Holliday junction trap shows how cells use recombination and a junction-guardian role of RecQ helicase.

DNA repair by homologous recombination (HR) underpins cell survival and fuels genome instability, cancer, and evolution. However, the main kinds and sources of DNA damage repaired by HR in somatic cells and the roles of important HR proteins remain elusive. We present engineered proteins that trap, map, and quantify Holliday junctions (HJs), a central DNA intermediate in HR, based on catalytically deficient mutant RuvC protein of Escherichia coli. We use RuvCDefGFP (RDG) to map genomic footprints of HR at defined DNA breaks in E. coli and demonstrate genome-scale directionality of double-strand break (DSB) repair along the chromosome. Unexpectedly, most spontaneous HR-HJ foci are instigated, not by DSBs, but rather by single-stranded DNA damage generated by replication. We show that RecQ, the E. coli ortholog of five human cancer proteins, nonredundantly promotes HR-HJ formation in single cells and, in a novel junction-guardian role, also prevents apparent non-HR-HJs promoted by RecA overproduction. We propose that one or more human RecQ orthologs may act similarly in human cancers overexpressing the RecA ortholog RAD51 and find that cancer genome expression data implicate the orthologs BLM and RECQL4 in conjunction with EME1 and GEN1 as probable HJ reducers in such cancers. Our results support RecA-overproducing E. coli as a model of the many human tumors with up-regulated RAD51 and provide the first glimpses of important, previously elusive reaction intermediates in DNA replication and repair in single living cells.

Roles of Nucleoid-Associated Proteins in Stress-Induced Mutagenic Break Repair in Starving Escherichia coli.

The mutagenicity of DNA double-strand break repair in Escherichia coli is controlled by DNA-damage (SOS) and general (RpoS) stress responses, which let error-prone DNA polymerases participate, potentially accelerating evolution during stress. Either base substitutions and indels or genome rearrangements result. Here we discovered that most small basic proteins that compact the genome, nucleoid-associated proteins (NAPs), promote or inhibit mutagenic break repair (MBR) via different routes. Of 15 NAPs, H-NS, Fis, CspE, and CbpA were required for MBR; Dps inhibited MBR; StpA and Hha did neither; and five others were characterized previously. Three essential genes were not tested. Using multiple tests, we found the following: First, Dps, which reduces reactive oxygen species (ROS), inhibited MBR, implicating ROS in MBR. Second, CbpA promoted F' plasmid maintenance, allowing MBR to be measured in an F'-based assay. Third, Fis was required for activation of the SOS DNA-damage response and could be substituted in MBR by SOS-induced levels of DinB error-prone DNA polymerase. Thus, Fis promoted MBR by allowing SOS activation. Fourth, H-NS represses ROS detoxifier sodB and was substituted in MBR by deletion of sodB, which was not otherwise mutagenic. We conclude that normal ROS levels promote MBR and that H-NS promotes MBR by maintaining ROS. CspE positively regulates RpoS, which is required for MBR. Four of five previously characterized NAPs promoted stress responses that enhance MBR. Hence, most NAPs affect MBR, the majority via regulatory functions. The data show that a total of six NAPs promote MBR by regulating stress responses, indicating the importance of nucleoid structure and function to the regulation of MBR and of coupling mutagenesis to stress, creating genetic diversity responsively.

Regulation of DNA Replication Initiation by Chromosome Structure.

Recent advancements in fluorescence imaging have shown that the bacterial nucleoid is surprisingly dynamic in terms of both behavior (movement and organization) and structure (density and supercoiling). Links between chromosome structure and replication initiation have been made in a number of species, and it is universally accepted that favorable chromosome structure is required for initiation in all cells. However, almost nothing is known about whether cells use changes in chromosome structure as a regulatory mechanism for initiation. Such changes could occur during natural cell cycle or growth phase transitions, or they could be manufactured through genetic switches of topoisomerase and nucleoid structure genes. In this review, we explore the relationship between chromosome structure and replication initiation and highlight recent work implicating structure as a regulatory mechanism. A three-component origin activation model is proposed in which thermal and topological structural elements are balanced with trans-acting control elements (DnaA) to allow efficient initiation control under a variety of nutritional and environmental conditions. Selective imbalances in these components allow cells to block replication in response to cell cycle impasse, override once-per-cell-cycle programming during growth phase transitions, and promote reinitiation when replication forks fail to complete.

DNA Replication Initiation Is Blocked by a Distant Chromosome-Membrane Attachment.

