The histone-like protein HU binds specifically to DNA recombination and repair intermediates

The heterodimeric HU protein associated with the Escherichia coli nucleoid shares some properties with histones and HMG proteins. HU binds DNA junctions and DNA containing a nick much more avidly than double-stranded (ds-) DNA. Cells lacking HU are extremely sensitive to gamma irradiation and we wondered how HU could play a role in maintaining the integrity of the bacterial chromosome. We show that HU binds with high affinity to DNA repair and recombination intermediates, including DNA invasions, DNA overhangs and DNA forks. The DNA structural motif that HU specifically recognizes in all these structures consists of a ds-DNA module joined to a second module containing either ds- or single-stranded (ss-) DNA. The two modules rotate freely relative to one another. Binding specificity results from the simultaneous interaction of HU with these two modules: HU arms bind the ds-DNA module whereas the HU body contacts the 'variable' module containing either ds- or ss-DNA. Both structural motifs are recognized by HU at least 1000-fold more avidly than duplex DNA.     

HU and IHF, two homologous histone-like proteins of Escherichia coli, form different protein-DNA complexes with short DNA fragments.

Using the gel retardation technique we have studied the protein-DNA complexes formed between HU--the major histone-like protein of Escherichia coli--and short DNA fragments. We show that several HU heterodimers bind DNA in a regularly spaced fashion with each heterodimer occupying about 9 base pairs. The alpha 2 and beta 2 HU homodimers form the same structure as the alpha beta heterodimer on double stranded DNA. However when compared to the heterodimer, they bind single stranded DNA with higher affinity. We also show that HU and the Integration Host Factor of E. coli (IHF) form different structures with the same DNA fragments. Moreover, HU seems to enhance the DNA-binding capacity of IHF to a DNA fragment which does not contain its consensus sequence.     

Evolutionary origin of the protozoan parasites histone-like proteins (HU)

The histone-like proteins (HU) belong to a family of DNA architectural proteins that stabilize nucleoprotein complexes. We found a putative HU protein (TgGlmHMM_3045) in Toxoplasma gondii genome that was homologous to the bacterial HU protein. This putative sequence was located in the scaffold TGG_995361 of the chromosome 10. The sequence included the prokaryotic bacterial histone-like domain, KFGSLGlRRRGERVARNPRT (ID number PS00045). HU protein sequences were also found in Plasmodium falciparum, Neospora caninum, Theileria parva and Theileria annulata. We found that the homology of the putative HU protein in Apicomplexa was greater with bacterial histone-like proteins than with eukaryotic histone proteins. The phylogenetic tree indicated that the putative HU protein genes were acquired in Apicomplexa by means of a secondary endosymbiotic event from red algae and later they were transferred from the apicoplast organelle to the nuclear genome.     

Role of SIRT1 in homologous recombination

The class III histone deacetylase (HDAC) SIRT1 plays a role in the metabolism, aging, and carcinogenesis of organisms and regulates senescence and apoptosis in cells. Recent reports revealed that SIRT1 also deacetylates several DNA double-strand break (DSB) repair proteins. However, its exact functions in DNA repair remained elusive. Using nuclear foci analysis and fluorescence-based, chromosomal DSB repair reporter, we find that SIRT1 activity promotes homologous recombination (HR) in human cells. Importantly, this effect is unrelated to functions of poly(ADP-ribose) polymerase 1 (PARP1), another NAD(+)-catabolic protein, and does not correlate with cell cycle changes or apoptosis. Interestingly, we demonstrate that inactivation of Rad51 does not eliminate the effect of SIRT1 on HR. By epistasis-like analysis through knockdown and use of mutant cells of distinct SIRT1 target proteins, we show that the non-homologous end joining (NHEJ) factor Ku70 as well as the Nijmegen Breakage Syndrome protein (nibrin) are not needed for this SIRT1-mediated effect, even though a partial contribution of nibrin cannot be excluded. Strikingly however, the Werner helicase (WRN), which in its mutated form causes premature aging and cancer and which was linked to the Rad51-independent single-strand annealing (SSA) DSB repair pathway, is required for SIRT1-mediated HR. These results provide first evidence that links SIRT1's functions to HR with possible implications for genomic stability during aging and tumorigenesis.     

