Search

While the molecular repertoire of the homologous recombination pathways is well studied, the search mechanism that enables recombination between distant homologous regions is poorly understood. Earlier work suggests that the recombinase RecA, an essential component for homology search, forms an elongated filament, nucleating at the break site. How this RecA structure carries out long-distance search remains unclear. Here, we follow the dynamics of RecA after induction of a single double-strand break on the Caulobacter chromosome. We find that the RecA-nucleoprotein filament, once formed, rapidly translocates in a directional manner in the cell, undergoing several pole-to-pole traversals, until homology search is complete. Concomitant with translocation, we observe dynamic variation in the length of the filament. Importantly in vivo, the RecA filament alone is incapable of such long-distance movement; both translocation and associated length variations are contingent on action of structural maintenance of chromosome (SMC)-like protein RecN, via its ATPase cycle. In summary, we have uncovered the three key elements of homology search driven by RecN: mobility of a finite segment of RecA, changes in filament length, and ability to conduct multiple pole-to-pole traversals, which together point to an optimal search strategy. [ABSTRACT FROM AUTHOR]

Zinc finger (ZnF) proteins represent one of the largest families of human proteins, although most remain uncharacterized. Given that numerous ZnF proteins are able to interact with DNA and poly(ADP ribose), there is growing interest in understanding their mechanism of action in the maintenance of genome integrity. We now report that the ZnF protein E4F transcription factor 1 (E4F1) is an actor in DNA repair. Indeed, E4F1 is rapidly recruited, in a poly(ADP ribose) polymerase (PARP)-dependent manner, to DNA breaks and promotes ATR/CHK1 signaling, DNA-end resection, and subsequent homologous recombination. Moreover, we identify E4F1 as a regulator of the ATP-dependent chromatin remodeling SWI/SNF complex in DNA repair. E4F1 binds to the catalytic subunit BRG1/SMARCA4 and together with PARP-1 mediates its recruitment to DNA lesions. We also report that a proportion of human breast cancers show amplification and overexpression of E4F1 or BRG1 that are mutually exclusive with BRCA1/2 alterations. Together, these results reveal a function of E4F1 in the DNA damage response that orchestrates proper signaling and repair of double-strand breaks and document a molecular mechanism for its essential role in maintaining genome integrity and cell survival. [ABSTRACT FROM AUTHOR]

Interhomolog recombination (IHR) occurs spontaneously in somatic human cells at frequencies that are low but sufficient to ameliorate some genetic diseases caused by heterozygous mutations or autosomal dominant mutations. Here we demonstrate that DNA nicks or double-strand breaks (DSBs) targeted by CRISPR-Cas9 to both homologs can stimulate IHR and associated copy-neutral loss of heterozygosity (cnLOH) in human cells. The frequency of IHR is 10-fold lower at nicks than at DSBs, but cnLOH is evident in a greater fraction of recombinants. IHR at DSBs occurs predominantly via reciprocal end joining. At DSBs, depletion of POLQ caused a dramatic increase in IHR and in the fraction of recombinants exhibiting cnLOH, suggesting that POLQ promotes end joining in cis, which limits breaks available for recombination in trans. These results define conditions that may produce cnLOH as a mutagenic signature in cancer and may, conversely, promote therapeutic correction of both compound heterozygous and dominant negative mutations associated with genetic disease. [ABSTRACT FROM AUTHOR]

One of the hallmarks of DNA damage is the rapid spreading of phosphorylated histone H2A (-H2AX) around a DNA doublestrand break (DSB). In the budding yeast Saccharomyces cerevisiae, nearly all H2A isoforms can be phosphorylated, either by Mec1ATR or Tel1ATM checkpoint kinases. We induced a site-specific DSB with HO endonuclease at the MAT locus on chromosome III and monitored the formation of ?-H2AX by chromatin immunoprecipitation (ChIP)- qPCR in order to uncover the mechanisms by which Mec1ATR and Tel1ATM propagate histone modifications across chromatin. With either kinase, ?-H2AX spreads as far as ~50 kb on both sides of the lesion within 1 h; but the kinetics and distribution of modification around the DSB are significantly different. The total accumulation of phosphorylation is reduced by about half when either of the two H2A genes is mutated to the nonphosphorylatable S129A allele. Mec1 activity is limited by the abundance of its ATRIP partner, Ddc2. Moreover, Mec1 is more efficient than Tel1 at phosphorylating chromatin in trans--at distant undamaged sites that are brought into physical proximity to the DSB. We compared experimental data to mathematical models of spreading mechanisms to determine whether the kinases search for target nucleosomes by primarily moving in three dimensions through the nucleoplasm or in one dimension along the chromatin. Bayesian model selection indicates that Mec1 primarily uses a three-dimensional diffusive mechanism, whereas Tel1 undergoes directed motion along the chromatin. [ABSTRACT FROM AUTHOR]

