DNA Daughter-strand Research Articles

How cells ensure symmetric inheritance

Molecular Biology Parental histones with modifications are recycled to newly replicated DNA strands during genome replication, but do the two sister chromatids inherit modified histones equally? Yu et al. and Petryk et al. found in mouse and yeast, respectively, that modified histones are segregated to both DNA daughter strands in a largely symmetric manner (see the Perspective by Ahmad and Henikoff). However, the mechanisms ensuring this symmetric inheritance in yeast and mouse were different. Yeasts use subunits of DNA polymerase to prevent the lagging-strand bias of parental histones, whereas in mouse cells, the replicative helicase MCM2 counters the leading-strand bias. Science , this issue p. [1386][1], p. [1389][2]; see also p. [1311][3] [1]: /lookup/doi/10.1126/science.aat8849 [2]: /lookup/doi/10.1126/science.aau0294 [3]: /lookup/doi/10.1126/science.aav0871

MCM2 promotes symmetric inheritance of modified histones during DNA replication.

During genome replication, parental histones are recycled to newly replicated DNA with their posttranslational modifications (PTMs). Whether sister chromatids inherit modified histones evenly remains unknown. We measured histone PTM partition to sister chromatids in embryonic stem cells. We found that parental histones H3-H4 segregate to both daughter DNA strands with a weak leading-strand bias, skewing partition at topologically associating domain (TAD) borders and enhancers proximal to replication initiation zones. Segregation of parental histones to the leading strand increased markedly in cells with histone-binding mutations in MCM2, part of the replicative helicase, exacerbating histone PTM sister chromatid asymmetry. This work reveals how histones are inherited to sister chromatids and identifies a mechanism by which the replication machinery ensures symmetric cell division.

Escherichia coli RarA commits cells to post‐replication repair pathways by facilitating replisome skipping

Members of the highly conserved RarA/Mgs1/WRNIP1 protein family have been implicated in the maintenance of genomic stability, but their exact functions remain unknown. Loss of one of these proteins rarely results in an altered phenotype, but a knockout mutation in genes encoding proteins of this family in tandem with the loss of other DNA metabolism functions can adversely affect cell survival. We hypothesize that the RarA protein of E. coli commits the cell to post‐replication repair pathways, namely translesion DNA synthesis (TLS) and daughter strand gap synthesis. RarA overexpression is toxic to the cell and knocking out DNA Polymerase IV and DNA Polymerase V, TLS‐competent polymerases, alleviates this toxicity. Furthermore, the presence of RarA induces sensitivity in cells lacking TLS polymerases to the DNA‐damaging agents nitrofurazone (NFZ) and 4‐nitroquinoline‐1‐oxide (4‐NQO). Deletion of RarA suppresses DNA Polymerase IV mediated sensitivity to NFZ and 4‐NQO. These data suggest that RarA can promote rapid replisome progress by committing cells to using post‐replication repair pathways and blocking access to other repair pathways when the replisome encounters certain types of lesions.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibition of Topoisomerase IIα by Metal Complexes of Selected Thiosemicarbazones

Uncontrolled cell proliferation is the main characteristic of all cancer types. To kill the over‐proliferated cells is one key to cure cancer. Before cell proliferation, the genetic material, DNA, needs to be replicated. Thus, DNA replication is the top target for many anti‐cancer drugs. Topoisomerase IIα (Topo IIα) is essential for DNA replication in order to release the supercoiled DNA daughter strands produced after replication (topology simplification) and reduce entanglements. Topo IIα breaks DNA strands, which make it one of the most important targets for anticancer drugs since the accumulation of DNA nicks from inhibition of Topo IIα induces apoptosis. We have synthesized and characterized several structurally different series of thiosemicarbazone ligands and their metal complexes, which show different inhibition activities towards Topo IIα. Our studies indicate that several of the new compounds exhibit high Topo IIα inhibition and are potential anticancer drugs.

