Fanconi anemia: at the crossroads of DNA repair.

Fanconi anemia (FA) is an autosomal disorder that causes genome instability. FA patients suffer developmental abnormalities, early-onset bone marrow failure, and a predisposition to cancer. The disease is manifested by defects in DNA repair, hypersensitivity to DNA crosslinking agents, and a high degree of chromosomal aberrations. The FA pathway comprises 13 disease-causing genes involved in maintaining genomic stability. The fast pace of study of the novel DNA damage network has led to the constant discovery of new FA-like genes involved in the pathway that when mutated lead to similar disorders. A majority of the FA proteins act as signal transducers and scaffolding proteins to employ other pathways to repair DNA. This review discusses what is known about the FA proteins and other recently linked FA-like proteins. The goal is to clarify how the proteins work together to carry out interstrand crosslink repair and homologous recombination-mediated repair of damaged DNA.

Rad54 protein is targeted to pairing loci by the Rad51 nucleoprotein filament.

Rad51 and Rad54 proteins are important for the repair of double-stranded DNA (dsDNA) breaks by homologous recombination in eukaryotes. Rad51 assembles on single-stranded DNA (ssDNA) to form a helical nucleoprotein filament that performs homologous pairing with dsDNA; Rad54 stimulates this pairing substantially. Here, we demonstrate that Rad54 acts in concert with the mature Rad51-ssDNA filament. Enhancement of DNA pairing by Rad54 is greatest at an equimolar ratio relative to Rad51 within the filament. Reciprocally, the Rad51-ssDNA filament enhances both the dsDNA-dependent ATPase and the dsDNA unwinding activities of Rad54. We conclude that Rad54 participates in the DNA homology search as a component of the Rad51-nucleoprotein filament and that the filament delivers Rad54 to the dsDNA pairing locus, thereby linking the unwinding of potential target DNA with the homology search process.

Tailed duplex DNA is the preferred substrate for Rad51 protein-mediated homologous pairing.

The repair of potentially lethal DNA double-stranded breaks (DSBs) by homologous recombination requires processing of the broken DNA into a resected DNA duplex with a protruding 3'-single-stranded DNA (ssDNA) tail. Accordingly, the canonical models for DSB repair require invasion of an intact homologous DNA template by the 3'-end of the ssDNA, a characteristic that the bacterial pairing protein RecA possesses. Unexpectedly, we find that for the eukaryotic homolog, Rad51 protein, the 5'-end of ssDNA is more invasive than the 3'-end. This pairing bias is unaffected by Rad52, Rad54 or Rad55-57 proteins. However, further investigation reveals that, in contrast to RecA protein, the preferred DNA substrate for Rad51 protein is not ssDNA but rather dsDNA with ssDNA tails. This important distinction permits the Rad51 proteins to promote DNA strand invasion using either 3'- or 5'-ends with similar efficiency.

A novel property of the RecA nucleoprotein filament: activation of double- stranded DNA for strand exchange in trans.

RecA protein catalyzes DNA strand exchange, a basic step of homologous recombination. Upon binding to single-stranded DNA (ssDNA), RecA protein forms a helical nucleoprotein filament. Normally, this nucleoprotein filament binds double-stranded DNA (dsDNA) and promotes exchange of base pairs between this dsDNA and the homologous ssDNA that is contained within this filament. Here, we demonstrate that this bound dsDNA can be activated by interaction with a heterologous RecA nucleoprotein filament for a novel type of strand exchange with homologous ssDNA that is external to, and, therefore, not within, the filament. We refer to this novel DNA strand exchange as being in trans. Thus, the RecA nucleoprotein filament is a protein scaffold that activates dsDNA for strand exchange with ssDNA either within the filament or external to it. This new property demonstrates that the RecA nucleoprotein filament makes dsDNA receptive for DNA strand exchange, and it defines an early step of the homology recognition mechanism.

The function of the secondary DNA-binding site of RecA protein during DNA strand exchange.

RecA protein features two distinct DNA-binding sites. During DNA strand exchange, the primary site binds to single-stranded DNA (ssDNA), forming the helical RecA nucleoprotein filament. The weaker secondary site binds double-stranded DNA (dsDNA) during the homology search process. Here we demonstrate that this site has a second important function. It binds the ssDNA strand that is displaced from homologous duplex DNA during DNA strand exchange, stabilizing the initial heteroduplex DNA product. Although the high affinity of the secondary site for ssDNA is essential for DNA strand exchange, it renders DNA strand exchange sensitive to an excess of ssDNA which competes with dsDNA for binding. We further demonstrate that single-stranded DNA-binding protein can sequester ssDNA, preventing its binding to the secondary site and thereby assisting at two levels: it averts the inhibition caused by an excess of ssDNA and prevents the reversal of DNA strand exchange by removing the displaced strand from the secondary site.