Topoisomerase I (Top1) resolves supercoils by nicking one DNA strand and facilitating religation after torsional stress has been relieved. During its reaction cycle, Top1 forms a covalent cleavage complex (Top1cc) with the nicked DNA, and this intermediate can be converted into a toxic double-strand break (DSB) during DNA replication. We previously reported that Top1cc trapping in yeast increases DSB-independent, short deletions at tandemly repeated sequences. In the current study, we report a type of DSBdependent mutation associated with Top1cc stabilization: large deletions (median size, ~100 bp) with little or no homology at deletion junctions. Genetic analyses demonstrated that Top1ccdependent large deletions are products of the nonhomologous end-joining (NHEJ) pathway and require Top1cc removal from DNA ends. Furthermore, these events accumulated in quiescent cells, suggesting that the causative DSBs may arise outside the context of replication. We propose a model in which the ends of different, Top1-associated DSBs are joined via NHEJ, which results in deletion of the intervening sequence. These findings have important implications for understanding the mutagenic effects of chemotherapeutic drugs that stabilize the Top1cc. [ABSTRACT FROM AUTHOR]

Histone methyltransferase G9a has critical roles in promoting cancer-cell growth and gene suppression, but whether it is also associated with the DNA damage response is rarely studied. Here, we report that loss of G9a impairs DNA damage repair and enhances the sensitivity of cancer cells to radiation and chemotherapeutics. In response to DNA double-strand breaks (DSBs), G9a is phosphorylated at serine 211 by casein kinase 2 (CK2) and recruited to chromatin. The chromatin-enriched G9a can then directly interact with replication protein A (RPA) and promote loading of the RPA and Rad51 recombinase to DSBs. This mechanism facilitates homologous recombination (HR) and cell survival. We confirmed the interaction between RPA and G9a to be critical for RPA foci formation and HR upon DNA damage. Collectively, our findings demonstrate a regulatory pathway based on CK2-G9a-RPA that permits HR in cancer cells and provide further rationale for the use of G9a inhibitors as a cancer therapeutic. [ABSTRACT FROM AUTHOR]

(-)-Lomaiviticin A (1) is a complex antiproliferative metabolite that inhibits the growth of many cultured cancer cell lines at low nanomolar-picomolar concentrations. (-)-Lomaiviticin A (1) possesses a C2-symmetric structure that contains two unusual diazotetrahydrobenzo[ b]fluorene (diazofluorene) functional groups. Nucleophilic activation of each diazofluorene within 1 produces vinyl radical intermediates that affect hydrogen atom abstraction from DNA, leading to the formation of DNA double-strand breaks (DSBs). Certain DNA DSB repair-deficient cell lines are sensitized toward 1, and 1 is under evaluation in preclinical models of these tumor types. However, the mode of binding of 1 to DNA had not been determined. Here we elucidate the structure of a 1:1 complex between 1 and the duplex d(GCTATAGC)2 by NMR spectroscopy and computational modeling. Unexpectedly, we show that both diazofluorene residues of 1 penetrate the duplex. This binding disrupts base pairing leading to ejection of the central AT bases, while placing the proreactive centers of 1 in close proximity to each strand. DNA binding may also enhance the reactivity of 1 toward nucleophilic activation through steric compression and conformational restriction (an example of shape-dependent catalysis). This study provides a structural basis for the DNA cleavage activity of 1, will guide the design of synthetic DNA-activated DNA cleavage agents, and underscores the utility of natural products to reveal novel modes of small molecule-DNA association. [ABSTRACT FROM AUTHOR]

