Conserved domains in DNA repair proteins and evolution of repair systems.

A detailed analysis of protein domains involved in DNA repair was performed by comparing the sequences of the repair proteins from two well-studied model organisms, the bacterium Escherichia coli and yeast Saccharomyces cerevisiae, to the entire sets of protein sequences encoded in completely sequenced genomes of bacteria, archaea and eukaryotes. Previously uncharacterized conserved domains involved in repair were identified, namely four families of nucleases and a family of eukaryotic repair proteins related to the proliferating cell nuclear antigen. In addition, a number of previously undetected occurrences of known conserved domains were detected; for example, a modified helix-hairpin-helix nucleic acid-binding domain in archaeal and eukaryotic RecA homologs. There is a limited repertoire of conserved domains, primarily ATPases and nucleases, nucleic acid-binding domains and adaptor (protein-protein interaction) domains that comprise the repair machinery in all cells, but very few of the repair proteins are represented by orthologs with conserved domain architecture across the three superkingdoms of life. Both the external environment of an organism and the internal environment of the cell, such as the chromatin superstructure in eukaryotes, seem to have a profound effect on the layout of the repair systems. Another factor that apparently has made a major contribution to the composition of the repair machinery is horizontal gene transfer, particularly the invasion of eukaryotic genomes by organellar genes, but also a number of likely transfer events between bacteria and archaea. Several additional general trends in the evolution of repair proteins were noticed; in particular, multiple, independent fusions of helicase and nuclease domains, and independent inactivation of enzymatic domains that apparently retain adaptor or regulatory functions.     

Conserved and nonconserved structures in the secretory proteins encoded in the Balbiani ring genes ofChironomus tentans

Summary The large, repetitive Balbiani ring (BR) genes, BR 1, 2, and 6, inChironomus tentans originated from a short ancestral sequence and have all evolved according to analogous amplification schemes. We analyzed the structures of the BR-encoded secretory proteins and defined the parts that have been conserved during the evolutionary process. The BR products show striking similarities, with the BR 1 and BR 2 products being more similar to each other than to the BR 6 product. In the constant (C) region of the repeat units, 7 of the 30 amino acid residues are strictly conserved; 4 of these are the cysteine residues. The subrepeat (SR) regions of all the BR products are dominated by repeated tripeptide elements rich in proline and charged amino acid residues. Most of the amino acid replacements in both regions are conservative. Secondary structure predictions suggested that the C regions of the BR 1 and BR2 products have several elements of secondary structure: an -helix, a -strand, and one or two reverse turns, as in globular structures. The prediction for the C region of the BR 6 product is similar but lacks a -strand. The predictions for the intervening SR regions appear less conclusive, but are clearly different from those for the C regions, and suggest regular structures not differing in their conformational elements. The SR regions evolved from an ancestor sequence similar to the C region; thus, the BR products seem to represent an example of evolution from one structure to two differently folded products. It is proposed that the alignment and polymerization of the long BR proteins could be promoted by the repetitive structure of the molecules, due to the possibility of forming disulfide bridges between half-cystine residues and electrostatic interactions between the charged residues of the SR regions. The divergence among the BR products is discussed in relation to possible functional differences among the members of the BR gene family.     

Separable roles for Exonuclease I in meiotic DNA double-strand break repair

Exo1 is a member of the Rad2 protein family and possesses both 5'-3' exonuclease and 5' flap endonuclease activities. In addition to performing a variety of functions during mitotic growth, Exo1 is also important for the production of crossovers during meiosis. However, its precise molecular role has remained ambiguous and several models have been proposed to account for the crossover deficit observed in its absence. Here, we present physical evidence that the nuclease activity of Exo1 is essential for normal 5'-3' resection at the Spo11-dependent HIS4 hotspot in otherwise wild-type cells. This same activity was also required for normal levels of gene conversion at the locus. However, gene conversions were frequently observed at a distance beyond that at which resection was readily detectable arguing that it is not the extent of the initial DNA end resection that limits heteroduplex formation. In addition to these nuclease-dependent functions, we found that an exo1-D173A mutant defective in nuclease activity is able to maintain crossing-over at wild-type levels in a number of genetic intervals, suggesting that Exo1 also plays a nuclease-independent role in crossover promotion.     