The specificity of the secondary DNA binding site of RecA protein defines its role in DNA strand exchange.

The RecA protein-single-stranded DNA (ssDNA) filament can bind a second DNA molecule. Binding of ssDNA to this secondary site shows specificity, in that polypyrimidinic DNA binds to the RecA protein-ssDNA filament with higher affinity than polypurinic sequences. The affinity of ssDNA, which is identical in sequence to that bound in the primary site, is not always greater than that of nonhomologous DNA. Moreover, this specificity of DNA binding does not depend on the sequence of the DNA bound to the RecA protein primary site. We conclude that the specificity reflects an intrinsic property of the secondary site of RecA protein rather than an interaction between DNa molecules within nucleoprotein filament--i.e., self-recognition. The secondary DNA binding site displays a higher affinity for ssDNA than for double-stranded DNA, and the binding of ssDNA to the secondary site strongly inhibits DNA strand exchange. We suggest that the secondary binding site has a dual role in DNA strand exchange. During the homology search, it binds double-stranded DNA weakly; upon finding local homology, this site binds, with higher affinity, the ssDNA strand that is displaced during DNA strand exchange. These characteristics facilitate homologous pairing, promote stabilization of the newly formed heteroduplex DNA, and contribute to the directionality of DNA strand exchange.

Dimerization of plasmid DNA accelerates selection for antibiotic resistance.

Dimerization of multicopy plasmids is widely assumed to be disadvantageous both for plasmid maintenance and for the host cell. It is known that dimerization causes plasmid instability; dimer-containing cells grow slower than their monomer-containing counterparts. However, as we demonstrate here, under conditions of selective stress, dimers provide an advantage for bacteria. Dimers facilitate segregation of mutants from numerous copies of the parental plasmid. Accelerated segregation greatly increases the rate of accumulation of plasmids carrying mutations that are adaptive for bacteria. In contrast, resolution of dimers by site-specific recombination decreases, 10(3)-10(5)-fold, the efficiency of selection of spontaneous reversions in the tet gene of pBR327.

DIROM: an experimental design interactive system for directed mutagenesis and nucleic acids engineering.

A computer system DIROM for oligonucleotide-directed mutagenesis and artificial gene design has been designed for better experimental planning and control. DIROM permits searching for optimal oligonucleotides with respect to certain important parameters, namely sufficient energy of oligonucleotide-target hybridization, the secondary structure of oligonucleotide and target DNA, the presence of alternate binding sites in the target DNA and terminal G/C pairs. It can also be used to plan polymerase chain reaction experiments, for optimal primer selection, in sequencing, etc. DIROM enables one to search for both existing and potential restriction sites, to perform vector + target sequence construction. The system consists of a set of original algorithms that formalize the empirical knowledge of oligonucleotide action as primers.

["DIROM"--an interactive system for planning experiments on directed mutagenesis and design of artificial genes]. / "DIROM"--interaktivnaia sistema dlia planirovaniia éksperimentov po napravlennomu mutagenezu i konstruirovaniiu iskusstvennykh genov.

We present a computer system "DIROM" for oligonucleotide-directed mutagenesis and artificial gene design experiments planning and support. "DIROM" allows to search for optimal oligonucleotides according to such parameters as sufficient energy of oligonucleotide-target hybridization, secondary structure of oligonucleotide and target DNA, presence of alternative attachment sites in target DNA, terminal G/C pairs presence. Both single-stranded and double-stranded vector mutagenesis methods are implemented. It can be also used for optimal primer selection for polymerase chain reaction, sequencing etc. "DIROM" can search for both existent and potential carry out vector+target sequence construction. Both amino acid and nucleotide sequences can be operated.

Molecular mechanisms of deletion formation in Escherichia coli plasmids. I. Deletion formation mediated by long direct repeats.