Defective homologous recombination (HR) DNA repair imposed by BRCA1 or BRCA2 deficiency sensitizes cells to poly (ADP-ribose) polymerase (PARP)-1 inhibition and is currently exploited in clinical treatment of HR-deficient tumors. Here we show that mild hyperthermia (41-42.5 °C) induces degradation of BRCA2 and inhibits HR. We demonstrate that hyperthermia can be used to sensitize innately HR-proficient tumor cells to PARP-1 inhibitors and that this effect can be enhanced by heat shock protein inhibition. Our results, obtained from cell lines and in vivo tumor models, enable the design of unique therapeutic strategies involving localized on-demand induction of HR deficiency, an approach that we term induced synthetic lethality. [ABSTRACT FROM AUTHOR]

Given its significant role in the maintenance of genomic stability, histone methylation has been postulated to regulate DNA repair. Histone methylation mediates localization of 53BP1 to a DNA double-strand break (DSB) during homologous recombination repair, but a role in DSB repair by nonhomologous end-joining (NHEJ) has not been defined. By screening for histone methylation after DSB induction by ionizing radiation we found that generation of dimethyl histone H3 lysine 36 (H3K36me2) was the major event. Using a novel human cell system that rapidly generates single defined DSB in the vast majority of cells, we found that the DNA repair protein Metnase (also SETMAR), which has a SET histone methylase domain, localized to an induced DSB and directly mediated the formation of H3K36me2 near the induced DSB. This dimethylation of H3K36 improved the association of early DNA repair components, including NBS1 and Ku70, with the induced DSB, and enhanced DSB repair. In addition, expression of JHDM1a (an H3K36me2 demethylase) or histone H3 in which K36 was mutated to A36 or R36 to prevent H3K36me2 formation decreased the association of early NHEJ repair components with an induced DSB and decreased DSB repair. Thus, these experiments define a histone methylation event that enhances DNA DSB repair by NHEJ. [ABSTRACT FROM AUTHOR]

Most carcinomas present some form of chromosome instability in combination with spindle defects. Numerical instability is likely caused by spindle aberrations, but the origin of breaks and translocations remains elusive. To determine whether one mechanism can bring about both types of instability, we studied the relationship between DNA damage and spindle defects. Although lacking apparent repair defects, primary Dido mutant cells formed micronuclei containing damaged DNA. The presence of centromeres showed that micronuclei were caused by spindle defects, and cell cycle markers showed that DNA damage was generated during mitosis. Although the micronuclei themselves persisted, the DNA damage within was repaired during S and G2 phases. DNA breaks in Dido mutant cells regularly colocalized with centromeres, which were occasionally distorted. Comparable defects were found in APC mutant cell lines, an independent system for spindle defects. On the basis of these results, we propose a model for break formation in which spindle defects lead to centromere shearing. [ABSTRACT FROM AUTHOR]

In both prokaryotes and eukaryotes. proteins involved in DNA repair often organize into multicomponent complexes that can be visualized as foci in living cells. We used a RecA-GFP fusion to examine the subcellular cues that direct RecA-GFP to assemble as foci in response to DNA damage. We used two different methods to inhibit initiation of DNA replication and determined that DNA replication is required for the cell to establish RecA-GFP foci after exposure to DNA-damaging agents. Furthermore, use of endonuclease cleavage to generate a site-specific double-strand break demonstrated that the replication machinery (replisome) and DNA synthesis are required for assembly of RecA-GFP foci during repair of a double-strand break. We monitored the cellular levels of RecA and found that focus formation does not require further induction of protein levels, suggesting that foci result from a redistribution of existing protein to sites of damage encountered by the replisome. Taken together, our results support the model that existing RecA protein is recruited to ssDNA generated by the replisome at sites of DNA damage. These results provide insight into the mechanisms that the cell uses to recruit repair proteins to damaged DNA in living cells. [ABSTRACT FROM AUTHOR]

The BRCA2 tumor suppressor plays an important role in the repair of DNA damage by homologous recombination, also termed homology-directed repair (HDR). Human BRCA2 is 3,418 aa and is composed of several domains. The central part of the protein contains multiple copies of a motif that binds the Rad51 recombinase (the BRC repeat), and the C terminus contains domains that have structural similarity to domains in the ssDNA-binding protein replication protein A (RPA). To gain insight into the role of BRCA2 in the repair of DNA damage, we fused a single (BRC3, BRC4) or multiple BRC motifs to the large RPA subunit. Expression of any of these protein fusions in Brca2 mutant cells substantially improved HDR while suppressing mutagenic repair. A fusion containing a Rad52 ssDNA-binding domain also was active in HDR. Mutations that reduced ssDNA or Rad51 binding impaired the ability of the fusion proteins to function in HDR. The high level of spontaneous chromosomal aberrations in Brca2 mutant cells was largely suppressed by the BRC-RPA fusion proteins, supporting the notion that the primary role of BRCA2 in maintaining genomic integrity is in HDR, specifically to deliver Rad51 to ssDNA. The fusion proteins also restored Rad51 focus formation and cellular survival in response to DNA damaging agents. Because as little as 2% of BRCA2 fused to RPA is sufficient to suppress cellular defects found in Brca2-mutant mammalian cells, these results provide insight into the recently discovered diversity of BRCA2 domain structures in different organisms. [ABSTRACT FROM AUTHOR]

