scholarly journals DNA polymerase   overexpression stimulates the Rad51-dependent homologous recombination in mammalian cells

2004 ◽  
Vol 32 (17) ◽  
pp. 5104-5112 ◽  
Author(s):  
Y. Canitrot
Keyword(s):  
Homologous Recombination ◽  
Dna Polymerase ◽  
Mammalian Cells

scholarly journals DNA polymerase theta suppresses mitotic crossing over

2020 ◽  
Author(s):  
Juan Carvajal-Garcia ◽  
K. Nicole Crown ◽  
Dale A. Ramsden ◽  
Jeff Sekelsky
Keyword(s):  
Homologous Recombination ◽  
Loss Of Heterozygosity ◽  
Dna Polymerase ◽  
Mammalian Cells ◽  
Holliday Junction ◽  
Crossing Over ◽  
Chromosome Break ◽  
Repair Pathway ◽  
Chromosome Breaks ◽  
Break Repair

AbstractPolymerase theta-mediated end joining (TMEJ) is a chromosome break repair pathway that is able to rescue the lethality associated with the loss of proteins involved in early steps in homologous recombination (e.g., BRCA1/2). This is due to the ability of polymerase theta (Pol θ) to use resected, 3’ single stranded DNA tails to repair chromosome breaks. These resected DNA tails are also the starting substrate for homologous recombination. However, it remains unknown if TMEJ can compensate for the loss of proteins involved in more downstream steps during homologous recombination. Here we expand the number of homologous recombination proteins synthetic lethal with Pol θ to the Holliday junction resolvases SLX4 and GEN1. SLX4 and GEN1 are required for viability in the absence of Pol θ in Drosophila melanogaster, and lack of all three proteins results in very high levels of apoptosis. We observe that flies deficient in Pol θ and SLX4 are extremely sensitive to DNA damaging agents, and mammalian cells require either Pol θ or SLX4 to survive. Our results suggest that TMEJ and Holliday junction formation/resolution share a common DNA substrate, likely a homologous recombination intermediate, that when left unrepaired leads to cell death. One major consequence of Holliday junction resolution by SLX4 and GEN1 is cancer-causing loss of heterozygosity due to mitotic crossing over. We measured mitotic crossovers in flies after a Cas9-induced chromosome break, and observed that this mutagenic form of repair is increased in the absence of Pol θ. This demonstrates that TMEJ can function upstream of the Holiday junction resolvases to protect cells from loss of heterozygosity. Our work argues that Pol θ can thus compensate for the loss of the Holliday junction resolvases by utilizing homologous recombination intermediates, suppressing mitotic crossing over and preserving the genomic stability of cells.Author summaryChromosome breaks are a common threat to the stability of DNA. Mutations in genes involved in the early steps of homologous recombination (BRCA1 and BRCA2), a mostly error-free chromosome break repair pathway, lead to hereditary breast cancer. Cells lacking BRCA1 and BRCA2 rely on DNA polymerase theta, a key protein for a more error-prone pathway, for survival. Using fruit flies and mammalian cells, we have shown that mutations in genes involved in later steps of homologous recombination (SLX4 and GEN1) also make cells reliant on polymerase theta. Moreover, we have shown that polymerase theta acts upstream of a type of homologous recombination that is error-prone and depends on SLX4 and GEN1. This form of homologous recombination, termed Holliday junction resolution, creates mitotic crossovers, which can lead to loss of heterozygosity and cancer. Our results expand the cellular contexts that make cells depend on polymerase theta for survival, and the substrates that this protein can use to repair chromosome breaks.

