Neil2-null Mice Accumulate Oxidized DNA Bases in the Transcriptionally Active Sequences of the Genome and Are Susceptible to Innate Inflammation.

Why mammalian cells possess multiple DNA glycosylases (DGs) with overlapping substrate ranges for repairing oxidatively damaged bases via the base excision repair (BER) pathway is a long-standing question. To determine the biological role of these DGs, null animal models have been generated. Here, we report the generation and characterization of mice lacking Neil2 (Nei-like 2). As in mice deficient in each of the other four oxidized base-specific DGs (OGG1, NTH1, NEIL1, and NEIL3), Neil2-null mice show no overt phenotype. However, middle-aged to old Neil2-null mice show the accumulation of oxidative genomic damage, mostly in the transcribed regions. Immuno-pulldown analysis from wild-type (WT) mouse tissue showed the association of NEIL2 with RNA polymerase II, along with Cockayne syndrome group B protein, TFIIH, and other BER proteins. Chromatin immunoprecipitation analysis from mouse tissue showed co-occupancy of NEIL2 and RNA polymerase II only on the transcribed genes, consistent with our earlier in vitro findings on NEIL2's role in transcription-coupled BER. This study provides the first in vivo evidence of genomic region-specific repair in mammals. Furthermore, telomere loss and genomic instability were observed at a higher frequency in embryonic fibroblasts from Neil2-null mice than from the WT. Moreover, Neil2-null mice are much more responsive to inflammatory agents than WT mice. Taken together, our results underscore the importance of NEIL2 in protecting mammals from the development of various pathologies that are linked to genomic instability and/or inflammation. NEIL2 is thus likely to play an important role in long term genomic maintenance, particularly in long-lived mammals such as humans.

Evidence for two different regulatory mechanisms linking replication and segregation of vibrio cholerae chromosome II.

Understanding the mechanisms that coordinate replication initiation with subsequent segregation of chromosomes is an important biological problem. Here we report two replication-control mechanisms mediated by a chromosome segregation protein, ParB2, encoded by chromosome II of the model multichromosome bacterium, Vibrio cholerae. We find by the ChIP-chip assay that ParB2, a centromere binding protein, spreads beyond the centromere and covers a replication inhibitory site (a 39-mer). Unexpectedly, without nucleation at the centromere, ParB2 could also bind directly to a related 39-mer. The 39-mers are the strongest inhibitors of chromosome II replication and they mediate inhibition by binding the replication initiator protein. ParB2 thus appears to promote replication by out-competing initiator binding to the 39-mers using two mechanisms: spreading into one and direct binding to the other. We suggest that both these are novel mechanisms to coordinate replication initiation with segregation of chromosomes.

Replication regulation of Vibrio cholerae chromosome II involves initiator binding to the origin both as monomer and as dimer.

The origin region of Vibrio cholerae chromosome II (chrII) resembles plasmid origins that have repeated initiator-binding sites (iterons). Iterons are essential for initiation as well as preventing over-initiation of plasmid replication. In chrII, iterons are also essential for initiation but over-initiation is prevented by sites called 39-mers. Both iterons and 39-mers are binding sites of the chrII specific initiator, RctB. Here, we have isolated RctB mutants that permit over-initiation in the presence of 39-mers. Characterization of two of the mutants showed that both are defective in 39-mer binding, which helps to explain their over-initiation phenotype. In vitro, RctB bound to 39-mers as monomers, and to iterons as both monomers and dimers. Monomer binding to iterons increased in both the mutants, suggesting that monomers are likely to be the initiators. We suggest that dimers might be competitive inhibitors of monomer binding to iterons and thus help control replication negatively. ChrII replication was found to be dependent on chaperones DnaJ and DnaK in vivo. The chaperones preferentially improved dimer binding in vitro, further suggesting the importance of dimer binding in the control of chrII replication.

Chromosome dynamics in multichromosome bacteria.

