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Cold Spring Harbor Perspectives in... Mar 2015Homologous recombination provides high-fidelity DNA repair throughout all domains of life. Live cell fluorescence microscopy offers the opportunity to image individual... (Review)
Review
Homologous recombination provides high-fidelity DNA repair throughout all domains of life. Live cell fluorescence microscopy offers the opportunity to image individual recombination events in real time providing insight into the in vivo biochemistry of the involved proteins and DNA molecules as well as the cellular organization of the process of homologous recombination. Herein we review the cell biological aspects of mitotic homologous recombination with a focus on Saccharomyces cerevisiae and mammalian cells, but will also draw on findings from other experimental systems. Key topics of this review include the stoichiometry and dynamics of recombination complexes in vivo, the choreography of assembly and disassembly of recombination proteins at sites of DNA damage, the mobilization of damaged DNA during homology search, and the functional compartmentalization of the nucleus with respect to capacity of homologous recombination.
Topics: Animals; Biochemical Phenomena; Cell Biology; DNA Breaks, Double-Stranded; DNA Repair; Humans; Mitosis; Models, Biological; Recombination, Genetic; Saccharomyces cerevisiae
PubMed: 25731763
DOI: 10.1101/cshperspect.a016535 -
PloS One Aug 2009The ability to express or deplete proteins in living cells is crucial for the study of biological processes. Viral vectors are often useful to deliver DNA constructs to...
The ability to express or deplete proteins in living cells is crucial for the study of biological processes. Viral vectors are often useful to deliver DNA constructs to cells that are difficult to transfect by other methods. Lentiviruses have the additional advantage of being able to integrate into the genomes of non-dividing mammalian cells. However, existing viral expression systems generally require different vector backbones for expression of cDNA, small hairpin RNA (shRNA) or microRNA (miRNA) and provide limited drug selection markers. Furthermore, viral backbones are often recombinogenic in bacteria, complicating the generation and maintenance of desired clones. Here, we describe a collection of 59 vectors that comprise an integrated system for constitutive or inducible expression of cDNAs, shRNAs or miRNAs, and use a wide variety of drug selection markers. These vectors are based on the Gateway technology (Invitrogen) whereby the cDNA, shRNA or miRNA of interest is cloned into an Entry vector and then recombined into a Destination vector that carries the chosen viral backbone and drug selection marker. This recombination reaction generates the desired product with >95% efficiency and greatly reduces the frequency of unwanted recombination in bacteria. We generated Destination vectors for the production of both retroviruses and lentiviruses. Further, we characterized each vector for its viral titer production as well as its efficiency in expressing or depleting proteins of interest. We also generated multiple types of vectors for the production of fusion proteins and confirmed expression of each. We demonstrated the utility of these vectors in a variety of functional studies. First, we show that the FKBP12 Destabilization Domain system can be used to either express or deplete the protein of interest in mitotically-arrested cells. Also, we generate primary fibroblasts that can be induced to senesce in the presence or absence of DNA damage. Finally, we determined that both isoforms of the AT-Rich Interacting Domain 4B (ARID4B) protein could induce G1 arrest when overexpressed. As new technologies emerge, the vectors in this collection can be easily modified and adapted without the need for extensive recloning.
Topics: Animals; Base Sequence; Cell Line; DNA Primers; DNA, Complementary; Electrophoresis, Polyacrylamide Gel; Flow Cytometry; Fluorescent Antibody Technique; Genetic Vectors; Humans; Proteins; RNA; RNA Interference; Recombination, Genetic; Retroviridae
PubMed: 19657394
DOI: 10.1371/journal.pone.0006529 -
Genetics Jun 2013Meiotic crossovers facilitate the segregation of homologous chromosomes and increase genetic diversity. The formation of meiotic crossovers was previously posited to... (Review)
Review
Meiotic crossovers facilitate the segregation of homologous chromosomes and increase genetic diversity. The formation of meiotic crossovers was previously posited to occur via two pathways, with the relative use of each pathway varying between organisms; however, this paradigm could not explain all crossovers, and many of the key proteins involved were unidentified. Recent studies that identify some of these proteins reinforce and expand the model of two meiotic crossover pathways. The results provide novel insights into the evolutionary origins of the pathways, suggesting that one is similar to a mitotic DNA repair pathway and the other evolved to incorporate special features unique to meiosis.
