Biochemical analysis of the N-terminal domain of human RAD54B

The human RAD54B protein is a paralog of the RAD54 protein, which plays important roles in homologous recombination. RAD54B contains an N-terminal region outside the SWI2/SNF2 domain that shares less conservation with the corresponding region in RAD54. The biochemical roles of this region of RAD54B are not known, although the corresponding region in RAD54 is known to physically interact with RAD51. In the present study, we have biochemically characterized an N-terminal fragment of RAD54B, consisting of amino acid residues 26–225 (RAD54B_26–225). This fragment formed a stable dimer in solution and bound to branched DNA structures. RAD54B_26–225 also interacted with DMC1 in both the presence and absence of DNA. Ten DMC1 segments spanning the entire region of the DMC1 sequence were prepared, and two segments, containing amino acid residues 153–214 and 296–340, were found to directly bind to the N-terminal domain of RAD54B. A structural alignment of DMC1 with the Methanococcus voltae RadA protein, a homolog of DMC1 in the helical filament form, indicated that these RAD54B-binding sites are located near the ATP-binding site at the monomer–monomer interface in the DMC1 helical filament. Thus, RAD54B binding may affect the quaternary structure of DMC1. These observations suggest that the N-terminal domain of RAD54B plays multiple roles of in homologous recombination.     

Crystal structure of archaeal recombinase RADA: a snapshot of its extended conformation

Homologous recombination of DNA plays crucial roles in repairing severe DNA damage and in generating genetic diversity. The process is facilitated by a superfamily of recombinases: bacterial RecA, archaeal RadA and Rad51, and eukaryal Rad51 and DMC1. These recombinases share a common ATP-dependent filamentous quaternary structure for binding DNA and facilitating strand exchange. We have determined the crystal structure of Methanococcus voltae RadA in complex with the ATP analog AMP-PNP at 2.0 A resolution. The RadA filament is a 106.7 A pitch helix with six subunits per turn. The DNA binding loops L1 and L2 are located in close proximity to the filament axis. The ATP analog is buried between two RadA subunits, a feature similar to that of the active filament of Escherichia coli RecA revealed by electron microscopy. The disposition of the N-terminal domain suggests a role of the Helix-hairpin-Helix motif in binding double-stranded DNA.     

FIGL1 coordinates with dosage-sensitive BRCA2 in modulating meiotic recombination in maize

Meiotic crossover(CO) formation between homologous chromosomes ensures their subsequent proper segregation and generates genetic diversity among offspring. In maize, however, the mechanisms that modulate CO formation remain poorly characterized. Here, we found that both maize BREAST CANCER SUSCEPTIBILITY PROTEIN 2(BRCA2) and AAA-ATPase FIDGETIN-LIKE-1(FIGL1)act as positive factors of CO formation by controlling the assembly or/and stability of two conserved DNA recombinases RAD51 and DMC1 filame...     

RAD51 supports DMC1 by inhibiting the SMC5/6 complex during meiosis

Meiosis is a fundamental process for sexual reproduction in most eukaryotes and the evolutionarily conserved recombinases RADiation sensitive51 (RAD51) and Disrupted Meiotic cDNA1 (DMC1) are essential for meiosis and thus fertility. The mitotic function of RAD51 is clear, but the meiotic function of RAD51 remains largely unknown. Here we show that RAD51 functions as an interacting protein to restrain the Structural Maintenance of Chromosomes5/6 (SMC5/6) complex from inhibiting DMC1. We unexpectedly found that loss of the SMC5/6 partially suppresses the rad51 knockout mutant in terms of sterility, pollen inviability, and meiotic chromosome fragmentation in a DMC1-dependent manner in Arabidopsis thaliana. Biochemical and cytological studies revealed that the DMC1 localization in meiotic chromosomes is inhibited by the SMC5/6 complex, which is attenuated by RAD51 through physical interactions. This study not only identified the long-sought-after function of RAD51 in meiosis but also discovered the inhibition of SMC5/6 on DMC1 as a control mechanism during meiotic recombination.

RAD51 functions as an interacting protein to restrain the SMC5/6 complex from inhibiting DMC1 during meiosis.     

