DNA damage-signaling, homologous recombination and genetic mutation induced by 5-azacytidine and DNA-protein crosslinks in Escherichia coli

ABSTRACT After unsuccessful attempts to recover a viable RecA-deficient mutant of the Lyme borreliosis agent Borrelia burgdorferi, we characterized the functional activities of RecA of B. burgdorferi, as well as RecA of the relapsing fever spirochete Borrelia hermsii and the free-living spirochete Leptospira biflexa, in a recA mutant of Escherichia coli. As a control, E. coli RecA was expressed from the same plasmid vector. DNA damage repair activity was assessed after exposure of the transgenic cells to UV light or the radiomimetic chemicals methyl methanesulfonate and mitomycin C. Recombination activity in the cells was assessed by using an assay for homologous recombination between repeats in the chromosome and by measuring the ability of the cells to foster lytic growth by red gam mutant bacteriophage λ. Overall, we found that transgenic cells with recA genes of B. burgdorferi, B. hermsii, and L. biflexa had approximately equivalent activities in promoting homologous recombination in the lacZ duplication assay, but cells with B. burgdorferi recA and, most notably, B. hermsii recA were significantly less capable than cells with L. biflexa recA or E. coli recA in responding to DNA damage or in facilitating plaque formation in the phage assay. The comparatively poor function of Borrelia recA in the latter set of assays may be the consequence of impaired coordination in the loading of the transgenic RecA by RecBCD and/or RecFOR in E. coli.

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TOPBP1 takes RADical command in recombinational DNA repair

The Journal of Cell Biology ◽

10.1083/jcb.201601028 ◽

2016 ◽

Vol 212 (3) ◽

pp. 263-266 ◽

Cited By ~ 6

Author(s):

Yi Liu ◽

Marcus B. Smolka

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Dna Replication ◽

Homologous Recombination ◽

Crucial Role ◽

Double Strand Breaks ◽

Strand Breaks ◽

Dna Damage Signaling ◽

Cell Biol ◽

Recombinational Dna Repair

TOPBP1 is a key player in DNA replication and DNA damage signaling. In this issue, Moudry et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201507042) uncover a crucial role for TOPBP1 in DNA repair by revealing its requirement for RAD51 loading during repair of double strand breaks by homologous recombination.

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HP1α recruitment to DNA damage by p150CAF-1 promotes homologous recombination repair

The Journal of Cell Biology ◽

10.1083/jcb.201101030 ◽

2011 ◽

Vol 193 (1) ◽

pp. 81-95 ◽

Cited By ~ 146

Author(s):

Céline Baldeyron ◽

Gaston Soria ◽

Danièle Roche ◽

Adam J. L. Cook ◽

Geneviève Almouzni

Keyword(s):

Dna Damage ◽

Homologous Recombination ◽

Mammalian Cells ◽

De Novo ◽

Heterochromatin Protein 1 ◽

Transient Event ◽

Dna Damage Signaling ◽

Recombination Repair ◽

Assembly Factor ◽

Laser Induced Damage

Heterochromatin protein 1 (HP1), a major component of constitutive heterochromatin, is recruited to DNA damage sites. However, the mechanism involved in this recruitment and its functional importance during DNA repair remain major unresolved issues. Here, by characterizing HP1α dynamics at laser-induced damage sites in mammalian cells, we show that the de novo accumulation of HP1α occurs within both euchromatin and heterochromatin as a rapid and transient event after DNA damage. This recruitment is strictly dependent on p150CAF-1, the largest subunit of chromatin assembly factor 1 (CAF-1), and its ability to interact with HP1α. We find that HP1α depletion severely compromises the recruitment of the DNA damage response (DDR) proteins 53BP1 and RAD51. Moreover, HP1α depletion leads to defects in homologous recombination–mediated repair and reduces cell survival after DNA damage. Collectively, our data reveal that HP1α recruitment at early stages of the DDR involves p150CAF-1 and is critical for proper DNA damage signaling and repair.

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Distribution of RecBCD and AddAB recombination-associated genes among bacteria in 33 phyla

Microbiology ◽

10.1099/mic.0.000980 ◽

2020 ◽

Vol 166 (11) ◽

pp. 1047-1064 ◽

Cited By ~ 1

Author(s):

Deepti Gurung ◽

Robert M. Blumenthal

Keyword(s):

Escherichia Coli ◽

Dna Damage ◽

Homologous Recombination ◽

Dna Sequences ◽

Type Species ◽

Bacterial Species ◽

Distribution Patterns ◽

Content Type ◽

Link Type ◽

Open Questions

Homologous recombination plays key roles in fundamental processes such as recovery from DNA damage and in bacterial horizontal gene transfer, yet there are still open questions about the distribution of recognized components of recombination machinery among bacteria and archaea. RecBCD helicase-nuclease plays a central role in recombination among Gammaproteobacteria like Escherichia coli ; while bacteria in other phyla, like the Firmicute Bacillus subtilis , use the related AddAB complex. The activity of at least some of these complexes is controlled by short DNA sequences called crossover hotspot instigator (Chi) sites. When RecBCD or AddAB complexes encounter an autologous Chi site during unwinding, they introduce a nick such that ssDNA with a free end is available to invade another duplex. If homologous DNA is present, RecA-dependent homologous recombination is promoted; if not (or if no autologous Chi site is present) the RecBCD/AddAB complex eventually degrades the DNA. We examined the distribution of recBCD and addAB genes among bacteria, and sought ways to distinguish them unambiguously. We examined bacterial species among 33 phyla, finding some unexpected distribution patterns. RecBCD and addAB are less conserved than recA, with the orthologous recB and addA genes more conserved than the recC or addB genes. We were able to classify RecB vs. AddA and RecC vs. AddB in some bacteria where this had not previously been done. We used logo analysis to identify sequence segments that are conserved, but differ between the RecBC and AddAB proteins, to help future differentiation between members of these two families.