pAMβ1 resolvase has an atypical recombination site and requires a histone-like protein HU

The broad-host-range plasmid pAMβ1 from Gram-positive bacteria encodes a resolvase, designated Resβ, which shares homology with the proteins of the resolvase—invertase family. Here we report the purification and in vitro characterization of Resβ. This resolvase is particular in two aspects: it has an atypical binding site and requires a cofactor to promote resolution in vitro . Resβ binds to two regions within its resolution site res . One contains two inverted repeats (R1 and R2), the other contains only one repeat (R3). The cofactor required for resolution in vitro is present in crude extracts of both Bacillus subtilis and Escherichia coli and can be substituted by the E. coli histone-like protein HU. The possible mode of action of HU in the resolution process is discussed.     

Role of enzymes of homologous recombination in illegitimate plasmid recombination in Bacillus subtilis.

The structural stability of plasmid pGP1, which encodes a fusion between the penicillinase gene (penP) of Bacillus licheniformis and the Escherichia coli lacZ gene, was investigated in Bacillus subtilis strains expressing mutated subunits of the ATP-dependent nuclease, AddAB, and strains lacking the major recombination enzyme, RecA. Strains carrying a mutation in the ATP-binding site of the AddB subunit exhibited high levels of plasmid instability, whereas a comparable mutation in the A subunit did not affect plasmid stability. Using an alternative plasmid system, pGP100, we were able to demonstrate that the differences in stability reflected differences in initial recombination frequencies. Based on a comparison of endpoint sequences observed in the various hosts, we speculate that at least two different mechanisms underlie the deletion events involved, the first (type I) occurring between nonrepeated sequences, and the second (type II) occurring between short direct repeats (DRs). The latter event was independent of single-strand replication intermediates and the mode of replication and possibly requires the introduction of double-strand breaks (DSBs) between the repeats. In the absence of functional AddAB complex, or the AddB subunit, DSBs are likely to be processed via a recA-independent mechanism, resulting in intramolecular recombination between the DRs. In wild-type cells, such DSBs are supposed to be either repaired by a mechanism involving AddAB-dependent recombination or degraded by the AddAB-associated exonuclease activity. Plasmid stability assays in a recA mutant showed that (i) the level of deletion formation was considerably higher in this host and (ii) that deletions between short DRs occurred at higher frequencies than those described previously for the parental strain. We propose that in wild-type cells, the recA gene product is involved in recombinational repair of DSBs.     

rec genes and homologous recombination proteins in Escherichia coli

The twenty-five years since the first published report of recA mutants in Escherichia coli has seen the identification of more than 12 other recombination genes. The genes are usually grouped into three pathways named RecBCD, RecE and RecF for prominent genes which function in each. A proposal is made here that there are two RecF pathways, one sensitive and one resistant to exonuclease I, the SbcB enzyme. Five methods of grouping the genes functionally are discussed: 1) by enzyme activity, 2) by common indirect suppressor, 3) by common phenotype, 4) by common regulation and 5) by epistasis. Five classes of enzyme activities implicated in recombination are discussed according to their involvement in presynapsis, synapsis or postsynapsis: 1) nucleases 2) helicases 3) DNA-binding proteins 4) topoisomerases and 5) ligases. Plausible presynaptic steps for the RecBCD, RecF (SbcBS) and RecE pathways show the common feature of generating 3'-terminated single-stranded DNA (ssDNA). On this ssDNA it is proposed that a RecA protein filament is generated discontinuously. This implies the existence of nucleation and possibly measurement and 3' end protection proteins. Specific proposals are made for which recombination genes might encode such products. Finally the generality of the RecA-ssDNA-filament mechanism of synapsis in the cellular biological world is discussed.     