Archaeal RecA homologues: different response to DNA-damaging agents in mesophilic and thermophilic Archaea

Two archaeal proteins, RadA and RadB, share similarity with the RecA/Rad51 family of recombinases, with RadA being the functional homologue. We have studied and compared the RadA and RadB proteins of mesophilic and thermophilic Archaea. In growing cells, RadA levels are similar in mesophilic Methanococcus species and the hyperthermophile Methanococcus jannaschii. Treatment of cells with mutagenic agents (methylmethane sulfonate or UV light) increased the expression of RadA (as evidenced by higher levels of both mRNA and protein) in all organisms tested, but the increase was greater in the mesophiles than in the thermophiles M. jannaschii and Sulfolobus solfataricus. Recombinantly expressed RadA proteins from the mesophile M. voltae and the thermophile M. jannaschii were similar in their ATPase- and DNA-binding activities. All the data are consistent with proposals that RadA plays the same role as eukaryotic Rad51. Surprisingly, the data also suggested that the thermophiles do not need more RadA protein or activity than the mesophiles. On the other hand, RadB is not coregulated with RadA, and its role remains unclear. Neither RadA nor RadB from a mesophile or from a thermophile rescued the UV-sensitive phenotype of an Escherichia coli recA- host.     

Comparison of bacteriophage T4 UvsX and human Rad51 filaments suggests that RecA-like polymers may have evolved independently

The UvsX protein from bacteriophage T4 is a member of the RecA/Rad51/RadA family of recombinases active in homologous genetic recombination. Like RecA, Rad51 and RadA, UvsX forms helical filaments on DNA. We have used electron microscopy and a novel method for image analysis of helical filaments to show that UvsX-DNA filaments exist in two different conformations: an ADP state and an ATP state. As with RecA protein, these two states have a large difference in pitch. Remarkably, even though UvsX is only weakly homologous to RecA, both UvsX filament states are more similar to the RecA crystal structure than are RecA-DNA filaments. We use this similarity to fit the RecA crystal structure into the UvsX filament, and show that two of the three previously described blocks of similarity between UvsX and RecA are involved in the subunit-subunit interface in both the UvsX filament and the RecA crystal filament. Conversely, we show that human Rad51-DNA filaments have a different subunit-subunit interface than is present in the RecA crystal, and this interface involves two blocks of sequence similarity between Rad51 and RecA that do not overlap with those found between UvsX and RecA. This suggests that helical filaments in the RecA/Rad51/RadA family may have arisen from convergent evolution, with a conserved core structure that has assembled into multimeric filaments in a number of different ways.     

RadA protein from Archaeoglobus fulgidus forms rings, nucleoprotein filaments and catalyses homologous recombination

Proteins that catalyse homologous recombination have been identified in all living organisms and are essential for the repair of damaged DNA as well as for the generation of genetic diversity. In bacteria homologous recombination is performed by the RecA protein, whereas in the eukarya a related protein called Rad51 is required to catalyse recombination and repair. More recently, archaeal homologues of RecA/Rad51 (RadA) have been identified and isolated. In this work we have cloned and purified the RadA protein from the hyperthermophilic, sulphate-reducing archaeon Archaeoglobus fulgidus and characterised its in vitro activities. We show that (i) RadA protein forms ring structures in solution and binds single- but not double-stranded DNA to form nucleoprotein filaments, (ii) RadA is a single-stranded DNA-dependent ATPase at elevated temperatures, and (iii) RadA catalyses efficient D-loop formation and strand exchange at temperatures of 60–70°C. Finally, we have used electron microscopy to visualise RadA-mediated joint molecules, the intermediates of homologous recombination. Intriguingly, RadA shares properties of both the bacterial RecA and eukaryotic Rad51 recombinases.     