On the basis of limited information, bacteria were once assumed to have no more than one chromosome. In the era of genomics, it has become clear that some, like eukaryotes, have more than one chromosome. Multichromosome bacteria provide opportunities to investigate how split genomes emerged, whether the individual chromosomes communicate to coordinate their replication and segregation, and what selective advantages split genomes might provide. Our current knowledge of these topics comes mostly from studies in Vibrio cholerae, which has two chromosomes, chr1 and chr2. Chr1 carries out most of the house-keeping functions and is considered the main chromosome, whereas chr2 appears to have originated from a plasmid and has acquired genes of mostly unknown origin and function. Nevertheless, unlike plasmids, chr2 replicates once and only once per cell cycle, like a bona fide chromosome. The two chromosomes replicate and segregate using separate programs, unlike eukaryotic chromosomes. They terminate replication synchronously, suggesting that there might be communication between them. Replication of the chromosomes is affected by segregation genes but in a chromosome specific fashion, a new development in the field of DNA replication control. The split genome allows genome duplication to complete in less time and with fewer replication forks, which could be beneficial for genome maintenance during rapid growth, which is the norm for V. cholerae in broth cultures and in the human host. In the latter, the expression of chr2 genes increases preferentially. Studies of chromosome maintenance in multichromosomal bacteria, although in their infancy, are already broadening our view of chromosome biology. This article is part of a Special Issue entitled: Chromatin in time and space.

A 29-mer site regulates transcription of the initiator gene as well as function of the replication origin of Vibrio cholerae chromosome II.

The region responsible for replication of Vibrio cholerae chromosome II (chrII) resembles those of plasmids that have repeated initiator binding sites (iterons) and an autorepressed initiator gene. ChrII has additional features: Its iterons require full methylation for initiator (RctB) binding, which makes them inactive for a part of the cell cycle when they are hemi-methylated. RctB also binds to a second kind of site, called 39-mers, in a methylation independent manner. This binding is inhibitory to chrII replication. The site that RctB uses for autorepression has not been identified. Here we show that a 29-mer sequence, similar to the 39-mers, serves as that site, as we find that it binds RctB in vitro and suffices to repress the rctB promoter in vivo. The site is not subject to methylation and is likely to be active throughout the cell cycle. The 29-mer, like the 39-mers, could inhibit RctB-dependent mini-chrII replication in Escherichia coli, possibly by coupling with iterons via RctB bridges, as was seen in vitro. The 29-mer thus appears to play a dual role in regulating chrII replication: one independent of the cell cycle, the other dependent upon iteron methylation, hence responsive to the cell cycle.

Transition from a plasmid to a chromosomal mode of replication entails additional regulators.

Plasmid origins of replication are rare in bacterial chromosomes, except in multichromosome bacteria. The replication origin of Vibrio cholerae chromosome II (chrII) closely resembles iteron-bearing plasmid origins. Iterons are repeated initiator binding sites in plasmid origins and participate both in replication initiation and its control. The control is mediated primarily by coupling of iterons via the bound initiators ("handcuffing"), which causes steric hindrance to the origin. The control in chrII must be different, since the timing of its replication is cell cycle-specific, whereas in plasmids it is random. Here we show that chrII uses, in addition to iterons, another kind of initiator binding site, named 39-mers. The 39-mers confer stringent control by increasing handcuffing of iterons, presumably via initiator remodeling. Iterons, although potential inhibitors of replication themselves, restrain the 39-mer-mediated inhibition, possibly by direct coupling ("heterohandcuffing"). We propose that the presumptive transition of a plasmid to a chromosomal mode of control requires additional regulators to increase the stringency of control, and as will be discussed, to gain the capacity to modulate the effectiveness of the regulators at different stages of the cell cycle.

IHF-dependent activation of P1 plasmid origin by dnaA.

In bacteria, many DNA-protein interactions that initiate transcription, replication and recombination require the mediation of DNA architectural proteins such as IHF and HU. For replication initiation, plasmid P1 requires three origin binding proteins: the architectural protein HU, a plasmid-specific initiator, RepA, and the Escherichia coli chromosomal initiator, DnaA. The two initiators bind in the origin of replication to multiple sites, called iterons and DnaA boxes respectively. We show here that all five known DnaA boxes can be deleted from the plasmid origin provided the origin is extended by about 120 bp. The additional DNA provides an IHF site and most likely a weak DnaA binding site, because replacing the putative site with an authentic DnaA box enhanced plasmid replication in an IHF-dependent manner. IHF most likely brings about interactions between distally bound DnaA and RepA by bending the intervening DNA. The role of IHF in activating P1 origin by allowing DnaA binding to a weak site is reminiscent of the role the protein plays in initiating the host chromosomal replication.