One of the hallmarks of DNA damage is the rapid spreading of phosphorylated histone H2A (γ-H2AX) around a DNA double-strand break (DSB). In the budding yeast Saccharomyces cerevisiae , nearly all H2A isoforms can be phosphorylated, either by Mec1 ATR or Tel1 ATM checkpoint kinases. We induced a site-specific DSB with HO endonuclease at the MAT locus on chromosome III and monitored the formation of γ-H2AX by chromatin immunoprecipitation (ChIP)-qPCR in order to uncover the mechanisms by which Mec1 ATR and Tel1 ATM propagate histone modifications across chromatin. With either kinase, γ-H2AX spreads as far as ∼50 kb on both sides of the lesion within 1 h; but the kinetics and distribution of modification around the DSB are significantly different. The total accumulation of phosphorylation is reduced by about half when either of the two H2A genes is mutated to the nonphosphorylatable S129A allele. Mec1 activity is limited by the abundance of its ATRIP partner, Ddc2. Moreover, Mec1 is more efficient than Tel1 at phosphorylating chromatin in trans -at distant undamaged sites that are brought into physical proximity to the DSB. We compared experimental data to mathematical models of spreading mechanisms to determine whether the kinases search for target nucleosomes by primarily moving in three dimensions through the nucleoplasm or in one dimension along the chromatin. Bayesian model selection indicates that Mec1 primarily uses a three-dimensional diffusive mechanism, whereas Tel1 undergoes directed motion along the chromatin., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Topoisomerase I (Top1) resolves supercoils by nicking one DNA strand and facilitating religation after torsional stress has been relieved. During its reaction cycle, Top1 forms a covalent cleavage complex (Top1cc) with the nicked DNA, and this intermediate can be converted into a toxic double-strand break (DSB) during DNA replication. We previously reported that Top1cc trapping in yeast increases DSB-independent, short deletions at tandemly repeated sequences. In the current study, we report a type of DSB-dependent mutation associated with Top1cc stabilization: large deletions (median size, ∼100 bp) with little or no homology at deletion junctions. Genetic analyses demonstrated that Top1cc-dependent large deletions are products of the nonhomologous end-joining (NHEJ) pathway and require Top1cc removal from DNA ends. Furthermore, these events accumulated in quiescent cells, suggesting that the causative DSBs may arise outside the context of replication. We propose a model in which the ends of different, Top1-associated DSBs are joined via NHEJ, which results in deletion of the intervening sequence. These findings have important implications for understanding the mutagenic effects of chemotherapeutic drugs that stabilize the Top1cc., Competing Interests: Competing interest: S.J.-R. and H.L.K. are coauthors on a 2019 review., (Copyright © 2019 the Author(s). Published by PNAS.)

Histone methyltransferase G9a has critical roles in promoting cancer-cell growth and gene suppression, but whether it is also associated with the DNA damage response is rarely studied. Here, we report that loss of G9a impairs DNA damage repair and enhances the sensitivity of cancer cells to radiation and chemotherapeutics. In response to DNA double-strand breaks (DSBs), G9a is phosphorylated at serine 211 by casein kinase 2 (CK2) and recruited to chromatin. The chromatin-enriched G9a can then directly interact with replication protein A (RPA) and promote loading of the RPA and Rad51 recombinase to DSBs. This mechanism facilitates homologous recombination (HR) and cell survival. We confirmed the interaction between RPA and G9a to be critical for RPA foci formation and HR upon DNA damage. Collectively, our findings demonstrate a regulatory pathway based on CK2-G9a-RPA that permits HR in cancer cells and provide further rationale for the use of G9a inhibitors as a cancer therapeutic., Competing Interests: The authors declare no conflict of interest.