Gene Targeting

1999 ◽  
Keyword(s):  
Homologous Recombination ◽  
Gene Targeting ◽  
Mammalian Cells ◽  
Es Cells ◽  
Practical Approach ◽  
Simple Method ◽  
Mouse Es Cells ◽  
Cell Clones ◽  
Previous Edition ◽  
Pros And Cons

Since the publication of the first edition of Gene Targeting: A Practical Approach in 1993 there have been many advances in gene targeting and this new edition has been thoroughly updated and rewritten to include all the major new techniques. It provides not only tried-and-tested practical protocols but detailed guidance on their use and applications. As with the previous edition Gene Targeting: A Practical Approach 2e concentrates on gene targeting in mouse ES cells, but the techniques described can be easily adapted to applications in tissue culture including those for human cells. The first chapter covers the design of gene targeting vectors for mammalian cells and describes how to distinguish random integrations from homologous recombination. It is followed by a chapter on extending conventional gene targeting manipulations by using site-specific recombination using the Cre-loxP and Flp-FRT systems to produce 'clean' germline mutations and conditionally (in)activating genes. Chapter 3 describes methods for introducing DNA into ES cells for homologous recombination, selection and screening procedures for identifying and recovering targeted cell clones, and a simple method for establishing new ES cell lines. Chapter 4 discusses the pros and cons or aggregation versus blastocyst injection to create chimeras, focusing on the technical aspects of generating aggregation chimeras and then describes some of the uses of chimeras. The next topic covered is gene trap strategies; the structure, components, design, and modification of GT vectors, the various types of GT screens, and the molecular analysis of GT integrations. The final chapter explains the use of classical genetics in gene targeting and phenotype interpretation to create mutations and elucidate gene functions. Gene Targeting: A Practical Approach 2e will therefore be of great value to all researchers studying gene function.

Gene replacement with one-sided homologous recombination

1992 ◽  
Vol 12 (1) ◽  
pp. 360-367
Author(s):  
N Berinstein ◽  
N Pennell ◽  
C A Ottaway ◽  
M J Shulman
Keyword(s):  
Homologous Recombination ◽  
Dna Sequences ◽  
Mammalian Cells ◽  
Target Gene ◽  
Gene Replacement ◽  
Immunoglobulin Genes ◽  
Nonhomologous Recombination ◽  
Immunoglobulin Locus ◽  
Chromosomal Sequences ◽  
Dna Vectors

Homologous recombination is now routinely used in mammalian cells to replace endogenous chromosomal sequences with transferred DNA. Vectors for this purpose are traditionally constructed so that the replacement segment is flanked on both sides by DNA sequences which are identical to sequences in the chromosomal target gene. To test the importance of bilateral regions of homology, we measured recombination between transferred and chromosomal immunoglobulin genes when the transferred segment was homologous to the chromosomal gene only on the 3' side. In each of the four recombinants analyzed, the 5' junction was unique, suggesting that it was formed by nonhomologous, i.e., random or illegitimate, recombination. In two of the recombinants, the 3' junction was apparently formed by homologous recombination, while in the other two recombinants, the 3' junction as well as the 5' junction might have involved a nonhomologous crossover. As reported previously, we found that the frequency of gene targeting increases monotonically with the length of the region of homology. Our results also indicate that targeting with fragments bearing one-sided homology can be as efficient as with fragments with bilateral homology, provided that the overall length of homology is comparable. The frequency of these events suggests that the immunoglobulin locus is particularly susceptible to nonhomologous recombination. Vectors designed for one-sided homologous recombination might be advantageous for some applications in genetic engineering.

Topological requirements for homologous recombination among DNA molecules transfected into mammalian cells

1985 ◽  
Vol 5 (8) ◽  
pp. 2080-2089
Author(s):  
C T Wake ◽  
F Vernaleone ◽  
J H Wilson
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Animal Cells ◽  
Linear Molecules ◽  
Sv40 Genome ◽  
Foreign Dna ◽  
The Individual ◽  
Sv40 Dna

Cultured animal cells rearrange foreign DNA very efficiently by homologous recombination. The individual steps that constitute the mechanism(s) of homologous recombination in transfected DNA are as yet undefined. In this study, we examined the topological requirements by using the genome of simian virus 40 (SV40) as a probe. By assaying homologous recombination between defective SV40 genomes after transfection into CV1 monkey cells, we showed that linear molecules are preferred substrates for homologous exchanges, exchanges are distributed around the SV40 genome, and the frequency of exchange is not diminished significantly by the presence of short stretches of non-SV40 DNA at the ends. These observations are considered in relation to current models of homologous recombination in mammalian cells, and a new model is proposed. The function of somatic cell recombination is discussed.

