intron RmInt1, which encodes a protein lacking a DNA endonuclease domain, similarly uses both the intron RNA and an intron-encoded protein with reverse transcriptase and maturase activities for mobility. However, while RmInt1 reverse splices into both single-stranded and double-stranded DNA target sites, it is unable to carry out site-specific antisense-strand cleavage due to the lack of a DNA endonuclease domain. Our results suggest that RmInt1 mobility involves reverse splicing into double-stranded or single-stranded DNA target sites, but due to the lack of DNA endonuclease function, it requires an alternate means of procuring a primer for target DNA-primed reverse transcription.

FANCI binds branched DNA and is monoubiquitinated by UBE2T-FANCL.

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.

Mechanism of eukaryotic homologous recombination.

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.

Bipartite stimulatory action of the Hop2-Mnd1 complex on the Rad51 recombinase.

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.

Promotion of homologous recombination and genomic stability by RAD51AP1 via RAD51 recombinase enhancement.

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.

Recombination mediator and Rad51 targeting activities of a human BRCA2 polypeptide.

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.

Genetic manipulation of Lactococcus lactis by using targeted group II introns: generation of stable insertions without selection.

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.