Organization of DNA replication.

The discovery of the DNA double helix structure half a century ago immediately suggested a mechanism for its duplication by semi-conservative copying of the nucleotide sequence into two DNA daughter strands. Shortly after, a second fundamental step toward the elucidation of the mechanism of DNA replication was taken with the isolation of the first enzyme able to polymerize DNA from a template. In the subsequent years, the basic mechanism of DNA replication and its enzymatic machinery components were elucidated, mostly through genetic approaches and in vitro biochemistry. Most recently, the spatial and temporal organization of the DNA replication process in vivo within the context of chromatin and inside the intact cell are finally beginning to be elucidated. On the one hand, recent advances in genome-wide high throughput techniques are providing a new wave of information on the progression of genome replication at high spatial resolution. On the other hand, novel super-resolution microscopy techniques are just starting to give us the first glimpses of how DNA replication is organized within the context of single intact cells with high spatial resolution. The integration of these data with time lapse microscopy analysis will give us the ability to film and dissect the replication of the genome in situ and in real time.

Recombinational DNA repair: the ignored repair systems

The recent finding of a role for the recA gene in DNA replication restart does not negate previous data showing the existence of recA-dependent recombinational DNA repair, which occurs when there are two DNA duplexes present, as in the case for recA-dependent excision repair, for postreplication repair (i.e., the repair of DNA daughter-strand gaps), and for the repair of DNA double-strand breaks. Recombinational DNA repair is critical for the survival of damaged cells.

The ultraviolet-sensitizing function of plasmid R391 interferes with a late step of postreplication repair in Escherichia coli

The conjugative plasmid R391 increases the UV radiation sensitivity of wild-type, uvrA, and lexA cells of Escherichia coli, but not recA strains. To investigate the UV-sensitizing function of R391, we examined the effect of R391 on the repair of DNA daughter-strand gaps and on the UV radiation sensitivities of various repair and/or recombination-deficient mutants. The presence of R391 did not significantly inhibit the repair of DNA daughter-strand gaps in uvrB cells. The presence of R391 increased the UV radiation sensitivity of uvrA, uvrA recF, uvrB, uvrB recF, uvrB recB, and uvrB ssb-113 cells to UV irradiation, but did not significantly increase the UV radiation sensitivity of uvrA ruvA and uvrA ruvC strains. Based on these results, we propose that the UV-sensitizing activity of R391 acts by inhibiting or interfering with the ruvABC-mediated postsynapsis step of recombinational repair.

Overexpression of the natural recO sequence and its effects on DNA repair of Escherichia coli

The recO gene is required for the RecF pathway of recombination and the repair of DNA daughter-strand gaps in Escherichia coli. In this work, the structural portion of recO was synthesized by the polymerase chain reaction (PCR) and cloned onto expression vectors at their Nco1 fusion cloning site, to eliminate the presence of mRNA leader sequence. While the plasmid carrying a Ptac promoter failed to overproduce RecO, the plasmid carrying a T7O10 promoter overproduced RecO in large quantity, indicating that the natural recO may be overexpressed. An increase of intracellular RecO, which may be due to the increased recO gene copies or to the induction of RecO synthesis, increased the UV resistance of recA, recF, and ssb cells, but did not increase the UV resistance of uvrB, uvrB recF, uvrB recA and uvrB ssb cells. We suggest that an increase of intracellular RecO may allow some recombination-deficient cells to perform more excision repair, thus increasing the survival. The possible causes for RecO overproduction on excision repair, and for the differential expression of recO by the Ptac and T7 promoter plasmids are discussed.