Evidence of a thermal unfolding dimeric intermediate for the Escherichia coli histone-like HU proteins: thermodynamics and structure

The Escherichia coli histone-like HU protein pool is composed of three dimeric forms: two homodimers, EcHUalpha(2) and EcHUbeta(2), and a heterodimer, EcHUalphabeta. The relative abundance of these dimeric forms varies during cell growth and in response to environmental changes, suggesting that each dimer plays different physiological roles. Here, differential scanning calorimetry and circular dichroism (CD) were used to study the thermal stability of the three E.coli HU dimers and show that each of them has its own thermodynamic signature. Unlike the other HU proteins studied so far, which melt through a single step (N(2)<-->2D), this present thermodynamic study shows that the three E.coli dimers melt according to a two-step mechanism (N(2)<-->I(2)<-->2D). The native dimer, N(2), melts partially into a dimeric intermediate, I(2), which in turn yields the unfolded monomers, D. In addition, the crystal structure of the EcHUalpha(2) dimer has been solved. Comparative thermodynamic and structural analysis between EcHUalpha(2) and the HU homodimer from Bacillus stearothermophilus suggests that the E.coli dimer is constituted by two subdomains of different energetic properties. The CD study indicates that the intermediate, I(2), corresponds to an HU dimer having partly lost its alpha-helices. The partially unfolded dimer I(2) is unable to complex with high-affinity, single-stranded break-containing DNA. These structural, thermodynamic and functional results suggest that the N(2)<-->I(2) equilibrium plays a central role in the physiology of E.coli HU. The I(2) molecular species seems to be the EcHUbeta(2) preferential conformation, possibly related to its role in the E.coli cold-shock adaptation. Besides, I(2) might be required in E.coli for the HU chain exchange, which allows the heterodimer formation from homodimers.     

The role of Deinococcus radiodurans RecFOR proteins in homologous recombination

Deinococcus radiodurans exhibits extraordinary resistance to the lethal effect of DNA-damaging agents, a characteristic attributed to its highly proficient DNA repair capacity. Although the D. radiodurans genome is clearly devoid of recBC and addAB counterparts as RecA mediators, the genome possesses all genes associated with the RecFOR pathway. In an effort to gain insights into the role of D. radiodurans RecFOR proteins in homologous recombination, we generated recF, recO and recR disruptant strains and characterized the disruption effects. All the disruptant strains exhibited delayed growth relative to the wild-type, indicating that the RecF, RecO and RecR proteins play an important role in cell growth under normal growth conditions. A slight reduction in transformation efficiency was observed in the recF and recO disruptant strains compared to the wild-type strain. Interestingly, disruption of recR resulted in severe reduction of the transformation efficiency. On the other hand, the recF disruptant strain was the most sensitive phenotype to γ rays, UV irradiation and mitomycin C among the three disruptants. In the recF disruptant strain, the intracellular level of the LexA1 protein did not decrease following γ irradiation, suggesting that a large amount of the RecA protein remains inactive despite being induced. These results demonstrate that the RecF protein plays a crucial role in the homologous recombination repair process by facilitating RecA activation in D. radiodurans. Thus, the RecF and RecR proteins are involved in the RecA activation and the stability of incoming DNA, respectively, during RecA-mediated homologous recombination processes that initiated the ESDSA pathway in D. radiodurans. Possible mechanisms that involve the RecFOR complex in homologous intermolecular recombination and homologous recombination repair processes are also discussed.     

