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Biomedical subjects

R Kanaar

Publications and source records attributed to R Kanaar.

At least 19 recordsLinked to original sources
PubMed2001

The structure-specific endonuclease Ercc1-Xpf is required for targeted gene replacement in embryonic stem cells.

The Ercc1-Xpf heterodimer, a highly conserved structure-specific endonuclease, functions in multiple DNA repair pathways that are pivotal for maintaining genome stability, including nucleotide excision repair, interstrand crosslink repair and homologous recombination. Ercc1-Xpf incises double-stranded DNA at double-strand/single-strand junctions, making it an ideal enzyme for processing DNA structures that contain partially unwound strands. Here we demonstrate that although Ercc1 is dispensable for recombination between sister chromatids, it is essential for targeted gene replacement in mouse embryonic stem cells. Surprisingly, the role of Ercc1-Xpf in gene targeting is distinct from its previously identified role in removing nonhomologous termini from recombination intermediates because it was required irrespective of whether the ends of the DNA targeting constructs were heterologous or homologous to the genomic locus. Our observations have implications for the mechanism of gene targeting in mammalian cells and define a new role for Ercc1-Xpf in mammalian homologous recombination. We propose a model for the mechanism of targeted gene replacement that invokes a role for Ercc1-Xpf in making the recipient genomic locus receptive for gene replacement.

Animals↗
PubMed2001

Repair of DNA interstrand cross-links.

DNA interstrand cross-links (ICLs) are very toxic to dividing cells, because they induce mutations, chromosomal rearrangements and cell death. Inducers of ICLs are important drugs in cancer treatment. We discuss the main properties of several classes of ICL agents and the types of damage they induce. The current insights in ICL repair in bacteria, yeast and mammalian cells are reviewed. An intriguing aspect of ICLs is that a number of multi-step DNA repair pathways including nucleotide excision repair, homologous recombination and post-replication/translesion repair all impinge on their repair. Furthermore, the breast cancer-associated proteins Brca1 and Brca2, the Fanconi anemia-associated FANC proteins, and cell cycle checkpoint proteins are involved in regulating the cellular response to ICLs. We depict several models that describe possible pathways for the repair or replicational bypass of ICLs.

Animals↗
PubMed2001

Growth inhibition and DNA damage induced by Cre recombinase in mammalian cells.

The use of Cre/loxP recombination in mammalian cells has expanded rapidly. We describe here that Cre expression in cultured mammalian cells may result in a markedly reduced proliferation and that this effect is dependent on the endonuclease activity of Cre. Chromosome analysis after Cre expression revealed numerous chromosomal aberrations and an increased number of sister chromatid exchanges. Titration experiments in mouse embryo fibroblasts with a ligand-regulatable Cre-ER(T) show that toxicity is dependent on the level of Cre activity. Prolonged, low levels of Cre activity permit recombination without concomitant toxicity. This urges for a careful titration of Cre activity in conditional gene modification in mammalian cells.

Aneuploidy↗
PubMed2001

The architecture of the human Rad54-DNA complex provides evidence for protein translocation along DNA.

Proper maintenance and duplication of the genome require accurate recombination between homologous DNA molecules. In eukaryotic cells, the Rad51 protein mediates pairing between homologous DNA molecules. This reaction is assisted by the Rad54 protein. To gain insight into how Rad54 functions, we studied the interaction of the human Rad54 (hRad54) protein with double-stranded DNA. We have recently shown that binding of hRad54 to DNA induces a change in DNA topology. To determine whether this change was caused by a protein-constrained change in twist, a protein-constrained change in writhe, or the introduction of unconstrained plectonemic supercoils, we investigated the hRad54--DNA complex by scanning force microscopy. The architecture of the observed complexes suggests that movement of the hRad54 protein complex along the DNA helix generates unconstrained plectonemic supercoils. We discuss how hRad54-induced superhelical stress in the target DNA may function to facilitate homologous DNA pairing by the hRad51 protein directly. In addition, the induction of supercoiling by hRad54 could stimulate recombination indirectly by displacing histones and/or other proteins packaging the DNA into chromatin. This function of DNA translocating motors might be of general importance in chromatin metabolism.

Adenosine Triphosphatases↗
PubMed2001

DNA repair: spot(light)s on chromatin.

Chromatin modifications regulate many nuclear processes. Recent studies on the phosphorylation of a histone 2A variant have revealed that this chromatin modification is a general and evolutionarily conserved cellular response to DNA double-strand breaks.

Animals↗
PubMed2001

DNA-binding and strand-annealing activities of human Mre11: implications for its roles in DNA double-strand break repair pathways.