Bacillus subtilis RadA/Sms contributes to chromosomal transformation and DNA repair in concert with RecA and circumvents replicative stress in concert with DisA

Bacillus subtilis radA is epistatic to disA and recA genes in response to methyl methane sulfonate- and 4-nitroquinoline-1-oxide-induced DNA damage. We show that ΔradA cells were sensitive to mitomycin C- and H₂O₂-induced damage and impaired in natural chromosomal transformation, whereas cells lacking DisA were not. RadA/Sms mutants in the conserved H1 (K104A and K104R) or KNRFG (K255A and K255R) motifs fail to rescue the sensitivity of ΔradA in response to the four different DNA damaging agents. A RadA/Sms H1 or KNRFG mutation impairs both chromosomal and plasmid transformation, but the latter defect was suppressed by inactivating RecA. RadA/Sms K255A, K255R and wild type RadA/Sms reduced the diadenylate cyclase activity of DisA, whereas RadA/Sms K104A and K104R blocked it. Single-stranded and Holliday junction DNA are preferentially bound over double-stranded DNA by RadA/Sms and its variants. Moreover, RadA/Sms ATPase activity was neither stimulated by a variety of DNA substrates nor by DisA. RadA/Sms possesses a 5´→3´ DNA helicase activity. The RadA/Sms mutants neither hydrolyze ATP nor unwind DNA. Thus, we propose that RadA/Sms has two activities: to modulate DisA and to promote RecA-mediated DNA strand exchange. Both activities are required to coordinate responses to replicative stress and genetic recombination.     

RAD51B plays an essential role during somatic and meiotic recombination in Physcomitrella

The eukaryotic RecA homologue Rad51 is a key factor in homologous recombination and recombinational repair. Rad51-like proteins have been identified in yeast (Rad55, Rad57 and Dmc1), plants and vertebrates (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3 and DMC1). RAD51 and DMC1 are the strand-exchange proteins forming a nucleofilament for strand invasion, however, the function of the paralogues in the process of homologous recombination is less clear. In yeast the two Rad51 paralogues, Rad55 and Rad57, have been shown to be involved in somatic and meiotic HR and they are essential to the formation of the Rad51/DNA nucleofilament counterbalancing the anti-recombinase activity of the SRS2 helicase. Here, we examined the role of RAD51B in the model bryophyte Physcomitrella patens. Mutant analysis shows that RAD51B is essential for the maintenance of genome integrity, for resistance to DNA damaging agents and for gene targeting. Furthermore, we set up methods to investigate meiosis in Physcomitrella and we demonstrate that the RAD51B protein is essential for meiotic homologous recombination. Finally, we show that all these functions are independent of the SRS2 anti-recombinase protein, which is in striking contrast to what is found in budding yeast where the RAD51 paralogues are fully dependent on the SRS2 anti-recombinase function.     

The association of ATR protein with mouse meiotic chromosome cores

The ATR (ataxia telangiectasia- and RAD3-related) protein is present on meiotic prophase chromosome cores and paired cores (synaptonemal complexes, SCs). Its striking characteristic is that the protein forms dense aggregates on the cores and SCs of the last chromosomes to pair at the zygotene-pachytene transition. It would appear that the ATR protein either signals delays in pairing or it is directly involved in the completion of the pairing phase. Atm-deficient spermatocytes, which are defective in the chromosome pairing phase, accumulate large amounts of ATR. The behaviour of ATR at meiotic prophase sets it apart from the distribution of the RAD51/DMC1 recombinase complex and our electron microscope observations confirm that they do not co-localize. We failed to detect ATM in association with cores/SCs and we have reported elsewhere that RAD1 protein does not co-localize with DMC1 foci. The expectation that putative DNA-damage checkpoint proteins, ATR, ATM and RAD1, are associated with RAD51/DMC1 recombination sites where DNA breaks are expected to be present, is therefore not supported by our observations. Received: 23 November 1998 / Accepted: 3 January 1999     

Domain analysis of an archaeal RadA protein for the strand exchange activity

Archaeal RadA, like eukaryotic Rad51 and bacterial RecA, promotes strand exchange between DNA strands with homologous sequences in vitro and is believed to participate in the homologous recombination in cells. The amino acid sequences of the archaeal RadA proteins are more similar to the eukaryotic Rad51s rather than the bacterial RecAs, and the N-terminal region containing domain I is conserved among the RadA and Rad51 proteins but is absent from RecA. To understand the structure-function relationship of RadA, we divided the RadA protein from Pyrococcus furiosus into two parts, the N-terminal one-third (RadA-n) and the residual C-terminal two-thirds (RadA-c), the latter of which contains the central core domain (domain II) of the RecA/Rad51 family proteins. RadA-c had the DNA-dependent ATPase activity and the strand exchange activity by itself, although much weaker (10%) than that of the intact RadA. These activities of RadA-c were restored to 60% of those of RadA by addition of RadA-n, indicating that the proper active structure of RadA was reconstituted in vitro. These results suggest that the basic activities of the RecA/Rad51 family proteins for homologous recombination are derived from domain II, and the N-terminal region may help to enhance the catalytic efficiencies.     