Role of the ruvB gene in homologous and homeologous recombination in Rhizobium etli

The Rhizobium etli ruvA and ruvB genes were cloned through a PCR-based approach, using degenerate primers matching conserved sectors in the amino acid sequences of RuvB from eight bacterial species. Comparative analysis of the predicted polypeptides for RuvA and RuvB of R. etli showed highly conserved blocks with the corresponding homologs in other bacteria; RuvB depicts characteristic motifs for DNA helicases (ATP-binding and DEXH-box motifs). An R. etli ruvB::loxP Sp mutant was constructed by interposon mutagenesis. This mutant was highly sensitive to DNA-damaging agents, such as methyl methanesulfonate and nitrofurantoin, implying a deficiency in DNA repair. Homologous and homeologous conjugational recombination was reduced almost tenfold in the ruvB::loxP Sp mutant; a recombination defect was also observed in assays employing recombination between small plasmids, albeit at a smaller magnitude. Although the ruvA and ruvB genes are contiguous in R. etli, complementation studies suggest that they are expressed independently.     

Genomic homologous recombination in planta.

A system for monitoring intrachromosomal homologous recombination in whole plants is described. A multimer of cauliflower mosaic virus (CaMV) sequences, arranged such that CaMV could only be produced by recombination, was integrated into Brassica napus nuclear DNA. This set-up allowed scoring of recombination events by the appearance of viral symptoms. The repeated homologous regions were derived from two different strains of CaMV so that different recombinant viruses (i.e. different recombination events) could be distinguished. In most of the transgenic plants, a single major virus species was detected. About half of the transgenic plants contained viruses of the same type, suggesting a hotspot for recombination. The remainder of the plants contained viruses with cross-over sites distributed throughout the rest of the homologous sequence. Sequence analysis of two recombinant molecules suggest that mismatch repair is linked to the recombination process.     

Role of the Rep helicase gene in homologous recombination in Neisseria gonorrhoeae

In Escherichia coli, the Rep helicase has been implicated in replication fork progression, replication restart, homologous recombination, and DNA repair. We show that a Neisseria gonorrhoeae rep mutant is deficient in the homologous-recombination-mediated processes of DNA transformation and pilus-based colony variation but not in DNA repair.     

Intermolecular homologous recombination in plants.

To study DNA topological requirements for homologous recombination in plants, we have constructed pairs of plasmids that contain nonoverlapping deletions in the neomycin phosphotransferase gene [APH(3')II], which, when intact, confers kanamycin resistance to plant cells. Protoplasts isolated from Nicotiana tabacum were cotransformed with complementary pairs of plasmids containing these truncated gene constructs. Homologous recombination or gene conversion within the homologous sequences (6 to 405 base pairs) of the protein-coding region of the truncated genes led to the restoration of the functional APH(3')II gene, rendering these cells resistant to kanamycin. Circular plasmid DNAs recombined very inefficiently, independent of the length of the homologous region. A double-strand break in one molecule only slightly increased the recombination frequency. The most favorable substrates for recombination were linear molecules. In this case, the recombination frequency was positively correlated with the length of the homologous regions. The recombination frequency of plasmids linearized at sites proximal to the deletion-homology junction was significantly higher than when linearization was distal to the homologous region. Vector homology within cotransformed plasmid sequences also increased the recombination frequency.     

Role of the AtRad1p endonuclease in homologous recombination in plants

Using a specific recombination assay, we show in the plant Arabidopsis thaliana that AtRad1 protein plays a role in the removal of non-homologous tails in homologous recombination. Recombination in the presence of non-homologous overhangs is reduced 11-fold in the atrad1 mutant compared with the wild-type plants. AtRad1p is the A. thaliana homologue of the human Xpf and Saccharomyces cerevisiae Rad1 proteins. Rad1p is a subunit of the Rad1p/Rad10p structure-specific endonuclease that acts in nucleotide excision repair and inter-strand crosslink repair. This endonuclease also plays a role in mitotic recombination to remove non-homologous, 3′-ended overhangs from recombination intermediates. The Arabidopsis atrad1 mutant (uvh1), unlike rad1 mutants known from other eukaryotes, is hypersensitive to ionizing radiation. This last observation may indicate a more important role for the Rad1/Rad10 endonuclease in recombination in plants. This is the first direct demonstration of the involvement of AtRad1p in homologous recombination in plants.     