DNA double-strand breaks (DSBs) in eukaryotic cells can be repaired by non-homologous end-joining or homologous recombination. The complex containing the Mre11, Rad50 and Nbs1 proteins has been implicated in both DSB repair pathways, even though they are mechanistically different. To get a better understanding of the properties of the human Mre11 (hMre11) protein, we investigated some of its biochemical activities. We found that hMre11 binds both double- and single-stranded (ss)DNA, with a preference for ssDNA. hMre11 does not require DNA ends for efficient binding. Interestingly, hMre11 mediates the annealing of complementary ssDNA molecules. In contrast to the annealing activity of the homologous recombination protein hRad52, the activity of hMre11 is abrogated by the ssDNA binding protein hRPA. We discuss the possible implications of the results for the role(s) of hMre11 in both DSB repair pathways.

Animals↗
PubMed2001

Human Rad50/Mre11 is a flexible complex that can tether DNA ends.

The human Rad50 protein, classified as a structural maintenance of chromosomes (SMC) family member, is complexed with Mre11 (R/M) and has important functions in at least two distinct double-strand break repair pathways. To find out what the common function of R/M in these pathways might be, we investigated its architecture. Scanning force microscopy showed that the complex architecture is distinct from the described SMC family members. R/M consisted of two highly flexible intramolecular coiled coils emanating from a central globular DNA binding domain. DNA end-bound R/M oligomers could tether linear DNA molecules. These observations suggest that a unified role for R/M in multiple aspects of DNA repair and chromosome metabolism is to provide a flexible, possibly dynamic, link between DNA ends.

DNA↗
PubMed2001

Chromosomal stability and the DNA double-stranded break connection.

Genome stability is of primary importance for the survival and proper functioning of all organisms. Double-stranded breaks in DNA are important threats to genome integrity because they can result in chromosomal aberrations that can affect, simultaneously, many genes, and lead to cell malfunctioning and cell death. These detrimental consequences are counteracted by two mechanistically distinct pathways of double-stranded break repair: homologous recombination and non-homologous end-joining. Recently, unexpected links between these double-stranded break-repair systems, and several human genome instability and cancer predisposition syndromes, have emerged. Now, interactions between both double-stranded break-repair pathways and other cellular processes, such as cell-cycle regulation and replication, are being unveiled.

Animals↗
PubMed2000

Homologous and non-homologous recombination differentially affect DNA damage repair in mice.

Ionizing radiation and interstrand DNA crosslinking compounds provide important treatments against cancer due to their extreme genotoxicity for proliferating cells. Both the efficacies of such treatments and the mutagenic potential of these agents are modulated by the ability of cells to repair the inflicted DNA damage. Here we demonstrate that homologous recombination-deficient mRAD54(-/-) mice are hypersensitive to ionizing radiation at the embryonic but, unexpectedly, not at the adult stage. However, at the adult stage mRAD54 deficiency dramatically aggravates the ionizing radiation sensitivity of severe combined immune deficiency (scid) mice that are impaired in DNA double-strand break repair through DNA end-joining. In contrast, regardless of developmental stage, mRAD54(-/-) mice are hypersensitive to the interstrand DNA crosslinking compound mitomycin C. These results demonstrate that the two major DNA double-strand break repair pathways in mammals have overlapping as well as specialized roles, and that the relative contribution of these pathways towards repair of ionizing radiation-induced DNA damage changes during development of the animal.

Animals↗
PubMed2000

Disruption of mouse SNM1 causes increased sensitivity to the DNA interstrand cross-linking agent mitomycin C.

DNA interstrand cross-links (ICLs) represent lethal DNA damage, because they block transcription, replication, and segregation of DNA. Because of their genotoxicity, agents inducing ICLs are often used in antitumor therapy. The repair of ICLs is complex and involves proteins belonging to nucleotide excision, recombination, and translesion DNA repair pathways in Escherichia coli, Saccharomyces cerevisiae, and mammals. We cloned and analyzed mammalian homologs of the S. cerevisiae gene SNM1 (PSO2), which is specifically involved in ICL repair. Human Snm1, a nuclear protein, was ubiquitously expressed at a very low level. We generated mouse SNM1(-/-) embryonic stem cells and showed that these cells were sensitive to mitomycin C. In contrast to S. cerevisiae snm1 mutants, they were not significantly sensitive to other ICL agents, probably due to redundancy in mammalian ICL repair and the existence of other SNM1 homologs. The sensitivity to mitomycin C was complemented by transfection of the human SNM1 cDNA and by targeting of a genomic cDNA-murine SNM1 fusion construct to the disrupted locus. We also generated mice deficient for murine SNM1. They were viable and fertile and showed no major abnormalities. However, they were sensitive to mitomycin C. The ICL sensitivity of the mammalian SNM1 mutant suggests that SNM1 function and, by implication, ICL repair are at least partially conserved between S. cerevisiae and mammals.

Amino Acid Sequence↗
PubMed2000

The Rad51 paralog Rad51B promotes homologous recombinational repair.