Although it has been recognized for several decades that chromosome structure regulates the capacity of replication origins to initiate, very little is known about how or if cells actively regulate structure to direct initiation. We report that a localized inducible protein tether between the chromosome and cell membrane in E. coli cells imparts a rapid and complete block to replication initiation. Tethers, composed of a trans-membrane and transcription repressor fusion protein bound to an array of operator sequences, can be placed up to 1 Mb from the origin with no loss of penetrance. Tether-induced initiation blocking has no effect on elongation at pre-existing replication forks and does not cause cell or DNA damage. Whole-genome and site-specific fluorescent DNA labeling in tethered cells indicates that global nucleoid structure and chromosome organization are disrupted. Gene expression patterns, assayed by RNA sequencing, show that tethering induces global supercoiling changes, which are likely incompatible with replication initiation. Parallels between tether-induced initiation blocking and rifampicin treatment and the role of programmed changes in chromosome structure in replication control are discussed.

MioC and GidA proteins promote cell division in E. coli.

The well-conserved genes surrounding the E. coli replication origin, mioC and gidA, do not normally affect chromosome replication and have little known function. We report that mioC and gidA mutants exhibit a moderate cell division inhibition phenotype. Cell elongation is exacerbated by a fis deletion, likely owing to delayed replication and subsequent cell cycle stress. Measurements of replication initiation frequency and origin segregation indicate that mioC and gidA do not inhibit cell division through any effect on oriC function. Division inhibition is also independent of the two known replication/cell division checkpoints, SOS and nucleoid occlusion. Complementation analysis indicates that mioC and gidA affect cell division in trans, indicating their effect is at the protein level. Transcriptome analysis by RNA sequencing showed that expression of a cell division septum component, YmgF, is significantly altered in mioC and gidA mutants. Our data reveal new roles for the gene products of gidA and mioC in the division apparatus, and we propose that their expression, cyclically regulated by chromatin remodeling at oriC, is part of a cell cycle regulatory program coordinating replication initiation and cell division.

A replication-inhibited unsegregated nucleoid at mid-cell blocks Z-ring formation and cell division independently of SOS and the SlmA nucleoid occlusion protein in Escherichia coli.

Chromosome replication and cell division of Escherichia coli are coordinated with growth such that wild-type cells divide once and only once after each replication cycle. To investigate the nature of this coordination, the effects of inhibiting replication on Z-ring formation and cell division were tested in both synchronized and exponentially growing cells with only one replicating chromosome. When replication elongation was blocked by hydroxyurea or nalidixic acid, arrested cells contained one partially replicated, compact nucleoid located mid-cell. Cell division was strongly inhibited at or before the level of Z-ring formation. DNA cross-linking by mitomycin C delayed segregation, and the accumulation of about two chromosome equivalents at mid-cell also blocked Z-ring formation and cell division. Z-ring inhibition occurred independently of SOS, SlmA-mediated nucleoid occlusion, and MinCDE proteins and did not result from a decreased FtsZ protein concentration. We propose that the presence of a compact, incompletely replicated nucleoid or unsegregated chromosome masses at the normal mid-cell division site inhibits Z-ring formation and that the SOS system, SlmA, and MinC are not required for this inhibition.

Engineered proteins detect spontaneous DNA breakage in human and bacterial cells.

Spontaneous DNA breaks instigate genomic changes that fuel cancer and evolution, yet direct quantification of double-strand breaks (DSBs) has been limited. Predominant sources of spontaneous DSBs remain elusive. We report synthetic technology for quantifying DSBs using fluorescent-protein fusions of double-strand DNA end-binding protein, Gam of bacteriophage Mu. In Escherichia coli GamGFP forms foci at chromosomal DSBs and pinpoints their subgenomic locations. Spontaneous DSBs occur mostly one per cell, and correspond with generations, supporting replicative models for spontaneous breakage, and providing the first true breakage rates. In mammalian cells GamGFP-labels laser-induced DSBs antagonized by end-binding protein Ku; co-localizes incompletely with DSB marker 53BP1 suggesting superior DSB-specificity; blocks resection; and demonstrates DNA breakage via APOBEC3A cytosine deaminase. We demonstrate directly that some spontaneous DSBs occur outside of S phase. The data illuminate spontaneous DNA breakage in E. coli and human cells and illustrate the versatility of fluorescent-Gam for interrogation of DSBs in living cells. DOI:http://dx.doi.org/10.7554/eLife.01222.001.

Regulation of sister chromosome cohesion by the replication fork tracking protein SeqA.

Analogously to chromosome cohesion in eukaryotes, newly replicated DNA in E. coli is held together by inter-sister linkages before partitioning into daughter nucleoids. In both cases, initial joining is apparently mediated by DNA catenation, in which replication-induced positive supercoils diffuse behind the fork, causing newly replicated duplexes to twist around each other. Type-II topoisomerase-catalyzed sister separation is delayed by the well-characterized cohesin complex in eukaryotes, but cohesion control in E. coli is not currently understood. We report that the abundant fork tracking protein SeqA is a strong positive regulator of cohesion, and is responsible for markedly prolonged cohesion observed at "snap" loci. Epistasis analysis suggests that SeqA stabilizes cohesion by antagonizing Topo IV-mediated sister resolution, and possibly also by a direct bridging mechanism. We show that variable cohesion observed along the E. coli chromosome is caused by differential SeqA binding, with oriC and snap loci binding disproportionally more SeqA. We propose that SeqA binding results in loose inter-duplex junctions that are resistant to Topo IV cleavage. Lastly, reducing cohesion by genetic manipulation of Topo IV or SeqA resulted in dramatically slowed sister locus separation and poor nucleoid partitioning, indicating that cohesion has a prominent role in chromosome segregation.