Derivatives of plasmid pBR327 with the tet gene interrupted by 165 pb or 401 bp direct repeats were constructed. In cells harboring these plasmids, deletions which restored the wild-type tet gene gave rise to tetracycline-resistant colonies, thereby allowing a simple phenotypic test for deletion formation. The frequencies of deletions in these plasmids were measured in Escherichia coli strains proficient or deficient in general recombination. The structure of plasmid DNA isolated from tetracycline-resistant transformants was analyzed by agarose gel electrophoresis, restriction mapping and sequencing. The data presented here demonstrate that deletion formation is always associated with dimerization of plasmid DNA. Dimeric plasmids were of two types. Those which carried both a deletion and a compensating duplication were the major type in a Rec+ background and were rare in recA, recF, recJ and recO backgrounds. Dimers of the second type contained deletions, but no compensating duplications, and their formation was RecA-independent. The data presented demonstrate that deletion formation mediated by long direct repeats is mainly the result of unequal crossing-over between two plasmid molecules.

Mechanisms of deletion formation in Escherichia coli plasmids. II. Deletions mediated by short direct repeats.

A set of plasmids containing 42, 21 and 31 bp direct repeats was used to analyze the effect of repeat length on the frequencies of deletion formation and the structure of the deleted derivatives of different recombination-deficient Escherichia coli strains. Agarose gel electrophoresis of plasmid DNA demonstrated that the formation of deletions in these plasmids was associated with dimerization of plasmid DNA. Restriction analysis of the dimers showed that deletions at short direct repeats arose non-conservatively, that is, the formation of a deletion in one monomeric plasmid unit was not associated with a duplication in the other. Mutations in the recA, recF, recJ and recO genes had no marked effect on either the frequencies of deletion formation or the structure of dimers. In contrast, recB recC mutations greatly increased the frequencies of deletion formation, 6-fold for 42 bp, and 115-fold for 21 bp direct repeats. Conversion of DNA replication to the rolling circle mode in a recB recC strain, resulting in the formation of double-stranded ends, is suggested as the stimulatory effector.

The chemical mutagen dimethyl sulphate induces homologous recombination of plasmid DNA by increasing the binding of RecA protein to duplex DNA.

The role of different DNA damages in the stimulation of homologous recombination was studied by using an in vivo plasmid recombination assay. Dimethyl sulphate (DMS) treatment of plasmid DNA induced a 20-50-fold increase in the frequency of recombinational events. DMS treatment also stimulated RecA protein binding to double-stranded DNA. In contrast, plasmid DNA containing uracil, which, like DMS, is also subject to repair, was less effective in stimulation of recombination. The ability of purified RecA protein to bind DMS-treated or uracil-containing DNA was tested by measuring its ATPase activity. The result indicates that DMS treatment, but not uracil incorporation, stimulates RecA protein binding to DNA. We conclude, that the main reason (or the first step) for stimulation of recombination by mutagens is activation of RecA binding to damaged DNA.

[Effectiveness of chemical mutagenesis in multiple damage to one or both strands of plasmid DNA]. / Effektivnost' khimicheskogo mutageneza pri mnozhestvennykh povrezhdeniiakh odnoi i dvukh nitei plazmidnoi DNK.

Methods for site-directed multiple modification of DNA have been developed and used for modification of either one or two strands of plasmid DNA. Plasmid DNAs modified in the region of the tet gene were transformed into Escherichia coli cells and Tet colonies were screened. It was shown that multiple lesions in one DNA strand performed using either N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or sodium bisulfite were effectively repaired in the cell by error-free mechanism. In contrast, modification of two DNA strands led to induction of mutations. The efficiency of mutagenesis in the case of modification of a local region of one DNA strand with sodium bisulfite and modification of the other strand with MNNG was 1.1-7.9%. Mutations were analysed by restriction mapping and sequencing. All of them were G----A transitions.

Site-directed insertion of long single-stranded DNA fragments into plasmid DNA.

A new site-directed method for inserting long single-stranded DNA fragments into any region of a duplex vector is described. Its major advantage is independence of the location of the restriction sites. The method involves the assembly of single-stranded DNA fragments by ligation to both ends of the inserted fragments of two cohesive flanks that are complementary to the target region. Short oligonucleotide templates are used to direct the ligation. The resulting fragments, designated further as omega fragments with cohesive flanks, are hybridized with a gapped DNA vector. The heteroduplexes are transformed into Escherichia coli cells without enzymatic filling and sealing of gapped DNA. As a consequence of intracellular repair and heteroduplex resolution, insertion mutants are recovered. To demonstrate the method's efficiency, we inserted a 51-nucleotide synthetic DNA fragment containing a modified glucocorticoid receptor binding site into the region of pBR322, near the transcription starting point of the tet gene. The method we developed makes possible site-directed insertion of synthetic and genome-derived DNA fragments at least 200 nucleotides long.