The highly conserved Saccharomyces cerevisiae Rad51 protein plays a central role in both mitotic and meiotic homologous DNA recombination. Seven members of the Rad51 family have been identified in vertebrate cells, including Rad51, Dmc1, and five Rad51-related proteins referred to as Rad51 paralogs, which share 20 to 30% sequence identity with Rad51. In chicken B lymphocyte DT40 cells, we generated a mutant with RAD51B/RAD51L1, a member of the Rad51 family, knocked out. RAD51B(-/-) cells are viable, although spontaneous chromosomal aberrations kill about 20% of the cells in each cell cycle. Rad51B deficiency impairs homologous recombinational repair (HRR), as measured by targeted integration, sister chromatid exchange, and intragenic recombination at the immunoglobulin locus. RAD51B(-/-) cells are quite sensitive to the cross-linking agents cisplatin and mitomycin C and mildly sensitive to gamma-rays. The formation of damage-induced Rad51 nuclear foci is much reduced in RAD51B(-/-) cells, suggesting that Rad51B promotes the assembly of Rad51 nucleoprotein filaments during HRR. These findings show that Rad51B is important for repairing various types of DNA lesions and maintaining chromosome integrity.

Amino Acid Sequence↗
PubMed2000

ATP-dependent chromatin remodeling by the Cockayne syndrome B DNA repair-transcription-coupling factor.

The Cockayne syndrome B protein (CSB) is required for coupling DNA excision repair to transcription in a process known as transcription-coupled repair (TCR). Cockayne syndrome patients show UV sensitivity and severe neurodevelopmental abnormalities. CSB is a DNA-dependent ATPase of the SWI2/SNF2 family. SWI2/SNF2-like proteins are implicated in chromatin remodeling during transcription. Since chromatin structure also affects DNA repair efficiency, chromatin remodeling activities within repair are expected. Here we used purified recombinant CSB protein to investigate whether it can remodel chromatin in vitro. We show that binding of CSB to DNA results in an alteration of the DNA double-helix conformation. In addition, we find that CSB is able to remodel chromatin structure at the expense of ATP hydrolysis. Specifically, CSB can alter DNase I accessibility to reconstituted mononucleosome cores and disarrange an array of nucleosomes regularly spaced on plasmid DNA. In addition, we show that CSB interacts not only with double-stranded DNA but also directly with core histones. Finally, intact histone tails play an important role in CSB remodeling. CSB is the first repair protein found to play a direct role in modulating nucleosome structure. The relevance of this finding to the interplay between transcription and repair is discussed.

Adenosine Triphosphate↗
PubMed2000

Mouse RAD54 affects DNA double-strand break repair and sister chromatid exchange.

Cells can achieve error-free repair of DNA double-strand breaks (DSBs) by homologous recombination through gene conversion with or without crossover. In contrast, an alternative homology-dependent DSB repair pathway, single-strand annealing (SSA), results in deletions. In this study, we analyzed the effect of mRAD54, a gene involved in homologous recombination, on the repair of a site-specific I-SceI-induced DSB located in a repeated DNA sequence in the genome of mouse embryonic stem cells. We used six isogenic cell lines differing solely in the orientation of the repeats. The combination of the three recombination-test substrates used discriminated among SSA, intrachromatid gene conversion, and sister chromatid gene conversion. DSB repair was most efficient for the substrate that allowed recovery of SSA events. Gene conversion with crossover, indistinguishable from long tract gene conversion, preferentially involved the sister chromatid rather than the repeat on the same chromatid. Comparing DSB repair in mRAD54 wild-type and knockout cells revealed direct evidence for a role of mRAD54 in DSB repair. The substrate measuring SSA showed an increased efficiency of DSB repair in the absence of mRAD54. The substrate measuring sister chromatid gene conversion showed a decrease in gene conversion with and without crossover. Consistent with this observation, DNA damage-induced sister chromatid exchange was reduced in mRAD54-deficient cells. Our results suggest that mRAD54 promotes gene conversion with predominant use of the sister chromatid as the repair template at the expense of error-prone SSA.

Animals↗
PubMed1999

Single-molecule manipulation of double-stranded DNA using optical tweezers: interaction studies of DNA with RecA and YOYO-1.