Involvement of RecF pathway recombination genes in postreplication repair in UV-irradiated Escherichia coli cells

Mutations affecting the RecF pathway of recombination (recF, recG, recJ, recN, recO, recQ, recR, ruvA, ruvC) were systematically introduced into two sets of strains: (a) uvrA and uvrA recA2020, (b) uvrA recBC sbcBC and uvrA recBC sbcBC recA2020. We examined: (i) the effect of these mutations on the repair of DNA daughter-strand gaps which are produced in the nascent DNA synthesized after UV irradiation, and (ii) the ability of recA2020 (a suppressor for the recF mutation) to suppress the UV radiation sensitivity caused by these mutations. In the uvrA cells, mutations in recF, recR or recO genes produced a major deficiency in the repair of daughter-strand gaps, whereas mutations in recJ, recG, recN, recQ, ruvA or ruvC genes had no effect on the repair of daughter-strand gaps. In both uvrA and uvrA recBC sbcBC backgrounds, the UV radiation sensitivity caused by recF, recG, recR, recO, ruvA, or ruvC mutation was partially suppressed by recA2020, whereas the UV radiation sensitivity caused by recJ, recN, or recQ mutations was not suppressed by recA2020. Partial suppression of the UV sensitivity of recG, ruvA and ruvC mutants was not observed with other suppressors for recF, i.e., recA441, recA720 and recA730. Taken together, these results further support the notion that the recF, recR and recO gene products (abbreviated as RecFOR) function at the same step in recombination repair, possible as a complex. It also suggests that this putative RecFOR) complex does not contain proteins encoded by other genes involved in the RecF pathway of recombination.

Alkali-labile sites in daughter DNA of damagedEscherichia coli do not reflect apyrimidinic sites

Apyrimidinic sites arising after excision of uracil incorporated into DNA daughter strands might constitute alkali-labile sites. The hypothesis was checked in the present paper and the data obtained do not support it.

Role of DNA polymerase I in postreplication repair: a reexamination with Escherichia coli delta polA

Using strains of Escherichia coli K-12 that are deleted for the polA gene, we have reexamined the role of DNA polymerase I (encoded by polA) in postreplication repair after UV irradiation. The polA deletion (in contrast to the polA1 mutation) made uvrA cells very sensitive to UV radiation; the UV radiation sensitivity of a uvrA delta polA strain was about the same as that of a uvrA recF strain, a strain known to be grossly deficient in postreplication repair. The delta polA mutation interacted synergistically with a recF mutation in UV radiation sensitization, suggesting that the polA gene functions in pathways of postreplication repair that are largely independent of the recF gene. When compared to a uvrA strain, a uvrA delta polA strain was deficient in the repair of DNA daughter strand gaps, but not as deficient as a uvrA recF strain. Introduction of the delta polA mutation into uvrA recF cells made them deficient in the repair of DNA double-strand breaks after UV irradiation. The UV radiation sensitivity of a uvrA polA546(Ts) strain (defective in the 5'----3' exonuclease of DNA polymerase I) determined at the restrictive temperature was very close to that of a uvrA delta polA strain. These results suggest a major role for the 5'----3' exonuclease activity of DNA polymerase I in postreplication repair, in the repair of both DNA daughter strand gaps and double-strand breaks.

RecA (Srf) suppression of recF deficiency in the postreplication repair of UV-irradiated Escherichia coli K-12.

The mechanism by which recA (Srf) mutations (recA2020 and recA801) suppress the deficiency in postreplication repair shown by recF mutants of Escherichia coli was studied in UV-irradiated uvrB and uvrA recB recC sbcB cells. The recA (Srf) mutations partially suppressed the UV radiation sensitivity of uvrB recF, uvrB recF recB, and uvrA recB recC sbcB recF cells, and they partially restored the ability of uvrB recF and uvrA recB recC sbcB recF cells to repair DNA daughter-strand gaps. In addition, the recA (Srf) mutations suppressed the recF deficiency in the repair of DNA double-strand breaks in UV-irradiated uvrA recB recC sbcB recF cells. The recA2020 and recA801 mutations do not appear to affect the synthesis of UV radiation-induced proteins, nor do they appear to produce an altered RecA protein, as detected by two-dimensional gel electrophoresis. These results are consistent with the suggestion (M. R. Volkert and M. A. Hartke, J. Bacteriol. 157:498-506, 1984) that the recA (Srf) mutations do not act by affecting the induction of SOS responses; rather, they allow the RecA protein to participate in the recF-dependent postreplication repair processes without the need of the RecF protein.