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DNA damage-signaling, homologous recombination and genetic mutation induced by 5-azacytidine and DNA-protein crosslinks in Escherichia coli

10.1101/2020.10.27.357855 ◽

2020 ◽

Author(s):

Julie A. Klaric ◽

David J. Glass ◽

Eli L. Perr ◽

Arianna D. Reuven ◽

Mason J. Towne ◽

...

Keyword(s):

Escherichia Coli ◽

Dna Damage ◽

Homologous Recombination ◽

Genetic Instability ◽

Tandem Repeats ◽

Sos Response ◽

Chemotherapeutic Agents ◽

Alternative Form ◽

Independent Effect ◽

List Type

ABSTRACTCovalent linkage between DNA and proteins produces highly toxic lesions and can be caused by commonly used chemotherapeutic agents, by internal and external chemicals and by radiation. In this study, using Escherichia coli, we investigate the consequences of 5-azacytidine (5-azaC), which traps covalent complexes between itself and the Dcm cytosine methyltransferase protein. DNA protein crosslink-dependent effects can be ascertained by effects that arise in wild-type but not in dcmΔ strains. We find that 5-azaC induces the bacterial DNA damage response and stimulates homologous recombination, a component of which is Dcm-dependent. Template-switching at an imperfect inverted repeat (“quasipalindrome”, QP) is strongly enhanced by 5-azaC and this enhancement was entirely Dcm-dependent. The SOS response helps ameliorate the mutagenic effect of 5-azaC but unbalanced expression of the SOS-induced DNA polymerases, especially PolIV, stimulates QP-associated mutagenesis. In the absence of Lon protease, Dcm-dependent QP-mutagenesis is elevated, suggesting it may play a role in 5-azaC tolerance. Deletions at short tandem repeats, which occur likewise by a replication template-switch, are elevated, but only modestly, by 5-azaC. We see evidence for Dcm-dependent and-independent killing by 5-azaC in sensitive mutants, such as recA, recB, and lon; homologous recombination and deletion mutations are also stimulated in part by a Dcm-independent effect of 5-azaC. Whether this occurs by a different protein/DNA crosslink or by an alternative form of DNA damage is unknown.Highlights5-azacytidine is broadly mutagenic and recombinogenicIn E. coli, 5-azaC promotes genetic instability through Dcm methyltransferase.There are other, unknown lesions induced by 5-azaC besides Dcm/DNA crosslinks5-azaC induces the SOS response, protecting cells from killing and genetic instability

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RAD52 Functions in Homologous Recombination and Its Importance on Genomic Integrity Maintenance and Cancer Therapy

Cancers ◽

10.3390/cancers11111622 ◽

2019 ◽

Vol 11 (11) ◽

pp. 1622 ◽

Cited By ~ 9

Author(s):

Augusto Nogueira ◽

Mara Fernandes ◽

Raquel Catarino ◽

Rui Medeiros

Keyword(s):

Dna Damage ◽

Cancer Therapy ◽

Homologous Recombination ◽

Signaling Pathways ◽

Chromosomal Abnormalities ◽

Cell Cycle Checkpoints ◽

Cytotoxic Agents ◽

Dna Damage Signaling ◽

Physiological Processes ◽

Repair Mechanisms

Genomes are continually subjected to DNA damage whether they are induced from intrinsic physiological processes or extrinsic agents. Double-stranded breaks (DSBs) are the most injurious type of DNA damage, being induced by ionizing radiation (IR) and cytotoxic agents used in cancer treatment. The failure to repair DSBs can result in aberrant chromosomal abnormalities which lead to cancer development. An intricate network of DNA damage signaling pathways is usually activated to eliminate these damages and to restore genomic stability. These signaling pathways include the activation of cell cycle checkpoints, DNA repair mechanisms, and apoptosis induction, also known as DNA damage response (DDR)-mechanisms. Remarkably, the homologous recombination (HR) is the major DSBs repairing pathway, in which RAD52 gene has a crucial repairing role by promoting the annealing of complementary single-stranded DNA and by stimulating RAD51 recombinase activity. Evidence suggests that variations in RAD52 expression can influence HR activity and, subsequently, influence the predisposition and treatment efficacy of cancer. In this review, we present several reports in which the down or upregulation of RAD52 seems to be associated with different carcinogenic processes. In addition, we discuss RAD52 inhibition in DDR-defective cancers as a possible target to improve cancer therapy efficacy.

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