scholarly journals Partial Depletion of Histone H4 Increases Homologous Recombination-Mediated Genetic Instability

2005 ◽  
Vol 25 (4) ◽  
pp. 1526-1536 ◽  
Author(s):  
Félix Prado ◽  
Andrés Aguilera
Keyword(s):  
Dna Replication ◽  
Homologous Recombination ◽  
Dna Sequences ◽  
Genetic Instability ◽  
Yellow Fluorescent Protein ◽  
Dna Lesions ◽  
Micrococcal Nuclease ◽  
Histone H4 ◽  
Nucleosome Assembly ◽  
Partial Depletion

ABSTRACT DNA replication can be a source of genetic instability. Given the tight connection between DNA replication and nucleosome assembly, we analyzed the effect of a partial depletion of histone H4 on genetic instability mediated by homologous recombination. A Saccharomyces cerevisiae strain was constructed in which the expression of histone H4 was driven by the regulated tet promoter. In agreement with defective nucleosome assembly, partial depletion of histone H4 led to subtle changes in plasmid superhelical density and chromatin sensitivity to micrococcal nuclease. Under these conditions, homologous recombination between ectopic DNA sequences was increased 20-fold above the wild-type levels. This hyperrecombination was not associated with either defective repair or transcription but with an accumulation of recombinogenic DNA lesions during the S and G2/M phases, as determined by an increase in the proportion of budded cells containing Rad52-yellow fluorescent protein foci. Consistently, partial depletion of histone H4 caused a delay during the S and G2/M phases. Our results suggest that histone deposition defects lead to the formation of recombinogenic DNA structures during replication that increase genomic instability.

scholarly journals Plant DNA Polymerases

2019 ◽  
Vol 20 (19) ◽  
pp. 4814 ◽  
Author(s):  
Jose-Antonio Pedroza-Garcia ◽  
Lieven De Veylder ◽  
Cécile Raynaud
Keyword(s):  
Dna Repair ◽  
Dna Replication ◽  
Dna Sequences ◽  
Nuclear Dna ◽  
Protein Complexes ◽  
Dna Polymerases ◽  
Variable Number ◽  
Dna Lesions ◽  
Plant Dna ◽  
Organellar Dna

Maintenance of genome integrity is a key process in all organisms. DNA polymerases (Pols) are central players in this process as they are in charge of the faithful reproduction of the genetic information, as well as of DNA repair. Interestingly, all eukaryotes possess a large repertoire of polymerases. Three protein complexes, DNA Pol α, δ, and ε, are in charge of nuclear DNA replication. These enzymes have the fidelity and processivity required to replicate long DNA sequences, but DNA lesions can block their progression. Consequently, eukaryotic genomes also encode a variable number of specialized polymerases (between five and 16 depending on the organism) that are involved in the replication of damaged DNA, DNA repair, and organellar DNA replication. This diversity of enzymes likely stems from their ability to bypass specific types of lesions. In the past 10–15 years, our knowledge regarding plant DNA polymerases dramatically increased. In this review, we discuss these recent findings and compare acquired knowledge in plants to data obtained in other eukaryotes. We also discuss the emerging links between genome and epigenome replication.

DNA replication-associated lesions: importance in early tumorigenesis and cancer therapy

2007 ◽  
Vol 35 (5) ◽  
pp. 1352-1354 ◽  
Author(s):  
E. Petermann ◽  
T. Helleday
Keyword(s):  
Dna Replication ◽  
Cancer Therapy ◽  
Genetic Instability ◽  
Cellular Response ◽  
Dna Lesions ◽  
Replication Forks ◽  
Associated Lesions ◽  
Repair Pathways

DNA lesions resulting from impaired progression of replication forks are implicated in genetic instability and tumorigenesis. Because the cellular response to these lesions poses an important tumorigenesis barrier, the responsible signalling and repair pathways are often mutated or inactive in tumours. Here, we discuss how such deficiencies can in turn be exploited for cancer therapy.

scholarly journals Recombinational Repair of DNA Damage inEscherichia coli and Bacteriophage λ

1999 ◽  
Vol 63 (4) ◽  
pp. 751-813 ◽  
Author(s):  
Andrei Kuzminov
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Replication ◽  
Homologous Recombination ◽  
Dna Lesions ◽  
Recombinational Repair ◽  
Recombination Proteins ◽  
Recombination System ◽  
Major Reaction