DNA strained breakage repair in ataxia telangiectasia fibroblast-like cells.

Human diploid fibroblast-like cells derived from four patients with the genetic disease ataxia telangiectasia and from two non-mutant donors were examined for the repair of X-ray induced strand breaks in DNA. The ataxia telangiectasia cultures showed no significant differences from the non-mutant cultures in the kinetics and extent of strand repair. This suggests that the increased spontaneous and X-ray induced chromatid aberrations observed in ataxia telangiectasia cells are not caused by a defect in the repair of single strand breaks as might be suspected from a general model of aberration production.     

Excess single-stranded DNA inhibits meiotic double-strand break repair

During meiosis, self-inflicted DNA double-strand breaks (DSBs) are created by the protein Spo11 and repaired by homologous recombination leading to gene conversions and crossovers. Crossover formation is vital for the segregation of homologous chromosomes during the first meiotic division and requires the RecA orthologue, Dmc1.We analyzed repair during meiosis of site-specific DSBs created by another nuclease, VMA1-derived endonuclease (VDE), in cells lacking Dmc1 strand-exchange protein. Turnover and resection of the VDE-DSBs was assessed in two different reporter cassettes that can repair using flanking direct repeat sequences, thereby obviating the need for a Dmc1-dependent DNA strand invasion step. Access of the single-strand binding complex replication protein A, which is normally used in all modes of DSB repair, was checked in chromatin immunoprecipitation experiments, using antibody against Rfa1. Repair of the VDE-DSBs was severely inhibited in dmc1Δ cells, a defect that was associated with a reduction in the long tract resection required to initiate single-strand annealing between the flanking repeat sequences. Mutants that either reduce Spo11-DSB formation or abolish resection at Spo11-DSBs rescued the repair block. We also found that a replication protein A component, Rfa1, does not accumulate to expected levels at unrepaired single-stranded DNA (ssDNA) in dmc1Δ cells. The requirement of Dmc1 for VDE-DSB repair using flanking repeats appears to be caused by the accumulation of large quantities of ssDNA that accumulate at Spo11-DSBs when Dmc1 is absent. We propose that these resected DSBs sequester both resection machinery and ssDNA binding proteins, which in wild-type cells would normally be recycled as Spo11-DSBs repair. The implication is that repair proteins are in limited supply, and this could reflect an underlying mechanism for regulating DSB repair in wild-type cells, providing protection from potentially harmful effects of overabundant repair proteins.     

A technique for the measurement of breakage and repair of DNA alkylated in vivo.

The alkaline elution technique has been adapted for use in the assessment of DNA damage induced in the livers and lungs of mice after administration of an alkylating agent, methylemthanesulfonat (MMS). At 4 h after administration of MMS, damage ot DNA was readily demonstrable; the damage was repaired in liver by 24 h. The lung, particularly of the A/J mouse, exhibited an increased alkaline elution rate when compared to C57BL/6J, and repair was not entirely complete (as judged from the rate of alkaline elution of DNA) by 24 h. The rate of elution was dependent upon temperature. It is believed that this adaptation should have great utility in examining DNA repair in vivo.     

Molecular-genetic analysis of dual-stranded DNA break repair in saccharomyces yeasts

DNA double-strand breaks (DSB) are the most dangerous damage to genetic material caused by ionizing radiation and some chemical agents. Nonrestored DSB lead to chromosomal rearrangements, genetic instability, and cell death. On the other hand, DSB normally occur in cells in the course of normal gene functioning. DSB repair not only protects cells from adverse consequences and maintains stability of genetic material but is directly involved in the most important processes of cell life, such as meiosis and humoral immunity in vertebrates. The diverse mechanisms of homologous and nonhomologous recombination underlie DSB repair. In this respect, yeast are the best-studied object. In this review, genetic control and molecular models of the recombination DNA DSB repair in Saccharomyces cerevisiae are considered. Evidence has accumulated that indicates the higher eukaryotes retained the basic set of the repair pathways characteristic of bacteria and lower eukaryotes. However, different repair mechanisms predominate in yeast as compared to higher eukaryotes. Therefore, the results obtained in yeast experiments may be applicable to higher eukaryotes.     

