The importance of repairing stalled replication forks

The bacterial SOS response to unusual levels of DNA damage has been recognized and studied for several decades. Pathways for re-establishing inactivated replication forks under normal growth conditions have received far less attention. In bacteria growing aerobically in the absence of SOS-inducing conditions, many replication forks encounter DNA damage, leading to inactivation. The pathways for fork reactivation involve the homologous recombination systems, are nonmutagenic, and integrate almost every aspect of DNA metabolism. On a frequency-of-use basis, these pathways represent the main function of bacterial DNA recombination systems, as well as the main function of a number of other enzymatic systems that are associated with replication and site-specific recombination.     

Spatial relationship of the Fis binding sites for Hin recombinational enhancer activity

Site-specific recombination reactions involve the joining or rearrangement of discrete DNA segments in a highly precise manner. A site-specific DNA inversion regulates the expression of flagellin genes in Salmonella by switching the orientation of a promoter. Analysis of the reaction has shown that, in addition to DNA sequences at the two boundaries of the 1-kilobase invertible segment where strand exchange occurs, another cis acting sequence is required for efficient inversion. This 60-base-pair enhancer-like sequence can function at many different locations and in either orientation in a plasmid substrate. It includes two binding sites for a host protein called Factor II or Fis (refs 4 and 5). Here we have investigated the importance of the spatial relationship between the two Fis binding sites for enhancer activity and have found that the correct helical positioning of the binding sites on the DNA is critical. However, this result could not be accounted for by effects on Fis binding. We propose a model for enhancer function in which the enhancer region acts to align the recombination sites into a specific conformation required for productive synapsis.     

Inversion in the H-2 complex of t-haplotypes in mice

Mouse t-haplotypes demonstrate strong linkage disequilibrium between t-lethal genes and specific H-2 types, presumably a result of recombination suppression between t and normal chromosomes. The observation of free recombination occurring between two complementary t-haplotypes suggested a chromosomal mismatch between t and normal chromosomes. Recent data showing the H-2 complex to be misplaced relative to two other markers, T and tf, in t-haplotypes suggested that chromosomal rearrangement in t-haplotypes might be the basis for their 'mismatch' with the normal chromosome. Here, to analyse the molecular nature of the rearrangement, we have cloned a polymorphic H-2 class I restriction fragment, which had previously been shown to map centromeric to the serologically defined H-2 complex in t-haplotypes. Genetic mapping studies show that this cloned t-DNA is homologous to the H-2 D region of wild-type chromosomes, and that the E alpha Ia gene maps telomeric to this DNA fragment in t-haplotypes, in contrast to its orientation in wild-type chromosomes. These results give molecular evidence for an inversion of H-2 in t-haplotypes, which may be at least partially responsible for recombination suppression and thus for linkage disequilibrium.     

Homology-dependent interactions in phage lambda site-specific recombination

General recombination shows a dependence on large regions of homology between the two participating segments of DNA. Many site-specific recombination systems also exhibit a dependence on homology, although in these systems the requirement is limited to a short region (less than 10 base pairs (bp]. We have used the in vitro phage lambda integration reaction to study the role of homology in this model site-specific recombination system. We find that certain non-homologous pairings which are strongly blocked for complete recombination, nevertheless make one pair of strand-exchanges to generate a joint molecule of the Holliday structure type. This result rules out recombination models in which the only homology-dependent step is synapsis (the juxtaposing of the two recombination sites). Our results also reveal a functional asymmetry in the recombination sites. We present models for bacteriophage lambda integrative recombination which accommodate these findings.     

Uncoupling of the recombination and topoisomerase activities of the gamma delta resolvase by a mutation at the crossover point

In several well-characterized site-specific recombination systems it has been shown that, for efficient recombination, the two recombining sites must have identical DNA sequences across the region between the staggered points of exchange. The precise DNA sequence of this overlap region, however, appears to be of little importance (with the exception of one position in the loxP site of bacteriophage P1 (ref. 6]. In this report we characterize a mutant recombination site for the site-specific recombination enzyme gamma delta resolvase (encoded by the gamma delta transposon), in which the dinucleotide at the crossover point is changed from AT to CT. Our results indicate that identity of the two overlap regions is not sufficient for recombination. Although resolvase binds normally to the mutant site and induces the structural deformation characteristic of the wild-type recombination site, catalysis at the crossover point (cutting and rejoining of DNA strands) is effectively limited to just one of the two strands, allowing resolvase to act as a topoisomerase but not as a recombinational enzyme.     