Purification and functional analysis of recombinant Acholeplasma laidlawii histone-like HU protein

HU is a most abundant DNA-binding protein in bacteria. This protein is conserved either in its heterodimeric form or in one of its homodimeric forms in all bacteria, in plant chloroplasts, and in some viruses. HU protein non-specifically binds and bends DNA as a hetero- or homodimer and can participate in DNA supercoiling and DNA condensation. It also takes part in some DNA functions such as replication, recombination, and repair. HU does not recognize any specific sequences but shows some specificity to cruciform DNA and to repair intermediates, e.g., nick, gap, bulge, 3′-overhang, etc. To understand the features of HU binding to DNA and repair intermediates, a fast and easy HU proteins purification procedure is required. Here we report overproduction and purification of the HU homodimers. The method of HU purification allows obtaining a pure recombinant non-tagged protein cloned in Escherichia coli. We applied this method for purification of Acholeplasma laidlawii HU and demonstrated that this protein possesses a DNA - binding activity and is free of contaminating nuclease activity. Besides that we have shown that expression of A. laidlawii ihf_hu gene in a slow-growing hupAB E. coli strain restores the wild-type growth indicating that aclHU can perform the basic functions of E. coli HU in vivo.     

Participation of the histone-like protein HU and of IHF in minichromosomal maintenance in Escherichia coli

The closely related Escherichia coli genes, hupA, hupB, himA and himD (hip), encode the bacterial histone-like protein subunits, HU-2, HU-1, IHF chi and IHF beta, respectively. We report here that E. coli minichromosomes [plasmids (2.7-12.2 kb) with oriC] carrying the intact mioC region were unable to transform mutants deficient in both HU and integration host factor (IHF), whereas they could transform mutants deficient in either HU or IHF as efficiently as the wild-type strain. Minichromosomes carrying a deletion of the proximal part of mioC or a DnaA box just upstream from mioC could not transform cells deficient in IHF, but could transform cells deficient in HU. These results suggested that HU and IHF participate in minichromosomal replication from oriC in E. coli.     

Exploring the pathways of homologous recombination.

There has been significant progress in elucidating the mechanisms by which meiotic and mitotic recombination occur. Double-strand breaks in particular have been the object of attention in studies on meiotic gene conversion, site-specific mitotic recombination, the repair of transposon excision and the transformation of cells with linearized DNA. A combination of genetic analysis and physical studies of molecular recombination intermediates have established that double-strand breaks can occur by two different mechanisms.     

A role for homologous recombination proteins in cell cycle regulation

Eukaryotic cells respond to DNA breaks, especially double-stranded breaks (DSBs), by activating the DNA damage response (DDR), which encompasses DNA repair and cell cycle checkpoint signaling. The DNA damage signal is transmitted to the checkpoint machinery by a network of specialized DNA damage-recognizing and signal-transducing molecules. However, recent evidence suggests that DNA repair proteins themselves may also directly contribute to the checkpoint control. Here, we investigated the role of homologous recombination (HR) proteins in normal cell cycle regulation in the absence of exogenous DNA damage. For this purpose, we used Chinese Hamster Ovary (CHO) cells expressing the Fluorescent ubiquitination-based cell cycle indicators (Fucci). Systematic siRNA-mediated knockdown of HR genes in these cells demonstrated that the lack of several of these factors alters cell cycle distribution, albeit differentially. The knock-down of MDC1, Rad51 and Brca1 caused the cells to arrest in the G2 phase, suggesting that they may be required for the G2/M transition. In contrast, inhibition of the other HR factors, including several Rad51 paralogs and Rad50, led to the arrest in the G1/G0 phase. Moreover, reduced expression of Rad51B, Rad51C, CtIP and Rad50 induced entry into a quiescent G0-like phase. In conclusion, the lack of many HR factors may lead to cell cycle checkpoint activation, even in the absence of exogenous DNA damage, indicating that these proteins may play an essential role both in DNA repair and checkpoint signaling.