Drosophila brca2 is required for mitotic and meiotic DNA repair and efficient activation of the meiotic recombination checkpoint

Heterozygous mutations in the tumor suppressor BRCA2 confer a high risk of breast and other cancers in humans. BRCA2 maintains genome stability in part through the regulation of Rad51-dependent homologous recombination. Much about its precise function in the DNA damage responses is, however, not yet known. We have made null mutations in the Drosophila homolog of BRCA2 and measured the levels of homologous recombination, non-homologous end-joining, and single-strand annealing in the pre-meiotic germline of Drosophila males. We show that repair by homologous recombination is dramatically decreased in Drosophila brca2 mutants. Instead, large flanking deletions are formed, and repair by the non-conservative single-strand annealing pathway predominates. We further show that during meiosis, Drosophila Brca2 has a dual role in the repair of meiotic double-stranded breaks and the efficient activation of the meiotic recombination checkpoint. The eggshell patterning defects that result from activation of the meiotic recombination checkpoint in other meiotic DNA repair mutants can be strongly suppressed by mutations in brca2. In addition, Brca2 co-immunoprecipitates with the checkpoint protein Rad9, suggesting a direct role for Brca2 in the transduction of the meiotic recombination checkpoint signal.     

Hijacked DNA repair proteins and unchained DNA polymerases

Somatic hypermutation of immunoglobulin (Ig) genes occurs at a frequency that is a million times greater than the mutation in other genes. Mutations occur in variable genes to increase antibody affinity, and in switch regions before constant genes to cause switching from IgM to IgG. Hypermutation is initiated in activated B cells when the activation-induced deaminase protein deaminates cytosine in DNA to uracil. Uracils can be processed by either a mutagenic pathway to produce mutations or a non-mutagenic pathway to remove mutations. In the mutagenic pathway, we first studied the role of mismatch repair proteins, MSH2, MSH3, MSH6, PMS2 and MLH1, since they would recognize mismatches. The MSH2–MSH6 heterodimer is involved in hypermutation by binding to U:G and other mismatches generated during repair synthesis, but the other proteins are not necessary. Second, we analysed the role of low-fidelity DNA polymerases η, ι and θ in synthesizing mutations, and conclude that polymerase η is the dominant participant by generating mutations at A:T base pairs. In the non-mutagenic pathway, we examined the role of the Cockayne syndrome B protein that interacts with other repair proteins. Mice deficient in this protein had normal hypermutation and class switch recombination, showing that it is not involved.     

MAD2 interacts with DNA repair proteins and negatively regulates DNA damage repair

MAD2 (mitotic arrest deficient 2) is a key regulator of mitosis. Recently, it had been suggested that MAD2-induced mitotic arrest mediates DNA damage response and that upregulation of MAD2 confers sensitivity to DNA-damaging anticancer drug-induced apoptosis. In this study, we report a potential novel role of MAD2 in mediating DNA nucleotide excision repair through physical interactions with two DNA repair proteins, XPD (xeroderma pigmentosum complementation group D) and ERCC1. First, overexpression of MAD2 resulted in decreased nuclear accumulation of XPD, a crucial step in the initiation of DNA repair. Second, immunoprecipitation experiments showed that MAD2 was able to bind to XPD, which led to competitive suppression of binding activity between XPD and XPA, resulting in the prevention of physical interactions between DNA repair proteins. Third, unlike its role in mitosis, the N-terminus domain seemed to be more important in the binding activity between MAD2 and XPD. Fourth, phosphorylation of H2AX, a process that is important for recruitment of DNA repair factors to DNA double-strand breaks, was suppressed in MAD2-overexpressing cells in response to DNA damage. These results suggest a negative role of MAD2 in DNA damage response, which may be accounted for its previously reported role in promoting sensitivity to DNA-damaging agents in cancer cells. However, the interaction between MAD2 and ERCC1 did not show any effect on the binding activity between ERCC1 and XPA in the presence or absence of DNA damage. Our results suggest a novel function of MAD2 by interfering with DNA repair proteins.     