Recombination occurs during telomere formation in yeast

Short stretches of cloned telomeric sequences are necessary and sufficient for telomere formation in yeast as long as the sequences are present in the same orientation as they are found in vivo. During telomere formation, DNA termini usually undergo RAD52-independent recombination with other DNA termini as would be predicted by models of recombination-mediated telomere replication.     

The BRCA2 homologue Brh2 nucleates RAD51 filament formation at a dsDNA-ssDNA junction

The BRCA2 tumour suppressor is essential for the error-free repair of double-strand breaks (DSBs) in DNA by homologous recombination. This is mediated by RAD51, which forms a nucleoprotein filament with the 3' overhanging single-stranded DNA (ssDNA) of the resected DSB, searches for a homologous donor sequence, and catalyses strand exchange with the donor DNA. The 3,418-amino-acid BRCA2 contains eight approximately 30-amino-acid BRC repeats that bind RAD51 (refs 5, 6) and a approximately 700-amino-acid DBD domain that binds ssDNA. The isolated BRC and DBD domains have the opposing effects of inhibiting and stimulating recombination, respectively, and the role of BRCA2 in repair has been unclear. Here we show that a full-length BRCA2 homologue (Brh2) stimulates Rad51-mediated recombination at substoichiometric concentrations relative to Rad51. Brh2 recruits Rad51 to DNA and facilitates the nucleation of the filament, which is then elongated by the pool of free Rad51. Brh2 acts preferentially at a junction between double-stranded DNA (dsDNA) and ssDNA, with strict specificity for the 3' overhang polarity of a resected DSB. These results establish a BRCA2 function in RAD51-mediated DSB repair and explain the loss of this repair capacity in BRCA2-associated cancers.     

A hybrid recognition sequence in a recombinant restriction enzyme and the evolution of DNA sequence specificity

Early attempts to generate new restriction specificities by recombination between allelic restriction-modification systems have been unsuccessful. Bullas et al. succeeded in isolating a new specificity, SQ, in Salmonella that they interpreted as being the result of a recombination event between the parental strains, Salmonella typhimurium and S. postdam, which encode the SB and SP restriction systems, respectively. This interpretation has recently been confirmed by DNA heteroduplex studies with the SB, SP and SQ structural genes. We have determined the DNA sequences recognized by the SB and SP enzymes and found that, like all type I restriction sequences, they are split into two specific domains by a spacer of nonspecific sequence that, for both SB and SP, is 6 base pairs (bp) long. We have now determined the sequence recognized by the recombinant SQ enzyme and find that it is a hybrid between the SB and SP sequences, containing one specific domain from each parental strain. This result implies that each of the two specific domains is recognized by a physically distinct part of the enzyme.     

The structural basis of Holliday junction resolution by T7 endonuclease I

The four-way (Holliday) DNA junction is the central intermediate in homologous recombination, a ubiquitous process that is important in DNA repair and generation of genetic diversity. The penultimate stage of recombination requires resolution of the DNA junction into nicked-duplex species by the action of a junction-resolving enzyme, examples of which have been identified in a wide variety of organisms. These enzymes are nucleases that are highly selective for the structure of branched DNA. The mechanism of this selectivity has, however, been unclear in the absence of structural data. Here we present the crystal structure of the junction-resolving enzyme phage T7 endonuclease I in complex with a synthetic four-way DNA junction. Although the enzyme is structure-selective, significant induced fit occurs in the interaction, with changes in the structure of both the protein and the junction. The dimeric enzyme presents two binding channels that contact the backbones of the junction's helical arms over seven nucleotides. These interactions effectively measure the relative orientations and positions of the arms of the junction, thereby ensuring that binding is selective for branched DNA that can achieve this geometry.     

Minisatellite repeat coding as a digital approach to DNA typing

Most DNA typing systems used in forensic and legal medicine assay allelic length variation at tandem repetitive DNA regions such as minisatellites. A simple alternative approach that displays patterns of variant repeat units along minisatellite alleles is described here. This produces DNA profiles as extraordinarily variable digital sequences appropriate for forensic investigations, including computer databasing, and for analysing allele diversity and the role of recombination in minisatellite instability.     

The Srs2 helicase prevents recombination by disrupting Rad51 nucleoprotein filaments

Homologous recombination is a ubiquitous process with key functions in meiotic and vegetative cells for the repair of DNA breaks. It is initiated by the formation of single-stranded DNA on which recombination proteins bind to form a nucleoprotein filament that is active in searching for homology, in the formation of joint molecules and in the exchange of DNA strands. This process contributes to genome stability but it is also potentially dangerous to cells if intermediates are formed that cannot be processed normally and thus are toxic or generate genomic rearrangements. Cells must therefore have developed strategies to survey recombination and to prevent the occurrence of such deleterious events. In Saccharomyces cerevisiae, genetic data have shown that the Srs2 helicase negatively modulates recombination, and later experiments suggested that it reverses intermediate recombination structures. Here we show that DNA strand exchange mediated in vitro by Rad51 is inhibited by Srs2, and that Srs2 disrupts Rad51 filaments formed on single-stranded DNA. These data provide an explanation for the anti-recombinogenic role of Srs2 in vivo and highlight a previously unknown mechanism for recombination control.     