SUMMARY Although homologous recombination and DNA repair phenomena in bacteria were initially extensively studied without regard to any relationship between the two, it is now appreciated that DNA repair and homologous recombination are related through DNA replication. In Escherichia coli, two-strand DNA damage, generated mostly during replication on a template DNA containing one-strand damage, is repaired by recombination with a homologous intact duplex, usually the sister chromosome. The two major types of two-strand DNA lesions are channeled into two distinct pathways of recombinational repair: daughter-strand gaps are closed by the RecF pathway, while disintegrated replication forks are reestablished by the RecBCD pathway. The phage λ recombination system is simpler in that its major reaction is to link two double-stranded DNA ends by using overlapping homologous sequences. The remarkable progress in understanding the mechanisms of recombinational repair in E. coli over the last decade is due to the in vitro characterization of the activities of individual recombination proteins. Putting our knowledge about recombinational repair in the broader context of DNA replication will guide future experimentation.

scholarly journals DNA polymerase ζ in DNA replication and repair

2019 ◽  
Vol 47 (16) ◽  
pp. 8348-8361 ◽  
Author(s):  
Sara K Martin ◽  
Richard D Wood
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Dna Sequences ◽  
Mammalian Cells ◽  
Translesion Synthesis ◽  
Dna Lesions ◽  
Replication Forks ◽  
Dna Replication And Repair ◽  
Radiation Induced ◽  
Break Induced Replication

Abstract Here, we survey the diverse functions of DNA polymerase ζ (pol ζ) in eukaryotes. In mammalian cells, REV3L (3130 residues) is the largest catalytic subunit of the DNA polymerases. The orthologous subunit in yeast is Rev3p. Pol ζ also includes REV7 subunits (encoded by Rev7 in yeast and MAD2L2 in mammalian cells) and two subunits shared with the replicative DNA polymerase, pol δ. Pol ζ is used in response to circumstances that stall DNA replication forks in both yeast and mammalian cells. The best-examined situation is translesion synthesis at sites of covalent DNA lesions such as UV radiation-induced photoproducts. We also highlight recent evidence that uncovers various roles of pol ζ that extend beyond translesion synthesis. For instance, pol ζ is also employed when the replisome operates sub-optimally or at difficult-to-replicate DNA sequences. Pol ζ also participates in repair by microhomology mediated break-induced replication. A rev3 deletion is tolerated in yeast but Rev3l disruption results in embryonic lethality in mice. Inactivation of mammalian Rev3l results in genomic instability and invokes cell death and senescence programs. Targeting of pol ζ function may be a useful strategy in cancer therapy, although chromosomal instability associated with pol ζ deficiency must be considered.

scholarly journals Conditional knockout of RAD51-related genes in Leishmania major reveals a critical role for homologous recombination during genome replication

2019 ◽  
Author(s):  
Jeziel D. Damasceno ◽  
João Reis-Cunha ◽  
Kathryn Crouch ◽  
Craig Lapsley ◽  
Luiz R. O. Tosi ◽  
...  
Keyword(s):  
Dna Replication ◽  
Homologous Recombination ◽  
Dna Synthesis ◽  
Leishmania Major ◽  
Critical Role ◽  
Dna Lesions ◽  
Replication Initiation ◽  
Conditional Knockout ◽  
Genome Replication ◽  
Genome Wide

AbstractHomologous recombination (HR) has an intimate relationship with genome replication, both during repair of DNA lesions that might prevent DNA synthesis and in tackling stalls to the replication fork. Recent studies led us to ask if HR might have a more central role in replicating the genome of Leishmania, a eukaryotic parasite. Conflicting evidence has emerged regarding whether or not HR genes are essential, and genome-wide mapping has provided evidence for an unorthodox organisation of DNA replication initiation sites, termed origins. To answer this question, we have employed a combined CRISPR/Cas9 and DiCre approach to rapidly generate and assess the effect of conditional ablation of RAD51 and three RAD51-related proteins in Leishmania major. Using this approach, we demonstrate that loss of any of these HR factors is not immediately lethal, but in each case growth slows with time and leads to DNA damage, accumulation of cells with aberrant DNA content, and genome-wide mutation. Despite these similarities, we show that only loss of RAD51 and RAD51-3 impairs DNA synthesis, and that the factors act in distinct ways. Finally, we reveal that loss of RAD51 has a profound effect on DNA replication, causing loss of initiation at the major origins and increased DNA synthesis at subtelomeres. Our work clarifies questions regarding the importance of HR to survival of Leishmania and reveals an unanticipated, central role for RAD51 in the programme of genome replication in a microbial eukaryote.

scholarly journals E2F1 facilitates DNA break repair by localizing to break sites and enhancing the expression of homologous recombination factors

2019 ◽  
Vol 51 (9) ◽  
pp. 1-12 ◽  
Author(s):  
Eui-Hwan Choi ◽  
Keun Pil Kim
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Replication ◽  
Homologous Recombination ◽  
Tumor Development ◽  
Dna Lesions ◽  
Dna Mutations ◽  
Dna Break ◽  
Risk Of Cancer ◽  
Fundamental Shift