Topics: Animals; Homologous Recombination; Humans; Meiosis; Mitosis; Mutation; Yeasts
PubMed: 23733849
DOI: 10.1534/genetics.113.150581 -
Genetics Nov 2014Homology-dependent exchange of genetic information between DNA molecules has a profound impact on the maintenance of genome integrity by facilitating error-free DNA... (Review)
Review
Homology-dependent exchange of genetic information between DNA molecules has a profound impact on the maintenance of genome integrity by facilitating error-free DNA repair, replication, and chromosome segregation during cell division as well as programmed cell developmental events. This chapter will focus on homologous mitotic recombination in budding yeast Saccharomyces cerevisiae. However, there is an important link between mitotic and meiotic recombination (covered in the forthcoming chapter by Hunter et al. 2015) and many of the functions are evolutionarily conserved. Here we will discuss several models that have been proposed to explain the mechanism of mitotic recombination, the genes and proteins involved in various pathways, the genetic and physical assays used to discover and study these genes, and the roles of many of these proteins inside the cell.
Topics: DNA Damage; DNA, Fungal; Genetic Techniques; Mitosis; Recombination, Genetic; Saccharomyces cerevisiae
PubMed: 25381364
DOI: 10.1534/genetics.114.166140 -
Trends in Cell Biology Jan 2022Failure to complete DNA replication is one of the major sources of genome instability leading to aneuploidy, chromosome breakage, and chromosome rearrangements that are... (Review)
Review
Failure to complete DNA replication is one of the major sources of genome instability leading to aneuploidy, chromosome breakage, and chromosome rearrangements that are associated with human cancer. One of the surprising revelations of the past decade is that the completion of replication at so-called common fragile sites (CFS) occurs very late in the cell cycle - at mitosis - through a process termed MiDAS (mitotic DNA synthesis). MiDAS is strongly related to another cancer-promoting phenomenon: the activation of alternative lengthening of telomeres (ALT). Our understanding of the mechanisms of ALT and MiDAS in mammalian cells has drawn heavily from recent advances in the study of break-induced replication (BIR), especially in budding yeast. We provide new insights into the BIR, MiDAS, and ALT pathways and their shared similarities.
Topics: Animals; DNA Repair; DNA Replication; Genomic Instability; Humans; Mammals; Recombination, Genetic; Telomere
PubMed: 34384659
DOI: 10.1016/j.tcb.2021.07.005 -
Current Opinion in Genetics &... Dec 2021Saccharomyces cerevisiae is at the forefront of defining the major recombination mechanisms/models that repair targeted double-strand breaks during mitosis. Each of... (Review)
Review
Saccharomyces cerevisiae is at the forefront of defining the major recombination mechanisms/models that repair targeted double-strand breaks during mitosis. Each of these models predicts specific molecular intermediates as well as genetic outcomes. Recent use of single-nucleotide polymorphisms to track the exchange of sequences in recombination products has provided an unprecedented level of detail about the corresponding intermediates and the extents to which different mechanisms are utilized. This approach also has revealed complexities that are not predicted by canonical models, suggesting that modifications to these models are needed. Current data are consistent with the initiation of most inter-homolog spontaneous mitotic recombination events by a double-strand break. In addition, the sister chromatid is preferred over the homolog as a repair template.
Topics: DNA Breaks, Double-Stranded; DNA Repair; Mitosis; Saccharomyces cerevisiae; Sister Chromatid Exchange
PubMed: 34311384
DOI: 10.1016/j.gde.2021.07.002 -
Cold Spring Harbor Perspectives in... Aug 2014A particularly relevant phenomenon in cell physiology and proliferation is the fact that spontaneous mitotic recombination is strongly enhanced by transcription. The... (Review)
Review
A particularly relevant phenomenon in cell physiology and proliferation is the fact that spontaneous mitotic recombination is strongly enhanced by transcription. The most accepted view is that transcription increases the occurrence of double-strand breaks and/or single-stranded DNA gaps that are repaired by recombination. Most breaks would arise as a consequence of the impact that transcription has on replication fork progression, provoking its stalling and/or breakage. Here, we discuss the mechanisms responsible for the cross talk between transcription and recombination, with emphasis on (1) the transcription-replication conflicts as the main source of recombinogenic DNA breaks, and (2) the formation of cotranscriptional R-loops as a major cause of such breaks. The new emerging questions and perspectives are discussed on the basis of the interference between transcription and replication, as well as the way RNA influences genome dynamics.