Rad54 protein promotes branch migration of Holliday junctions

Homologous recombination has a crucial function in the repair of DNA double-strand breaks and in faithful chromosome segregation. The mechanism of homologous recombination involves the search for homology and invasion of the ends of a broken DNA molecule into homologous duplex DNA to form a cross-stranded structure, a Holliday junction (HJ). A HJ is able to undergo branch migration along DNA, generating increasing or decreasing lengths of heteroduplex. In both prokaryotes and eukaryotes, the physical evidence for HJs, the key intermediate in homologous recombination, was provided by electron microscopy. In bacteria there are specialized enzymes that promote branch migration of HJs. However, in eukaryotes the identity of homologous recombination branch-migration protein(s) has remained elusive. Here we show that Rad54, a Swi2/Snf2 protein, binds HJ-like structures with high specificity and promotes their bidirectional branch migration in an ATPase-dependent manner. The activity seemed to be conserved in human and yeast Rad54 orthologues. In vitro, Rad54 has been shown to stimulate DNA pairing of Rad51, a key homologous recombination protein. However, genetic data indicate that Rad54 protein might also act at later stages of homologous recombination, after Rad51 (ref. 13). Novel DNA branch-migration activity is fully consistent with this late homologous recombination function of Rad54 protein.     

DNA helicase Srs2 disrupts the Rad51 presynaptic filament

Mutations in the Saccharomyces cerevisiae gene SRS2 result in the yeast's sensitivity to genotoxic agents, failure to recover or adapt from DNA damage checkpoint-mediated cell cycle arrest, slow growth, chromosome loss, and hyper-recombination. Furthermore, double mutant strains, with mutations in DNA helicase genes SRS2 and SGS1, show low viability that can be overcome by inactivating recombination, implying that untimely recombination is the cause of growth impairment. Here we clarify the role of SRS2 in recombination modulation by purifying its encoded product and examining its interactions with the Rad51 recombinase. Srs2 has a robust ATPase activity that is dependent on single-stranded DNA (ssDNA) and binds Rad51, but the addition of a catalytic quantity of Srs2 to Rad51-mediated recombination reactions causes severe inhibition of these reactions. We show that Srs2 acts by dislodging Rad51 from ssDNA. Thus, the attenuation of recombination efficiency by Srs2 stems primarily from its ability to dismantle the Rad51 presynaptic filament efficiently. Our findings have implications for the basis of Bloom's and Werner's syndromes, which are caused by mutations in DNA helicases and are characterized by increased frequencies of recombination and a predisposition to cancers and accelerated ageing.     

Human meiotic recombinase Dmc1 promotes ATP-dependent homologous DNA strand exchange

Homologous recombination is crucial for the repair of DNA breaks and maintenance of genome stability. In Escherichia coli, homologous recombination is dependent on the RecA protein. In the presence of ATP, RecA mediates the homologous DNA pairing and strand exchange reaction that links recombining DNA molecules. DNA joint formation is initiated through the nucleation of RecA onto single-stranded DNA (ssDNA) to form helical nucleoprotein filaments. Two RecA-like recombinases, Rad51 and Dmc1, exist in eukaryotes. Whereas Rad51 is needed for both mitotic and meiotic recombination events, the function of Dmc1 is restricted to meiosis. Here we examine human Dmc1 protein (hDmc1) for the ability to promote DNA strand exchange, and show that hDmc1 mediates strand exchange between paired DNA substrates over at least several thousand base pairs. DNA strand exchange requires ATP and is strongly dependent on the heterotrimeric ssDNA-binding molecule replication factor A (RPA). We present evidence that hDmc1-mediated DNA recombination initiates through the nucleation of hDmc1 onto ssDNA to form a helical nucleoprotein filament. The DNA strand exchange activity of hDmc1 is probably indispensable for repair of DNA double-strand breaks during meiosis and for maintaining the ploidy of meiotic chromosomes.     