New reagent for discrimination of single- and double-stranded regions in DNA.

It was found that DNA alkylation at the N-7 guanine with the bulky alkylating reagent, N,N,N'-tri-(beta-chloroethyl)-N'-(p-formylphenyl)propylene diamine-1,3 (TFP) is much diminished when DNA is double-stranded. We report here an application of this reaction for probing the hairpin structure in the palindrome-containing single-stranded (ss) DNA fragment of 377 bases prepared from the Eco-RI-BaMHI fragment of plasmid pBR322. 5'-Labeled ss fragment was modified with TFP and cleaved by piperidine hydrolysis at the alkylated guanine residues according to the Maxam-Gilbert procedure. Guanines in the hairpin formed by palindrome of 9 bp were protected from TFP action, while dimethyl sulfate modified all guanines.

[Introduction of new DNA sequences into previously selected regions of a plasmid genome by means of the formation of heteroduplexes]. / Vvedenie novykh posledovatel'nostei DNK v zaranee izbrannye uchatki genoma plazmidy putem obrazovaniia geterodupleksov.

A new method for obtaining the recombinant DNA based on heteroduplex-initiated site-directed insertion of alien nucleotide sequences is proposed. To generate a single-stranded region, plasmid DNA was nicked with restriction endonuclease in the presence of ethidium bromide with subsequent exonuclease III controlled digestion. The inserted DNA sequences flanked by nucleotide sequences complementary to single-stranded region were annealed with plasmid DNA and E. coli cells were transformed by the resulting heteroduplex molecules. The presented data show the possibility to insert as many as 200 nucleotides. The yield of recombinant DNA varied from 16 to 0.7% as the number of nucleotides inserted correspondingly varied from 15 to 200. The site of insertion does not depend crucially on the localization of the restriction site used.

[Mutations predetermined by the primary structure of DNA]. / Mutatsii, predeterminirovannye pervichnoi strukturoi DNK.

This study is concerned with an experimental verification of hypotheses postulating the involvement of self-complementary nucleotide sequences in the formation of deletions and insertions. It was suggested that deletions can arise in the regions of self-complementary nucleotide sequences, which allows the formation of the hairpin structures in a single-stranded DNA, arising during excision repair. These hairpin structures can be eliminated by nucleases or during DNA replication. Insertions can arise as a result of homologous recombination, when a migrating DNA strand contains a self-complementary sequence which forms hairpin structure. Model experiments were carried out with the pBR322 plasmid. A plasmid DNA with premutational damage in the palindrome-containing region was constructed by in vitro dimethylsulfate modification of one strand of EcoRI-BamHI restriction fragment. The plasmid was used for transformation of Escherichia coli. Restriction mapping and nucleotide analysis of the mutant DNAs demonstrated that they all contained deletions. The end points of the deletions coincide with the palindrome. To model homologous recombination, a plasmid with D-loop was constructed. A single-stranded DNA fragment containing palindrome forming a hairpin structure was introduced into the plasmid DNA and covalently fixed in the complex. When E. coli cells were transfected with this DNA, plasmid mutants containing insertions predetermined by palindromic structure arose. The evolutionary role of mutations predetermined by primary DNA structure is discussed.

[A new method of studying DNA conformation by chemical modification]. / Novyi metod izucheniia konformatsii DNK s pomoshch'iu khimicheskoi modifikatsii.

A new method of discrimination of double-stranded (ds) and single-stranded (ss) regions in DNA molecules has been developed. It makes use of two alkylating reagents, a voluminous and a small-sized, the former being sensitive to the DNA conformation. A bulky reagent, N,N,N'-tri(beta-chloroethyl)-N'-(p-formylphenyl) propylendiamine-1,3 (TFP), was used to detect the hairpin structure in the palindrome-containing DNA fragment 373 nucleotides long prepared from the ds EcoRI-BamHI fragment of the plasmid pBR322. The fragment was modified by TFP and cleaved by piperidine at the alkylated guanine residues according to the Maxam-Gilbert procedure. Guanine residues in the hairpin formed by palindrome were protected from the TFP action, while dimethylsulfate modified all guanines. Application of the method for the identification of loops, stem-and-loop structures, and unwinded regions of DNA is discussed.