Structural and Functional Characterisation of a Conserved Archaeal RadA Paralog with Antirecombinase Activity

DNA recombinases (RecA in bacteria, Rad51 in eukarya and RadA in archaea) catalyse strand exchange between homologous DNA molecules, the central reaction of homologous recombination, and are among the most conserved DNA repair proteins known. RecA is the sole protein responsible for this reaction in bacteria, whereas there are several Rad51 paralogs that cooperate to catalyse strand exchange in eukaryotes. All archaea have at least one (and as many as four) RadA paralog, but their function remains unclear. Herein, we show that the three RadA paralogs encoded by the Sulfolobus solfataricus genome are expressed under normal growth conditions and are not UV inducible. We demonstrate that one of these proteins, Sso2452, which is representative of the large archaeal RadC subfamily of archaeal RadA paralogs, functions as an ATPase that binds tightly to single-stranded DNA. However, Sso2452 is not an active recombinase in vitro and inhibits D-loop formation by RadA. We present the high-resolution crystal structure of Sso2452, which reveals key structural differences from the canonical RecA family recombinases that may explain its functional properties. The possible roles of the archaeal RadA paralogs in vivo are discussed.     

Crystal structure of an ATPase-active form of Rad51 homolog from Methanococcus voltae. Insights into potassium dependence

Homologous gene recombination is crucial for the repair of DNA. A superfamily of recombinases facilitate a central strand exchange reaction in the repair process. This reaction is initiated by coating single-stranded DNA (ssDNA) with recombinases in the presence of ATP and Mg(2+) co-factors to form helical nucleoprotein filaments with elevated ATPase and strand invasion activities. At the amino acid sequence level, archaeal RadA and Rad51 and eukaryal Rad51 and meiosis-specific DMC1 form a closely related group of recombinases distinct from bacterial RecA. Unlike the extensively studied Escherichia coli RecA (EcRecA), increasing evidences on yeast and human recombinases imply that their optimal activities are dependent on the presence of a monovalent cation, particularly potassium. Here we present the finding that archaeal RadA from Methanococcus voltae (MvRadA) is a stringent potassium-dependent ATPase, and the crystal structure of this protein in complex with the non-hydrolyzable ATP analog adenosine 5'-(beta,gamma-iminotriphosphate), Mg(2+), and K(+) at 2.4 A resolution. Potassium triggered an in situ conformational change in the ssDNA-binding L2 region concerted with incorporation of two potassium ions at the ATPase site in the RadA crystals preformed in K(+)-free medium. Both potassium ions were observed in contact with the gamma-phosphate of the ATP analog, implying a direct role by the monovalent cations in stimulating the ATPase activity. Cross-talk between the ATPase site and the ssDNA-binding L2 region visualized in the MvRadA structure provides an explanation to the co-factor-induced allosteric effect on RecA-like recombinases.     

Improper chromosome synapsis is associated with elongated RAD51 structures in the maize<Emphasis Type="Italic"> desynaptic2</Emphasis> mutant

The RecA homolog, RAD51, performs a central role in catalyzing the DNA strand exchange event of meiotic recombination. During meiosis, RAD51 complexes develop on pairing chromosomes and then most disappear upon synapsis. In the maize meiotic mutant desynaptic2 (dsy2), homologous chromosome pairing and recombination are reduced by ~70% in male meiosis. Fluorescent in situ hybridization studies demonstrate that a normal telomere bouquet develops but the pairing of a representative gene locus is still only 25%. Chromosome synapsis is aberrant as exemplified by unsynapsed regions of the chromosomes. In the mutant, we observed unusual RAD51 structures during chromosome pairing. Instead of spherical single and double RAD51 structures, we saw long thin filaments that extended along or around a single chromosome or stretched between two widely separated chromosomes. Mapping with simple sequence repeat (SSR) markers places the dsy2 gene to near the centromere on chromosome 5, therefore it is not an allele of rad51. Thus, the normal dsy2 gene product is required for both homologous chromosome synapsis and proper RAD51 filament behavior when chromosomes pair. Edited by: P. Moens     