scholarly journals Replication Past the -Radiation-Induced Guanine-Thymine Cross-Link G[8,5-Me]T by Human and Yeast DNA Polymerase

2010 ◽  
Vol 2010 ◽  
pp. 1-10 ◽  
Author(s):  
Paromita Raychaudhury ◽  
Ashis K. Basu
Keyword(s):  
Dna Polymerase ◽  
Mammalian Cells ◽  
Translesion Synthesis ◽  
Kidney Cells ◽  
Equal Frequency ◽  
Cross Link ◽  
Dominant Mutation ◽  
Human Embryonic Kidney Cells ◽  
Radiation Induced

-Radiation-induced intrastrand guanine-thymine cross-link, G[8,5-Me]T, hinders replicationin vitroand is mutagenic in mammalian cells. Herein we reportin vitrotranslesion synthesis of G[8,5-Me]T by human and yeast DNA polymerase (hPol and yPol ). dAMP misincorporation opposite the cross-linked G by yPol was preferred over correct incorporation of dCMP, but further extension was 100-fold less efficient for :A compared to :C. For hPol , both incorporation and extension were more efficient with the correct nucleotides. To evaluate translesion synthesis in the presence of all four dNTPs, we have developed a plasmid-based DNA sequencing assay, which showed that yPol was more error-prone. Mutational frequencies of yPol and hPol were 36% and 14%, respectively. Targeted was the dominant mutation by both DNA polymerases. But yPol induced targeted in 23% frequency relative to 4% by hPol . For yPol , targeted and constituted 83% of the mutations. By contrast, with hPol , semi-targeted mutations (7.2%), that is, mutations at bases near the lesion, occurred at equal frequency as the targeted mutations (6.9%). The kind of mutations detected with hPol showed significant similarities with the mutational spectrum of G[8,5-Me]T in human embryonic kidney cells.

scholarly journals Topological requirements for homologous recombination among DNA molecules transfected into mammalian cells.

1985 ◽  
Vol 5 (8) ◽  
pp. 2080-2089 ◽  
Author(s):  
C T Wake ◽  
F Vernaleone ◽  
J H Wilson
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Animal Cells ◽  
Linear Molecules ◽  
Sv40 Genome ◽  
Foreign Dna ◽  
The Individual ◽  
Sv40 Dna

Cultured animal cells rearrange foreign DNA very efficiently by homologous recombination. The individual steps that constitute the mechanism(s) of homologous recombination in transfected DNA are as yet undefined. In this study, we examined the topological requirements by using the genome of simian virus 40 (SV40) as a probe. By assaying homologous recombination between defective SV40 genomes after transfection into CV1 monkey cells, we showed that linear molecules are preferred substrates for homologous exchanges, exchanges are distributed around the SV40 genome, and the frequency of exchange is not diminished significantly by the presence of short stretches of non-SV40 DNA at the ends. These observations are considered in relation to current models of homologous recombination in mammalian cells, and a new model is proposed. The function of somatic cell recombination is discussed.

scholarly journals Amifostine Metabolite WR-1065 Disrupts Homologous Recombination in Mammalian Cells

2010 ◽  
Vol 173 (2) ◽  
pp. 175-183 ◽  
Author(s):  
Jaroslaw Dziegielewski ◽  
Wilfried Goetz ◽  
Jeffrey S. Murley ◽  
David J. Grdina ◽  
William F. Morgan ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells

Mutations and Homologous Recombination Induced by N-Substituted Aryl Compounds in Mammalian Cells

Nitroarenes ◽  
1990 ◽  
pp. 149-156
Author(s):  
Veronica M. Maher ◽  
M. Chia-Miao Mah ◽  
Jia-Ling Yang ◽  
Nitai P. Bhattacharyya ◽  
J. Justin McCormick
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells
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