By using optical tweezers and a specially designed flow cell with an integrated glass micropipette, we constructed a setup similar to that of Smith et al. (Science 271:795-799, 1996) in which an individual double-stranded DNA (dsDNA) molecule can be captured between two polystyrene beads. The first bead is immobilized by the optical tweezers and the second by the micropipette. Movement of the micropipette allows manipulation and stretching of the DNA molecule, and the force exerted on it can be monitored simultaneously with the optical tweezers. We used this setup to study elongation of dsDNA by RecA protein and YOYO-1 dye molecules. We found that the stability of the different DNA-ligand complexes and their binding kinetics were quite different. The length of the DNA molecule was extended by 45% when RecA protein was added. Interestingly, the speed of elongation was dependent on the external force applied to the DNA molecule. In experiments in which YOYO-1 was added, a 10-20% extension of the DNA molecule length was observed. Moreover, these experiments showed that a change in the applied external force results in a time-dependent structural change of the DNA-YOYO-1 complex, with a time constant of approximately 35 s (1/e2). Because the setup provides an oriented DNA molecule, we determined the orientation of the transition dipole moment of YOYO-1 within DNA by using fluorescence polarization. The angle of the transition dipole moment with respect to the helical axis of the DNA molecule was 69 degrees +/- 3.

Bacteriophage lambda↗
PubMed1999

Absence of interdomain contacts in the crystal structure of the RNA recognition motifs of Sex-lethal.

By binding specific RNA transcripts, the Sex-lethal protein (SXL) governs sexual differentiation and dosage compensation in Drosophila melanogaster. To investigate the basis for RNA binding specificity, we determined the crystal structure of the tandem RNA recognition motifs (RRMs) of SXL. Both RRMs adopt the canonical RRM fold, and the 10-residue, interdomain linker shows significant disorder. In contrast to the previously determined structure of the two-RRM fragment of heterogeneous nuclear ribonucleoprotein Al, SXL displays no interdomain contacts between RRMs. These results suggest that the SXL RRMs are flexibly tethered in solution, and RNA binding restricts the orientation of RRMs. Therefore, the observed specificity for single-stranded, U-rich sequences does not arise from a predefined, rigid architecture of the isolated SXL RRMs.

Animals↗
PubMed1999

Mouse Rad54 affects DNA conformation and DNA-damage-induced Rad51 foci formation.

Error-free repair by homologous recombination of DNA double-strand breaks induced by ionizing radiation (IR) requires the Rad52 group proteins, including Rad51 and Rad54, in the yeast Saccharomyces cerevisiae [1]. The formation of a 'joint' molecule between the damaged DNA and the homologous repair template is a key step in recombination mediated by Rad51 and stimulated by Rad54 [2] [3] [4] [5]. Mammalian homologs of Rad51 and Rad54 have been identified [2] [3] [6]. Here, we demonstrate that mouse Rad54 (mRad54) formed IR-induced nuclear foci that colocalized with mRad51. Interaction between mRad51 and mRad54 was induced by genotoxic stress, but only when lesions that required mRad54 for their repair were formed. Interestingly, mRad54 was essential for the formation of IR-induced mRad51 foci. Rad54 belongs to the SWI2/SNF2 protein family, members of which modulate protein-DNA interactions in an ATP-driven manner [7]. Results of a topological assay suggested that purified human Rad54 (hRad54) protein can unwind double-stranded (ds) DNA at the expense of ATP hydrolysis. Unwinding of the homologous repair template could promote the formation or stabilization of hRad51-mediated joint molecules. Rad54 appears to be required downstream of other Rad52 group proteins, such as Rad52 and the Rad55-Rad57 heterodimer, that assist Rad51 in interacting with the broken DNA [2] [3] [4].

Adenosine Triphosphate↗
PubMed1999

Rec8p, a meiotic recombination and sister chromatid cohesion phosphoprotein of the Rad21p family conserved from fission yeast to humans.

Our work and that of others defined mitosis-specific (Rad21 subfamily) and meiosis-specific (Rec8 subfamily) proteins involved in sister chromatid cohesion in several eukaryotes, including humans. Mutation of the fission yeast Schizosaccharomyces pombe rec8 gene was previously shown to confer a number of meiotic phenotypes, including strong reduction of recombination frequencies in the central region of chromosome III, absence of linear element polymerization, reduced pairing of homologous chromosomes, reduced sister chromatid cohesion, aberrant chromosome segregation, defects in spore formation, and reduced spore viability. Here we extend the description of recombination reduction to the central regions of chromosomes I and II. We show at the protein level that expression of rec8 is meiosis specific and that Rec8p localizes to approximately 100 foci per prophase nucleus. Rec8p was present in an unphosphorylated form early in meiotic prophase but was phosphorylated prior to meiosis I, as demonstrated by analysis of the mei4 mutant blocked before meiosis I. Evidence for the persistence of Rec8p beyond meiosis I was obtained by analysis of the mutant mes1 blocked before meiosis II. A human gene, which we designate hrec8, showed significant primary sequence similarity to rec8 and was mapped to chromosome 14. High mRNA expression of mouse and human rec8 genes was found only in germ line cells, specifically in testes and, interestingly, in spermatids. hrec8 was also expressed at a low level in the thymus. Sequence similarity and testis-specific expression indicate evolutionarily conserved functions of Rec8p in meiosis. Possible roles of Rec8p in the integration of different meiotic events are discussed.

Amino Acid Sequence↗