Abstract The human genome is constantly exposed to both endogenous and exogenous stresses, which can lead to errors in DNA replication and the accumulation of DNA mutations, thereby increasing the risk of cancer development. The transcription factor E2F1 is a key regulator of DNA repair. E2F1 also has defined roles in the replication of many cell cycle-related genes and is highly expressed in cancer cells, and its abundance is strongly associated with poor prognosis in cancers. Studies on colon cancer have demonstrated that the depletion of E2F1 leads to reduced levels of homologous recombination (HR), resulting in interrupted DNA replication and the subsequent accumulation of DNA lesions. Our results demonstrate that the depletion of E2F1 also causes reduced RAD51-mediated DNA repair and diminished cell viability resulting from DNA damage. Furthermore, the extent of RAD51 and RPA colocalization is reduced in response to DNA damage; however, RPA single-stranded DNA (ssDNA) nucleofilament formation is not affected following the depletion of E2F1, implying that ssDNA gaps accumulate when RAD51-mediated DNA gap filling or repair is diminished. Surprisingly, we also demonstrate that E2F1 forms foci with RAD51 or RPA at DNA break sites on damaged DNA. These findings provide evidence of a molecular mechanism underlying the E2F1-mediated regulation of HR activity and predict a fundamental shift in the function of E2F1 from regulating cell division to accelerating tumor development.

Inhibitors of topoisomerases I and II arrest DNA replication, but do not prevent nucleosome assembly in vivo

1989 ◽  
Vol 93 (4) ◽  
pp. 593-603
Author(s):  
A.T. Annunziato
Keyword(s):  
Dna Replication ◽  
Chromatin Assembly ◽  
Micrococcal Nuclease ◽  
Nucleosome Assembly ◽  
Protein Synthesis Inhibitors ◽  
Experimental Protocol ◽  
Eukaryotic Dna ◽  
Replication Intermediates ◽  
Experimental Strategies

Specific inhibitors of eukaryotic DNA topoisomerases I and II (camptothecin and VM-26, respectively) were used to examine the involvement of topoisomerases in DNA replication and chromatin assembly in vivo. When used singly, either camptothecin or VM-26 inhibited DNA synthesis in HeLa cells by more than 80%; when used simultaneously, the inhibitors effectively stopped replication, demonstrating that at least one class of topoisomerase must be active for fork propagation in vivo. To study nucleosome assembly during topoisomerase inhibition, three experimental strategies were employed: (1) pulse-chase experiments; (2) analyses of chromatin synthesized during residual replication in the presence of either camptothecin or VM-26; and (3) the assembly of previously replicated, unassembled DNA, generated in the presence of protein synthesis inhibitors. Using sensitivity to micrococcal nuclease and the maturation of non-nucleosomal replication intermediates as criteria, neither camptothecin nor VM-26, alone or in concert, inhibited nucleosome assembly under any experimental protocol tested. These data provide evidence that, although topoisomerase activity is essential for DNA replication, neither continuous fork propagation nor topoisomerase activity is required for chromatin assembly on new DNA.

UV Stimulation of Chromosomal Marker Exchange in Sulfolobus acidocaldarius: Implications for DNA Repair, Conjugation and Homologous Recombination at Extremely High Temperatures

Genetics ◽  
1999 ◽  
Vol 152 (4) ◽  
pp. 1407-1415 ◽  
Author(s):  
Katherine J Schmidt ◽  
Kristen E Beck ◽  
Dennis W Grogan
Keyword(s):  
Homologous Recombination ◽  
Incubation Temperature ◽  
Uv Light ◽  
Dna Lesions ◽  
Sulfolobus Acidocaldarius ◽  
Chromosomal Marker ◽  
Marker Exchange ◽  
Chromosomal Markers ◽  
Rate Of Decay ◽  
Uv Photoproducts

Abstract The hyperthermophilic archaeon Sulfolobus acidocaldarius exchanges and recombines chromosomal markers by a conjugational mechanism, and the overall yield of recombinants is greatly increased by previous exposure to UV light. This stimulation was studied in an effort to clarify its mechanism and that of marker exchange itself. A variety of experiments failed to identify a significant effect of UV irradiation on the frequency of cell pairing, indicating that subsequent steps are primarily affected, i.e., transfer of DNA between cells or homologous recombination. The UV-induced stimulation decayed rather quickly in parental cells during preincubation at 75°, and the rate of decay depended on the incubation temperature. Preincubation at 75° decreased the yield of recombinants neither from unirradiated parental cells nor from parental suspensions subsequently irradiated. We interpret these results as evidence that marker exchange is stimulated by recombinogenic DNA lesions formed as intermediates in the process of repairing UV photoproducts in the S. acidocaldarius chromosome.

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