Transcriptional inactivation of a regulatory site for replication of Vibrio cholerae chromosome II.

The bacterium Vibrio cholerae has two chromosomes. The origin of replication of chromosome I is similar to that of Escherichia coli. The origin-containing region of chromosome II (oriCII) resembles replicons of plasmids such as P1, except for the presence of an additional gene, rctA [Egan, E. S. & Waldor, M. K. (2003) Cell 114, 521-530]. The oriCII region that includes the initiator gene, rctB, can function as a plasmid in E. coli. Here we show that RctB suffices for the oriCII-based plasmid replication, and rctA in cis or trans reduces the plasmid copy number, thereby serving as a negative regulator. The inhibitory activity could be overcome by increasing the concentration of RctB, suggesting that rctA titrates the initiator. Purified RctB bound to a DNA fragment carrying rctA, confirming that the two can interact. Although rctA apparently works as a titrating site, it is nonetheless transcribed. We find that the transcription attenuates the inhibitory activity of the gene, presumably by interfering with RctB binding. RctB, in turn, repressed the rctA promoter and, thereby, could control its own titration by modulating the transcription of rctA. This control circuit appears to be a putative novel mechanism for homeostasis of initiator availability.

Multipartite regulation of rctB, the replication initiator gene of Vibrio cholerae chromosome II.

Replication initiator proteins in bacteria not only allow DNA replication but also often regulate the rate of replication initiation as well. The regulation is mediated by limiting the synthesis or availability of initiator proteins. The applicability of this principle is demonstrated here for RctB, the replication initiator for the smaller of the two chromosomes of Vibrio cholerae. A strong promoter for the rctB gene named rctBp was identified and found to be autoregulated in Escherichia coli. Promoter activity was lower in V. cholerae than in E. coli, and a part of this reduction is likely to be due to autorepression. Sequences upstream of rctBp, implicated earlier in replication control, enhanced the repression. The action of the upstream sequences required that they be present in cis, implying long-range interactions in the control of the promoter activity. A second gene specific for chromosome II replication, rctA, reduced rctB translation, most likely by antisense RNA control. Finally, optimal rctBp activity was found to be dependent on Dam. Increasing RctB in trans increased the copy number of a miniplasmid carrying oriCII(VC), implying that RctB can be rate limiting for chromosome II replication. The multiple modes of control on RctB are expected to reduce fluctuations in the initiator concentration and thereby help maintain chromosome copy number homeostasis.

An antisense RNA plays a central role in the replication control of a repC plasmid.

The widespread replicons of repABC and repC families from alpha-proteobacteria share high similarity in their replication initiator proteins (RepC). Here we describe the minimal region required for stable replication of a member of the repC family, the low copy-number plasmid pRmeGR4a from Sinorizobium meliloti GR4. This region contains only two genes: one encoding the initiator protein RepC (46.8 kDa) and other, an antisense RNA (67 nt). Mapping of transcriptional start sites and promoter regions of both genes showed that the antisense RNA is nested within the repC mRNA leader. The constitutively expressed countertranscribed RNA (ctRNA) forms a single stem-loop structure that acts as an intrinsic rho-independent terminator. The ctRNA is a strong trans-incompatibility factor and negative regulator of repC expression. Based on structural and functional similarities between members of the repC and repABC families we propose a model of their evolutionary relationship.

Two discrete elements are required for the replication of a repABC plasmid: an antisense RNA and a stem-loop structure.

The repABC replicons contain an operon encoding the initiator protein (RepC) and partitioning proteins (RepA and RepB). The latter two proteins negatively regulate the transcription of the operon. In this article we have identified two novel regulatory elements, located within the conserved repB-repC intergenic sequence, which negatively modulate the expression of repC, in plasmid p42d of Rhizobium etli. One of them is a small antisense RNA and the other is a stem-loop structure in the repABC mRNA that occludes the Shine-Dalgarno sequence of repC. According to in vivo and in vitro analyses, the small antisense RNA (57-59 nt) resembles canonical negative regulators of replication because: (i) it is transcribed from a strong constitutive promoter (P2), (ii) the transcript overlaps untranslated region upstream of the RepC coding sequences, (iii) the RNA forms one secondary structure acting as a rho-independent terminator, (iv) the antisense RNA is a strong trans-incompatibility factor and (v) its presence reduces the level of repC expression. Surprisingly, both of these seemingly negative regulators are required for efficient plasmid replication.