Isolation of a LIM15/DMC1 homolog from the basidiomycete Coprinus cinereus and its expression in relation to meiotic chromosome pairing

The Escherichia coli gene recA is essential for homologous recombination and DNA repair, and homologs have been identified in eukaryotes. A basidiomycete, Coprinus cinereus, which has many advantages for the study of meiosis, was recently reported to have a homolog of one of these, RAD51. In the yeast Saccharomyces, mutations in the RAD5I gene cause defects in both somatic and meiotic cells. Based on this finding, we screened for a meiosis-specific homolog of recA, equivalent to Lilium LIM15 or Saccharomyces DMC1, in C. cinereus, and isolated a clone containing a 1.2-kb DNA fragment from a cDNA library constructed with Coprinus poly(A)⁺ RNA isolated from cells undergoing meiosis. The predicted amino acid sequence was 52% identical to the putative gene product of the lily cDNA clone LIM15 and 61% identical to Saccharomyces DMC1, and showed limited sequence similarity to the products of RAD52, 55, and 57. The synchrony of meiosis in Coprinus provides an ideal system for the investigation of differential gene expression in relation to meiosis and fruiting body development. Northern analysis indicated that Coprinus LIM15/DMC1 was expressed at meiotic prophase within 8 h after the onset of karyogamy, suggesting that the gene functions mostly at the stage at which the homologous chromosomes pair, but may not be essential at the point at which they recombine. The gene is not expressed in somatic cells. Received: 8 October 1998 / Accepted: 22 July 1999     

超嗜热古菌Sulfolobus tokodaii RadA蛋白的克隆表达及 其辐射可诱导性

RecA/Rad51/RadA家族蛋白是细胞内重要的重组修复蛋白,在功能上非常保守.研究发现在细菌、真核生物、甲烷古菌和嗜盐古菌细胞内RecA/Rad51/RadA均可以受紫外线辐射诱导转录.而对极端嗜热古菌中的RadA辐射可诱导性仍存在争议.通过体外表达极端嗜热古菌Sulfolobus tokodaii的RadA蛋白,制备抗体,利用免疫学方法并结合RT-PCR分析,对嗜热古菌S.tokodaii中RadA的辐射诱导进行了研究.经过100J/m2和200J/m2 UV辐照处理,radA基因的转录分别上调了2倍和3倍,同时RadA蛋白的表达分别上升了1.5倍和1倍.实验结果表明S.tokodaii中RadA可以被紫外线辐射诱导表达,证实了极端嗜热古菌S.tokodaii细胞中存在DNA损伤诱导反应的观点.     

Meiotic Recombination in Arabidopsis Is Catalysed by DMC1, with RAD51 Playing a Supporting Role

Recombination establishes the chiasmata that physically link pairs of homologous chromosomes in meiosis, ensuring their balanced segregation at the first meiotic division and generating genetic variation. The visible manifestation of genetic crossing-overs, chiasmata are the result of an intricate and tightly regulated process involving induction of DNA double-strand breaks and their repair through invasion of a homologous template DNA duplex, catalysed by RAD51 and DMC1 in most eukaryotes. We describe here a RAD51-GFP fusion protein that retains the ability to assemble at DNA breaks but has lost its DNA break repair capacity. This protein fully complements the meiotic chromosomal fragmentation and sterility of Arabidopsis rad51, but not rad51 dmc1 mutants. Even though DMC1 is the only active meiotic strand transfer protein in the absence of RAD51 catalytic activity, no effect on genetic map distance was observed in complemented rad51 plants. The presence of inactive RAD51 nucleofilaments is thus able to fully support meiotic DSB repair and normal levels of crossing-over by DMC1. Our data demonstrate that RAD51 plays a supporting role for DMC1 in meiotic recombination in the flowering plant, Arabidopsis.