Temporal analysis of meiotic DNA double-strand break formation and repair in Drosophila females

Using an antibody against the phosphorylated form of His2Av (γ-His2Av), we have described the time course for the series of events leading from the formation of a double-strand break (DSB) to a crossover in Drosophila female meiotic prophase. MEI-P22 is required for DSB formation and localizes to chromosomes prior to γ-His2Av foci. Drosophila females, however, are among the group of organisms where synaptonemal complex (SC) formation is not dependent on DSBs. In the absence of two SC proteins, C(3)G and C(2)M, the number of DSBs in oocytes is significantly reduced. This is consistent with the appearance of SC protein staining prior to γ-His2Av foci. However, SC formation is incomplete or absent in the neighboring nurse cells, and γ-His2Av foci appear with the same kinetics as in oocytes and do not depend on SC proteins. Thus, competence for DSB formation in nurse cells occurs with a specific timing that is independent of the SC, whereas in the oocytes, some SC proteins may have a regulatory role to counteract the effects of a negative regulator of DSB formation. The SC is not sufficient for DSB formation, however, since DSBs were absent from the heterochromatin even though SC formation occurs in these regions. All γ-His2Av foci disappear before the end of prophase, presumably as repair is completed and crossovers are formed. However, oocytes in early prophase exhibit a slower response to X-ray–induced DSBs compared to those in the late pachytene stage. Assuming all DSBs appear as γ-His2Av foci, there is at least a 3:1 ratio of noncrossover to crossover products. From a comparison of the frequency of γ-His2Av foci and crossovers, it appears that Drosophila females have only a weak mechanism to ensure a crossover in the presence of a low number of DSBs.     

Pre-ribosomal RNA reorganizes DNA damage repair factors in nucleus during meiotic prophase and DNA damage response

In response to DNA double-strand breaks(DSBs),DNA damage repair factors are recruited to DNA lesions and form nuclear foci.However,the underlying molecular mech...     

Impaired DNA double strand break repair in cells from Nijmegen breakage syndrome patients

Nijmegen breakage syndrome, caused by mutations in the NBS1 gene, is an autosomal recessive chromosomal instability disorder characterized by cancer predisposition. Cells isolated from Nijmegen breakage syndrome patients display increased levels of spontaneous chromosome aberrations and sensitivity to ionizing radiation. Here, we have investigated DNA double strand break repair pathways of homologous recombination, including single strand annealing, and non-homologous end-joining in Nijmegen breakage syndrome patient cells. We used recently developed GFP-YFP-based plasmid substrates to measure the efficiency of DNA double strand break repair. Both single strand annealing and non-homologous end-joining processes were markedly impaired in NBS1-deficient cells, and repair proficiency was restored upon re-introduction of full length NBS1 cDNA. Despite the observed defects in the repair efficiency, no apparent differences in homologous recombination or non-homologous end-joining effector proteins RAD51, KU70, KU86, or DNA-PK(CS) were observed. Furthermore, comparative analysis of junction sequences of plasmids recovered from NBS1-deficient and NBS1-complemented cells revealed increased dependence on microhomology-mediated end-joining DNA repair process in NBS1-complemented cells.     

Structural bases for substrate recognition and repair system of base-excision DNA repair proteins.

The model structure of Escherichia coli AlkA (3-methyladenine-DNA glycosylase II) protein complexed with the double helical DNA is elucidated from X-ray structures of related DNA glycosylase enzymes and mutagenic studies. The free enzyme structure has no difficulty in building the platform to afford the bended and wedge DNA with the flipped out nucleotide. The helix-hairpin-helix motif and the insertion residue L125 in free structure can be located without severe contacts. The alkylated base is surrounded with a variety of aromatic rings, such as W218, W272, Y273 and F18. The aromatic indole ring of tryptophan is a good candidate for forming the stacking with the positively charged base moiety pi-cation interaction). Some hydrophobic residues, such as V128 and L240, also attend to substrate recognition.     