Repetitive shuttling of a motor protein on DNA

Many helicases modulate recombination, an essential process that needs to be tightly controlled. Mutations in some human disease helicases cause increased recombination, genome instability and cancer. To elucidate the potential mode of action of these enzymes, here we developed a single-molecule fluorescence assay that can visualize DNA binding and translocation of Escherichia coli Rep, a superfamily 1 DNA helicase homologous to Saccharomyces cerevisiae Srs2. Individual Rep monomers were observed to move on single-stranded (ss)DNA in the 3' to 5' direction using ATP hydrolysis. Strikingly, on hitting a blockade, such as duplex DNA or streptavidin, the protein abruptly snapped back close to its initial position, followed by further cycles of translocation and snapback. This repetitive shuttling is likely to be caused by a blockade-induced protein conformational change that enhances DNA affinity for the protein's secondary DNA binding site, thereby resulting in a transient DNA loop. Repetitive shuttling was also observed on ssDNA bounded by a stalled replication fork and an Okazaki fragment analogue, and the presence of Rep delayed formation of a filament of recombination protein RecA on ssDNA. Thus, the binding of a single Rep monomer to a stalled replication fork can lead to repetitive shuttling along the single-stranded region, possibly keeping the DNA clear of toxic recombination intermediates.     

A protein binding to the J kappa recombination sequence of immunoglobulin genes contains a sequence related to the integrase motif

Site-specific recombination requires conserved DNA sequences specific to each system, and system-specific proteins that recognize specific DNA sequences. The site-specific recombinases seem to fall into at least two families, based on their protein structure and chemistry of strand breakage. One of these is the resolvase-invertase family, members of which seem to form a serine-phosphate linkage with DNA. Members of the other family, called the integrase family, contain a conserved tyrosine residue that forms a covalent linkage with the 3'-phosphate of DNA at the site of recombination. Structural comparison of integrases shows that these proteins share a highly conserved 40-residue motif. V-(D)-J recombination of the immunoglobulin gene requires conserved recombination signal sequences (RS) of a heptamer CACTGTG and a T-rich nonamer GGTTTTTGT, which are separated by a spacer sequence of either 12 or 23 bases We have recently purified, almost to homogeneity, a protein that specifically binds to the immunoglobulin J kappa RS containing the 23-base-pair spacer sequence. By synthesizing probes on the basis of partial amino-acid sequences of the purified protein, we have now isolated and characterized the complementary DNA of this protein. The amino-acid sequence deduced from the cDNA sequence reveals that the J kappa RS-binding protein has a sequence similar to the 40-residue motif of integrases of phages, bacteria and yeast, indicating that this protein could be involved in V-(D)-J recombination as a recombinase.     

A primitive Y chromosome in papaya marks incipient sex chromosome evolution

Many diverse systems for sex determination have evolved in plants and animals. One involves physically distinct (heteromorphic) sex chromosomes (X and Y, or Z and W) that are homozygous in one sex (usually female) and heterozygous in the other (usually male). Sex chromosome evolution is thought to involve suppression of recombination around the sex determination genes, rendering permanently heterozygous a chromosomal region that may then accumulate deleterious recessive mutations by Muller's ratchet, and fix deleterious mutations by hitchhiking as nearby favourable mutations are selected on the Y chromosome. Over time, these processes may cause the Y chromosome to degenerate and to diverge from the X chromosome over much of its length; for example, only 5% of the human Y chromosome still shows X-Y recombination. Here we show that papaya contains a primitive Y chromosome, with a male-specific region that accounts for only about 10% of the chromosome but has undergone severe recombination suppression and DNA sequence degeneration. This finding provides direct evidence for the origin of sex chromosomes from autosomes.     

DNA end resection, homologous recombination and DNA damage checkpoint activation require CDK1

A single double-strand break (DSB) induced by HO endonuclease triggers both repair by homologous recombination and activation of the Mec1-dependent DNA damage checkpoint in budding yeast. Here we report that DNA damage checkpoint activation by a DSB requires the cyclin-dependent kinase CDK1 (Cdc28) in budding yeast. CDK1 is also required for DSB-induced homologous recombination at any cell cycle stage. Inhibition of homologous recombination by using an analogue-sensitive CDK1 protein results in a compensatory increase in non-homologous end joining. CDK1 is required for efficient 5' to 3' resection of DSB ends and for the recruitment of both the single-stranded DNA-binding complex, RPA, and the Rad51 recombination protein. In contrast, Mre11 protein, part of the MRX complex, accumulates at unresected DSB ends. CDK1 is not required when the DNA damage checkpoint is initiated by lesions that are processed by nucleotide excision repair. Maintenance of the DSB-induced checkpoint requires continuing CDK1 activity that ensures continuing end resection. CDK1 is also important for a later step in homologous recombination, after strand invasion and before the initiation of new DNA synthesis.