Role of the radB gene in postreplication repair in UV-irradiated Escherichia coli uvrB

In UV-irradiated Escherichia coli, the radB101 mutation sensitized uvrB recF cells 4-fold and uvrB recB cells 1.2-fold, but did not sensitize uvrB recB recF cells. The radB mutation had very little effect (1.2-fold or less) on the repair of UV radiation-induced DNA daughter-strand gaps in uvrB cells, but it did cause about a 3-fold deficiency in the repair of the DNA double-strand breaks that arise in association with nonrepaired daughter-strand gaps in UV-irradiated uvrB recF cells. Thus, the radB gene does not appear to be involved in the recF-dependent or recF recB-independent processes for the repair of DNA daughter-strand gaps, but is involved in the recB-dependent postreplication repair of DNA double-strannd breaks.

Postreplicational formation and repair of DNA double-strand breaks in UV-irradiated Escherichia coli uvrB cells

The number of DNA double-strand breaks formed in UV-irradiated uvrB rec F recB cells correlates with the number of unrepaired DNA daughter-strand gaps, and is dependent on DNA synthesis after UV-irradiation. These results are consistent with the model that the DNA double-strand breaks that are produced in UV-irradiated excision-deficient cells occur as the results of breaks in the parental DNA opposite unrepaired DNA daughter-strand gaps. By employing a temperature-sensitive recA200 mutation, breaks. Possible mechanisms for the postreplication repair of DNA double-strand breaks are discussed.

Mechanism of sbcB-suppression of the recBC-deficiency in postreplication repair in UV-irradiated Escherichia coli K-12.

The mechanism by which an sbcB mutation suppresses the deficiency in postreplication repair shown by recB recC mutants of Escherichia coli was studied. The presence of an sbcB mutation in uvrA recB recC cells increased their resistance to UV radiation. This enhanced resistance was not due to a suppression of the minor deficiency in the repair of DNA daughter-strand gaps or to an inhibition of the production of DNA double-strand breaks in UV-irradiated uvrA recB recC cells; rather, the presence of an sbcB mutation enabled uvrA recB recC cells to carry out the repair of DNA double-strand breaks. In the uvrA recB recC sbcB background, a mutation at recF produced a huge sensitization to UV radiation, and it rendered cells deficient in the repair of both DNA daughter-strand gaps and DNA double-strand breaks. Thus, an additional sbcB mutation in uvrA recB recC cells restored their ability to perform the repair of DNA double-strand breaks, but the further addition of a recF mutation blocked this repair capacity.

A minor pathway of postreplication repair in Escherichia coli is independent of the recB, recC and recF genes

After ultraviolet (UV) irradiation, an Escherichia coli K12 uvrB5 recB12 recF143 strain (SR1203) was able to perform a limited amount of postreplication repair when incubated in minimal growth medium (MM), but not if incubated in a rich growth medium. Similarly, this strain showed a higher survival after UV irradiation if plated on MM versus rich growth medium (i.e., it showed minimical medium recovery (MMR)). In fact, its survival after UV irradiation on rich growth medium was similar to that of a uvB5 recA56 strain, which does not show MMR or postreplication repair. The results obtained with a uvB5 recF332::Tn3 Δ recBC strain and a uvB5 recF332::Tn3 recB21 recC22 strain were similar to those obtained for strain SR1203, suggesting that the recB21 and recF143 alleles are not leaky in strain SR1203. The treatment of UV-irradiated uvB5 recB21 recF143 and uvrB5 recF332::Tn3 δ recBC cells with rifampicin for 2 h had no effect on survival or the repair of DNA daughter-strand gaps. Therefore, a pathway of postreplication repair has been demonstrated that is constitutive in nature, is inhibited by postirradiation incubation in rich growth medium, and does not require the recB recC and recF gene products, which control the major pathways of postreplication repair.