Atmnd1-delta1 is sensitive to gamma-irradiation and defective in meiotic DNA repair

The efficient repair of double-strand breaks (DSBs) in genomic DNA is crucial for the survival of all organisms. Mnd1 is suggested to promote the strand invasion step during meiotic recombination. We used a forward genetics approach, through the search for mutants, to characterize the Arabidopsis homologue of Mnd1. Atmnd1 null mutants exhibit normal vegetative and flower development. In contrast, during prophase I, chromosomes become fragmented resulting in random distribution of the fragments between polyads. Male and female meiosis are defective and strong accumulation of AtRAD51 was observed in atmnd1-delta1 nuclei. These results suggest that similarly to its yeast and animal homologues, AtMnd1 plays a role in DSB repair during meiosis. In addition, like its human homologue AtMnd1 is expressed in somatic cells. AtMnd1 expression is strongly induced by gamma-rays and null mutants show increased sensibility to gamma-rays. Taken together, these results suggest that AtMnd1 is also required for DSB repair in somatic cells.     

Conserved interactions of the splicing factor Ntr1/Spp382 with proteins involved in DNA double-strand break repair and telomere metabolism

The ligation of DNA double-strand breaks in the process of non-homologous end-joining (NHEJ) is accomplished by a heterodimeric enzyme complex consisting of DNA ligase IV and an associated non-catalytic factor. This DNA ligase also accounts for the fatal joining of unprotected telomere ends. Hence, its activity must be tightly controlled. Here, we describe interactions of the DNA ligase IV-associated proteins Lif1p and XRCC4 of yeast and human with the putatively orthologous G-patch proteins Ntr1p/Spp382p and NTR1/TFIP11 that have recently been implicated in mRNA splicing. These conserved interactions occupy the DNA ligase IV-binding sites of Lif1p and XRCC4, thus preventing the formation of an active enzyme complex. Consistently, an excess of Ntr1p in yeast reduces NHEJ efficiency in a plasmid ligation assay as well as in a chromosomal double-strand break repair (DSBR) assay. Both yeast and human NTR1 also interact with PinX1, another G-patch protein that has dual functions in the regulation of telomerase activity and telomere stability, and in RNA processing. Like PinX1, NTR1 localizes to telomeres and associates with nucleoli in yeast and human cells, suggesting a function in localized control of DSBR.     

Rec25 and Rec27, novel linear-element components, link cohesin to meiotic DNA breakage and recombination

Meiosis is a specialized nuclear division by which sexually reproducing diploid organisms generate haploid gametes. Recombination between homologous chromosomes facilitates accurate meiotic chromosome segregation and is initiated by DNA double-strand breaks (DSBs) made by the conserved topoisomerase-like protein Spo11 (Rec12 in fission yeast), but DSBs are not evenly distributed across the genome. In Schizosaccharomyces pombe, proteinaceous structures known as linear elements (LinEs) are formed during meiotic prophase. The meiosis-specific cohesin subunits Rec8 and Rec11 are essential for DSB formation in some regions of the genome, as well as for formation of LinEs or the related synaptonemal complex (SC) in other eukaryotes. Proteins required for DSB formation decorate LinEs, and mutants lacking Rec10, a major component of LinEs, are completely defective for recombination. Although recombination may occur in the context of LinEs, it is not well understood how Rec10 is loaded onto chromosomes. We describe two novel components of LinEs in fission yeast, Rec25 and Rec27. Comparisons of rec25Delta, rec27Delta, and rec10Delta mutants suggest multiple pathways to load Rec10. In the major pathway, Rec10 is loaded, together with Rec25 and Rec27, in a Rec8-dependent manner with subsequent region-specific effects on recombination.