Escherichia coli sister chromosome separation includes an abrupt global transition with concomitant release of late-splitting intersister snaps.

The basis for segregation of sister chromosomes in bacteria is not established. We show here that two discrete ~150-kb regions, both located early in the right replichore, exhibit prolonged juxtaposition of sister loci, for 20 and 30 min, respectively, after replication. Flanking regions, meanwhile, separate. Thus, the two identified regions comprise specialized late-splitting intersister connections or snaps. Sister snap loci separate simultaneously in both snap regions, concomitant with a major global nucleoid reorganization that results in emergence of a bilobed nucleoid morphology. Split snap loci move rapidly apart to a separation distance comparable with one-half the length of the nucleoid. Concomitantly, at already split positions, sister loci undergo further separation to a comparable distance. The overall consequence of these and other effects is that thus far replicated sister chromosomes become spatially separated (individualized) into the two nucleoid lobes, while the terminus region (and likely, all unreplicated portions of the chromosome) moves to midcell. These and other findings imply that segregation of Escherichia coli sister chromosomes is not a smooth continuous process but involves at least one and likely, two major global transition(s). The presented patterns further suggest that accumulation of internal intranucleoid forces and constraining of these forces by snaps play central roles in global chromosome dynamics. They are consistent with and supportive of our previous proposals that individualization of sisters in E. coli is driven primarily by internally generated pushing forces and is directly analogous to sister individualization at the prophase to prometaphase transition of the eukaryotic cell cycle.

The mouse frizzy (fr) and rat 'hairless' (frCR) mutations are natural variants of protease serine S1 family member 8 (Prss8).

Please cite this paper as: The mouse frizzy (fr) and rat 'hairless' (fr(CR)) mutations are natural variants of protease serine S1 family member 8 (Prss8). Experimental Dermatology 2010; 19: 527-532. Abstract: We have previously suggested (based on genetic mapping analysis) that the allelic 'fuzzy' and 'hairless' mutations in the rat are likely orthologues of the mouse frizzy mutation (fr). Here, we analysed three large intraspecific backcross panels that segregated for mouse fr to restrict this locus to a 0.6-Mb region that includes fewer than 30 genes. DNA sequencing of one of these candidates known to be expressed in skin, protease serine S1 family member 8 (Prss8), revealed a T to A transversion associated with the fr allele that would result in a valine to aspartate substitution at residue 170 in the gene product. To test whether this missense mutation might be the molecular basis of this frizzy variant, we crossed fr/fr mice with mice that carried a recessive perinatal lethal mutation in Prss8. Hybrid offspring that inherited both fr and the Prss8 null allele displayed abnormal hair and skin, showing that these two mutations are allelic, and suggesting strongly that the T to A mutation in Prss8 is responsible for the mutant frizzy phenotype. Sequence analysis of all Prss8 coding regions in the 'hairless' rat identified a 12-bp deletion in the third exon, indicating that mouse fr and the rat 'hairless' mutations are indeed orthologues. However, this analysis failed to detect any alterations to Prss8 coding sequences in the allelic 'fuzzy' rat variant.

The mouse frizzy mutation (fr) maps between D7Csu5 and D7Mit165.

We have previously shown that the rat fuzzy and Charles River 'hairless' mutations are defects in the same gene on rat Chr 1, and are likely orthologues of the frizzy mutation (fr) on mouse Chr 7. To test the hypothesis that these variants could result from defects in Fgfr2, we crossed fr/fr mice (from the inbred FS/EiJ strain) with mice that carry a recessive lethal mutation in Fgfr2. Mice inheriting both mutations were phenotypically normal, indicating that fr is not an allele of Fgfr2. To genetically map fr, we crossed these hybrid mice, or F(1) mice made by crossing FS/EiJ with the wild-type C57BL/6J or BALB/cBy strains, back to the FS/EiJ strain. The resulting 546 backcross progeny were typed for linked markers to position fr centromeric of Fgfr2, between D7Csu5 and D7Mit165; an interval that contains only 2.7 Mb and fewer than 70 genes. Further characterization of regional recombinants for sequence-level polymorphisms should allow sufficient refinement of fr's location to facilitate an eventual molecular assignment for this classical mutation.