Role of the umuC gene in postreplication repair in UV-irradiated Escherichia coli K-12 uvrB

The role of the umuC gene product in postreplication repair was studied in UV-irradiated Escherichia coli K-12 uvrB cells. A mutation at umuC increased the UV radiation sensitivities of uvrB, uvrB recF, uvrB recB, and uvrB recF recB cells; it also increased the deficiencies in the repair of DNA daughter-strand gaps in these strains, but it did not affect the repair of DNA double-strand breaks that arose from unrepaired DNA daughter-strand gaps. We suggest that the umuC gene product is involved in a minor system for the repair of DNA daughter-strand gaps, possibly the repair of overlapping DNA daughter-strand gaps.

RecF-dependent and recF recB-independent DNA gap-filling repair processes transfer dimer-containing parental strands to daughter strands in Escherichia coli K-12 uvrB.

The processes for repairing DNA daughter-strand gaps were studied in UV-irradiated uvrB, uvrB recB, uvrB recF, and uvrB recB recF cells of Escherichia coli K-12. The dimer-containing parental DNA was found to be joined to daughter strands during postreplication repair in all four strains examined. Therefore, both the major (recF-dependent) and the minor (recF recB-independent) gap-filling processes repair DNA daughter-strand gaps by transferring parental strands into daughter strands.

Mechanisms for recF-dependent and recB-dependent pathways of postreplication repair in UV-irradiated Escherichia coli uvrB.

The molecular mechanisms for the recF-dependent and recB-dependent pathways of postreplication repair were studied by sedimentation analysis of DNA from UV-irradiated Escherichia coli cells. When the ability to repair DNA daughter strand gaps was compared, uvrB recF cells showed a gross deficiency, whereas uvrB recB cells showed only a small deficiency. Nevertheless, the uvrB recF cells were able to perform some limited repair of daughter strand gaps compared with a "repairless" uvrB recA strain. The introduction of a recB mutation into the uvrB recF strain greatly increased its UV radiation sensitivity, yet decreased only slightly its ability to repair daughter strand gaps. Kinetic studies of DNA repair with alkaline and neutral sucrose gradients indicated that the accumulation of unrepaired daughter strand gaps led to the formation of low-molecular-weight DNA duplexes (i.e., DNA double-strand breaks were formed). The uvrB recF cells were able to regenerate high-molecular-weight DNA from these low-molecular-weight DNA duplexes, whereas the uvrB recF recB and uvrB recA cells were not. A model for the recB-dependent pathway of postreplication repair is presented.

New mutation (mmrA1) in Escherichia coli K-12 that affects minimal medium recovery and postreplication repair after UV irradiation.

After UV irradiation, Escherichia coli uvrA mutant cells show higher survival on minimal than on rich growth medium, i.e., they show minimal-medium recovery. This effect of rich growth medium on survival is not observed in a uvrA mutant carrying an mmrA1 mutation, and the uvrA mmrA strain showed the same survival rate on minimal and rich growth media as the uvrA strain did on minimal medium plates. The mmrA1 mutation was isolated as a hidden mutation from a uvrA polA mutant strain and shown to map at 84.3 min on the E. coli K-12 linkage map. In contrast to the uvrA strain, the repair of DNA daughter strand gaps was not inhibited in the uvrA mmrA strain by rich growth medium after irradiation. However, the uvrA and uvrA mmrA strains were similar in their ability to repair DNA when compared in minimal medium. These data are consistent with the idea that the mmr gene product is not involved directly in the repair of UV radiation-induced DNA damage, but rather allows rich growth medium to inhibit a portion of postreplication repair.