Incompatibility and the partitioning site of the repABC basic replicon of the symbiotic plasmid from Rhizobium etli.

The basic replicon of the symbiotic plasmid (p42d) of Rhizobium etli CE3 is constituted by the repABC operon. Whereas RepC is essential for plasmid replication, RepA and RepB are involved in plasmid partitioning. Three incompatibility regions have been previously identified in this plasmid: the first one encodes RepA, a partitioning protein that also down-regulates the repABC transcription. The second region is situated within the repB-repC intergenic sequence (inc(alpha)), and the last one, inc(beta), is located in a 502 bp EcoRI fragment spanning the last 72-bp of the coding region of repC and the following downstream sequence. In this paper we show that: (1) The inc(beta) region is required for plasmid partitioning. (2) A 16-bp palindrome sequence, located 40 bp downstream of the repC gene of plasmid p42d, is necessary and sufficient to induce incompatibility towards the parental plasmid, and accounts for all the incompatibility properties of this region (inc(beta)). (3). The palindrome is the DNA target site for RepB binding. With these findings we propose that inc(beta) contains the partitioning site (par site) of the basic replicon of plasmid p42d, and that the 16-bp palindrome is the core sequence to nucleate the RepB binding.

Characterization of the cryptic plasmid pCC1 from Corynebacterium callunae and its use for vector construction.

The complete nucleotide sequence of the cryptic plasmid pCC1 from Corynebacterium callunae (4109 bp) was determined. DNA sequence analysis revealed five open reading frames longer than 200 bp. One of the deduced polypeptides showed homology with the Rep proteins encoded by plasmids of the pIJ101/pJV1 family of plasmids replicating by the rolling-circle (RC) mechanism. Within this plasmid family, the Rep protein of pCC1 showed the highest degree of similarity to the Rep proteins of corynebacterial plasmids pAG3 and pBL1. These data suggest that the plasmid pCC1 replicates by the RC mechanism. The Escherichia coli/Corynebacterium glutamicum shuttle cloning vector pSCCD1, carrying the pCC1 rep gene on the 2.1-kb DNA fragment and the streptomycin/spectinomycin resistance determinant, was constructed. This vector is stably maintained in population of C. glutamicum cells grown in the absence of selection pressure and it is compatible with plasmid vectors based on corynebacterial plasmids pBL1 and pSR1.

Control of rep gene expression in plasmid pGA1 from Corynebacterium glutamicum.

The cryptic multicopy plasmid pGA1 (4,826 bp) from Corynebacterium glutamicum LP-6 belongs to the fifth group of rolling-circle-replicating plasmids. A determinant, which negatively controls pGA1 replication, was localized in the leader region of the rep gene coding for the initiator of plasmid replication. This region, when cloned into the compatible vector pEC6, was found to cause decrease of segregational stability of the pGA1 derivative pKG48. A promoter and a single transcriptional start site were found in the rep leader region in orientation opposite to the rep gene. These results suggest that a small countertranscribed RNA (ctRNA) (ca. 89 nucleotides in length), which might inhibit translation of pGA1 rep gene, is formed. Analysis of predicted secondary structure of the pGA1-encoded ctRNA revealed features common with the known ctRNAs in bacteria. Inactivation of the promoter P-ctRNA caused a dramatic increase of copies of the respective plasmid, which proved a negative role of the ctRNA in control of pGA1 copy number. A region between the promoters Prep and P-ctRNA with a potential to form secondary structures on both ctRNA and rep mRNA was found to cause low activity of the rep promoter even when promoter P-ctRNA was deleted. Thus, the sequence within the rep leader region itself seems to act, in addition to the ctRNA, as a second regulatory element of a novel type, negatively influencing expression of the pGA1 rep gene.