Topics: DNA; DNA Breaks, Double-Stranded; DNA Replication; Humans; Models, Genetic; RNA; Recombination, Genetic; Transcription, Genetic
PubMed: 25085910
DOI: 10.1101/cshperspect.a016543 -
Cold Spring Harbor Perspectives in... Mar 2015Homologous recombination (HR) and mismatch repair (MMR) are inextricably linked. HR pairs homologous chromosomes before meiosis I and is ultimately responsible for... (Review)
Review
Homologous recombination (HR) and mismatch repair (MMR) are inextricably linked. HR pairs homologous chromosomes before meiosis I and is ultimately responsible for generating genetic diversity during sexual reproduction. HR is initiated in meiosis by numerous programmed DNA double-strand breaks (DSBs; several hundred in mammals). A characteristic feature of HR is the exchange of DNA strands, which results in the formation of heteroduplex DNA. Mismatched nucleotides arise in heteroduplex DNA because the participating parental chromosomes contain nonidentical sequences. These mismatched nucleotides may be processed by MMR, resulting in nonreciprocal exchange of genetic information (gene conversion). MMR and HR also play prominent roles in mitotic cells during genome duplication; MMR rectifies polymerase misincorporation errors, whereas HR contributes to replication fork maintenance, as well as the repair of spontaneous DSBs and genotoxic lesions that affect both DNA strands. MMR suppresses HR when the heteroduplex DNA contains excessive mismatched nucleotides, termed homeologous recombination. The regulation of homeologous recombination by MMR ensures the accuracy of DSB repair and significantly contributes to species barriers during sexual reproduction. This review discusses the history, genetics, biochemistry, biophysics, and the current state of studies on the role of MMR in homologous and homeologous recombination from bacteria to humans.
Topics: Animals; Biological Evolution; DNA Breaks, Double-Stranded; DNA Mismatch Repair; Homologous Recombination; Humans; Meiosis; Models, Biological; Species Specificity
PubMed: 25731766
DOI: 10.1101/cshperspect.a022657 -
DNA Repair Aug 2017Eukaryotic genomes contain many repetitive DNA sequences that exhibit size instability. Some repeat elements have the added complication of being able to form secondary... (Review)
Review
Eukaryotic genomes contain many repetitive DNA sequences that exhibit size instability. Some repeat elements have the added complication of being able to form secondary structures, such as hairpin loops, slipped DNA, triplex DNA or G-quadruplexes. Especially when repeat sequences are long, these DNA structures can form a significant impediment to DNA replication and repair, leading to DNA nicks, gaps, and breaks. In turn, repair or replication fork restart attempts within the repeat DNA can lead to addition or removal of repeat elements, which can sometimes lead to disease. One important DNA repair mechanism to maintain genomic integrity is recombination. Though early studies dismissed recombination as a mechanism driving repeat expansion and instability, recent results indicate that mitotic recombination is a key pathway operating within repetitive DNA. The action is two-fold: first, it is an important mechanism to repair nicks, gaps, breaks, or stalled forks to prevent chromosome fragility and protect cell health; second, recombination can cause repeat expansions or contractions, which can be deleterious. In this review, we summarize recent developments that illuminate the role of recombination in maintaining genome stability at DNA repeats.
Topics: Animals; DNA; DNA Repair; DNA Repeat Expansion; DNA Replication; Genomic Instability; Humans; Nucleic Acid Conformation; Recombination, Genetic; Yeasts
PubMed: 28641941
DOI: 10.1016/j.dnarep.2017.06.018 -
Nature Reviews. Molecular Cell Biology Mar 2010Mitotic homologous recombination promotes genome stability through the precise repair of DNA double-strand breaks and other lesions that are encountered during normal... (Review)
Review
Mitotic homologous recombination promotes genome stability through the precise repair of DNA double-strand breaks and other lesions that are encountered during normal cellular metabolism and from exogenous insults. As a result, homologous recombination repair is essential during proliferative stages in development and during somatic cell renewal in adults to protect against cell death and mutagenic outcomes from DNA damage. Mutations in mammalian genes encoding homologous recombination proteins, including BRCA1, BRCA2 and PALB2, are associated with developmental abnormalities and tumorigenesis. Recent advances have provided a clearer understanding of the connections between these proteins and of the key steps of homologous recombination and DNA strand exchange.
Topics: Animals; DNA Breaks, Double-Stranded; DNA Breaks, Single-Stranded; DNA Repair; Genomic Instability; Humans; Mitosis; Models, Biological; Neoplasms; Recombination, Genetic
PubMed: 20177395
DOI: 10.1038/nrm2851